A Clinical Study to Assess the Feasibility of a Controlled Human Plasmodium Vivax Malaria Infection Model Through Sporozoite Infection in Thai Adults
Phase: NA
Conditions: Plasmodium Vivax Infection
Sponsor: University of Oxford

MIST 1 protocol V 10.0 dated 14 February 2022                                               Page 1 of 85 
 
A clinical study to assess the feasibility of a controlled human Plasmodium vivax 
malaria infection model through experimental sporozoite infection in Thai 
Adults 
Short Study Title: Vivax malaria human infection studies in Thailand 
Protocol Number: MAL19002 
OxTREC Ref: 43-19 
Trial Registry Name: ClinicalTrials.gov; ClinicalTrials.gov ID: NCT04083508 
Date and Version No:  14/02/2022 Version 10.0 
Co-Principal Investigator 
 
 
 
 
 
 
 
 
 
Professor Nicholas Day  
Mahidol-Oxford Tropical Medicine Research Unit (MORU) 
Faculty of Tropical Medicine 
Mahidol University 
420/6 Rajvithi Road 
Bangkok 10400 
Thailand 
Tel. +66-(0)2-3549170 
Email: nickd@tropmedres.ac 
Mobile Thailand: +66 9 2648 6322 
Co-Principal Investigator 
Research Professor Dr. Jetsumon Sattabongkot Prachumsri 
Mahidol Vivax Research Unit ( MVRU) 
Faculty of Tropical Medicine 
Mahidol University 
420/6 Rajvithi Road 
Bangkok 10400 
Thailand 
Tel. +66-(0)2-35491000 ext 2022 
Email: jetsumon.pra@mahidol.ac.th 

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Mobile Thailand: +66 8 1611 4241 
Site-Principal Investigator 
Assist. Professor Podjanee Jittamala 
Mahidol-Oxford Tropical Medicine Research Unit (MORU) 
Faculty of Tropical Medicine 
Mahidol University 
420/6 Rajvithi Road 
Bangkok 10400 
Thailand 
Tel. +66-(0)2-3549170 
Email: Podjanee@tropmedres.ac 
Mobile Thailand: +66 8 1956 3371 
Co-Investigator 
Professor Sasithon Pukrittayakamee 
Mahidol-Oxford Tropical Medicine Research Unit (MORU) 
Faculty of Tropical Medicine 
Mahidol University 
420/6 Rajvithi Road 
Bangkok 10400 
Thailand 
Tel. +66-(0)2-3549170 
Email: yon@tropmedres.ac 
Mobile Thailand: +66 8 2360 8261 
Co-Investigator 
Assist. Professor Borimas Hanboonkunupakarn 
Mahidol-Oxford Tropical Medicine Research Unit (MORU) 
Faculty of Tropical Medicine 
Mahidol University 
420/6 Rajvithi Road 
Bangkok 10400 
Thailand 

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Tel. +66-(0)2-3549170 
Email: Borimas@tropmedres.ac 
Mobile Thailand: +66 8 6970 5705 
Co-Investigator 
Associate Professor Kittiyod Poovorawan 
Mahidol-Oxford Tropical Medicine Research Unit (MORU) 
Faculty of Tropical Medicine 
Mahidol University 
420/6 Rajvithi Road 
Bangkok 10400 
Thailand 
Tel. +66-(0)2-3549170 
Email: kittiyod.poo@mahidol.ac.th 
Mobile Thailand: +66  8 3149 6864 
Co-Investigator 
Professor Sir Nick White 
Mahidol-Oxford Tropical Medicine Research Unit (MORU) 
Faculty of Tropical Medicine 
Mahidol University 
420/6 Rajvithi Road 
Bangkok 10400 
Thailand 
Tel. +66-(0)2-3549170 
Email: nickwdt@tropmedres.ac  
Mobile Thailand: +66 9 203 7200 
Co-Investigator 
Professor Arjen Dondorp 
Mahidol-Oxford Tropical Medicine Research Unit (MORU) 
Faculty of Tropical Medicine 
Mahidol University 
420/6 Rajvithi Road 

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Bangkok 10400 
Thailand 
Tel. +66-(0)2-3549170 
Email: arjen@tropmedres.ac  
Co-Investigator 
Dr. Angela M Minassian 
Centre for Clinical Vaccinology and Tropical Medicine, 
Jenner Institute, University of Oxford, 
Churchill Hospital, Old Road, Headington, 
Oxford, OX3 7LE 
Email: angela.minassian@ndm.ox.ac.uk 
Co-Investigator 
Professor Philip Bejon 
Kemri-Wellcome Trust research program 
Kilifi, Kenya 
Email: pbejon@kemri-wellcome.org 
Co-Investigator 
Professor Mallika Imwong 
Molecular Tropical Medicine and Genetics 
Faculty of Tropical Medicine 
Mahidol University 
420/6 Rajvithi Road 
Bangkok 10400 Thailand 
Email: mallika.imw@mahidol.ac.th 
Co-Investigator 
Professor Kesinee Chotivanich 
Mahidol-Oxford Tropical Medicine Research Unit (MORU) 
Faculty of Tropical Medicine 
Mahidol University 
420/6 Rajvithi Road 
Bangkok 10400 
Thailand 

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Tel. +66-(0)2-3549170 
Email: Nok@tropmedres.ac 
Mobile Thailand: +668 1821 4263 
Co-Investigator 
Dr. Wanlapa Roobsoong 
Mahidol Vivax Research Unit ( MVRU) 
Faculty of Tropical Medicine 
Mahidol University 
420/6 Rajvithi Road 
Bangkok 10400 
Thailand 
Tel. +66-(0)2-35491000 ext 2020 
Email: wanlapa.ros@mahidol.edu 
Co-Investigator 
Associate Professor Jiraporn Ruangsittichai 
The Department of Medical Entomology 
Faculty of Tropical Medicine 
Mahidol University 
420/6 Rajvithi Road 
Bangkok 10400 
Thailand 
Tel. +66-(0)2 2400056 
Email: Jiraporn.ru@mahidol.ac.th 
Co-Investigator 
Associate Professor Pornsawan Leaungwutiwong 
Tropical Medicine Diagnostic Reference laboratory 
Department of Microbiology and Immunology 
Faculty of Tropical Medicine 
Mahidol University 
420/6 Rajvithi Road 
Bangkok 10400 

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Thailand 
Tel. +66-(0)2 3069100 ext 3082 
Email: Pornsawan.lea@mahidol.ac.th 
Statistician 
Dr. Mavuto Mukata 
Mahidol-Oxford Tropical Medicine Research Unit (MORU) 
Faculty of Tropical Medicine 
Mahidol University 
420/6 Rajvithi Road 
Bangkok 10400 
Thailand 
Tel. +66-(0) 2-3549170 
Email: mavuto@tropmedres.ac 
Mobile Thailand: +66 9 9640 3249  
Data manager 
Ms. Naomi Waitira 
Mahidol-Oxford Tropical Medicine Research Unit (MORU) 
Faculty of Tropical Medicine 
Mahidol University 
420/6 Rajvithi Road 
Bangkok 10400 
Thailand 
Tel. +66-(0) 2-3549170 
Email: naomi@tropmedres.ac 

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Project manager 
Dr. Pongphaya Pongsuwan 
Mahidol-Oxford Tropical Medicine Research Unit (MORU) 
Faculty of Tropical Medicine 
Mahidol University 
420/6 Rajvithi Road 
Bangkok 10400 
Thailand 
Tel. +66-(0)2-3549170 
Email: pongphaya@tropmedres.ac 
Mobile Thailand: +66 8 1172 3170 
Sponsor  
University of Oxford 
Clinical Trials and Research Governance  
Joint Research Office, Block 60, Churchill Hospital,  
Old Road, Headington, Oxford, OX3 7LE, United Kingdom 
Tel: 01865 572224 
Fax: 01865 572228 
Email: ctrg@admin.ox.ac.uk 
Funding partner  
Wellcome Trust 
Medical Monitoring Team Leader 
Dr. Lorenz Von Seidlein MD 
 
Confidentiality Statement 
This document contains confidential information that must not be disclosed to anyone other than the 
sponsor, the investigator team, regulatory authorities, and members of the research ethics committees. 
This information cannot be used for any purpose other than the evaluation or conduct of the clinical 
investigation without the prior written consent of Site Principal Investigator, Assist Professor Dr. Podjanee 
Jittamala AND both Co-PIs, Professor Dr. Nicholas Day and Research Professor Dr. Jetsumon Sattabongkot 
Prachumsri. 
 

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Investigator Agreement and Conflict of Interest 
 
We have read this protocol and agree to abide by all provisions set forth therein. We agree to comply with the 
principles of the International Conference on Harmonisation Tripartite Guideline on Good Clinical Practice.  
 
We declare no conflict of interest, according to the current version of the Declaration of Helsinki. 
 
 
Professor Nicholas Day 
Co-Principal Investigator 
 
Signature 
 
Date  
 
 
Research Professor Dr. Jetsumon Sattabongkot Prachumsri 
Co-Principal Investigator 
 
Signature 
 
Date  
 
 
Assist. Professor Podjanee Jittamala 
Site Principal Investigator 
 
Signature 
 
Date  

MIST 1 protocol V 10.0 dated 14 February 2022                                               Page 9 of 85 
TABLE OF CONTENTS        
1. 
SYNOPSIS ........................................................................................................................................ 15 
2. 
BACKGROUND, OVERVIEW AND RATIONALE .................................................................................... 17 
2.1 
PLASMODIUM VIVAX MALARIA ...................................................................................................................... 17 
2.2 
THE IMPACT OF PLASMODIUM VIVAX INFECTION .............................................................................................. 17 
2.3 
CHALLENGES IN P. VIVAX DRUG AND VACCINE DEVELOPMENT ............................................................................. 18 
2.4 
VACCINE DEVELOPMENT .............................................................................................................................. 18 
2.5 
CONTROLLED HUMAN INFECTION STUDIES ....................................................................................................... 19 
2.6 
CONTROLLED HUMAN MALARIA INFECTION STUDIES.......................................................................................... 20 
2.7 
CONTROLLED HUMAN MALARIA INFECTION WITH PLASMODIUM FALCIPARUM ....................................................... 20 
2.8 
CONTROLLED HUMAN MALARIA INFECTION WITH PLASMODIUM VIVAX ................................................................ 21 
2.9 
BACKGROUND TO FIRST VIVAX CHMI STUDY IN THAILAND (COVERED BY CURRENT PROTOCOL) ................................ 27 
2.10 
OVER VIEW OF THE WHOLE PROJECTS (POSITION OF CURRENT STUDY PROTOCOL WITHIN THE OVERALL THAILAND VIVAX 
CONTROLLED HUMAN CHALLENGE INFECTION PROGRAMME) .......................................................................................... 28 
3. 
OBJECTIVES ..................................................................................................................................... 30 
3.1 
PRIMARY OBJECTIVES: ................................................................................................................................. 30 
3.2 
SECONDARY OBJECTIVES: ............................................................................................................................. 30 
4. 
STUDY END POINTS ......................................................................................................................... 30 
4.1 
PRIMARY ENDPOINTS:.................................................................................................................................. 30 
4.2 
SECONDARY ENDPOINTS:.............................................................................................................................. 30 
5. 
STUDY DESIGN ................................................................................................................................ 30 
5.1 
SUMMARY OF TRIAL DESIGN ......................................................................................................................... 31 
6. 
PARTICIPANT IDENTIFICATION AND RECRUITMENT .......................................................................... 31 
6.1 
TRIAL PARTICIPANTS .................................................................................................................................... 31 
6.2 
INCLUSION CRITERIA .................................................................................................................................... 31 

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6.3 
EXCLUSION CRITERIA ................................................................................................................................... 31 
7. 
STUDY PROCEDURES ....................................................................................................................... 33 
7.1 
RECRUITMENT OF VOLUNTEERS ..................................................................................................................... 34 
7.2 
INFORMED CONSENT ................................................................................................................................... 35 
7.3 
SCREENING AND ELIGIBILITY ASSESSMENT ........................................................................................................ 36 
7.3.1 
SCREENING VISIT 1 .................................................................................................................................... 36 
7.3.2 
SCREENING VISIT 2: 7 DAYS BEFORE CHALLENGE (D-7) .................................................................................... 37 
7.3.3 
SCREENING VISIT 3/ A DAY BEFORE THE CHALLENGE DAY (1 DAY BEFORE CHALLENGE, D-1) ................................... 37 
7.4 
INTERVENTIONS IN VOLUNTEERS .................................................................................................................... 39 
7.4.1 
INPATIENT PHASE UNTIL END OF ANTIMALARIAL TREATMENT: ........................................................................... 39 
7.4.2 
FOLLOW UP ASSESSMENT PHASE (AFTER COMPLETION OF ANTIMALARIAL TREATMENT) ......................................... 44 
7.5 
DETAILS OF STUDY CLINICAL PROCEDURES/LABORATORY TESTS ........................................................................... 57 
7.5.1 
CLINICAL PROCEDURES ............................................................................................................................... 57 
7.5.2 
LABORATORY PROCEDURES ......................................................................................................................... 57 
7.5.3 
BLOOD DONATION, PROCESSING AND STORAGE.............................................................................................. 59 
7.6 
DEFINITION OF START AND END OF TRIAL ........................................................................................................ 60 
7.7 
WITHDRAWAL OF VOLUNTEERS ............................................................................................................. 60 
8. 
DESCRIPTION AND ADMINISTRATION OF THE STUDY AGENTS (WHICH IS THE P. VIVAX INFECTED 
MOSQUITOES) ........................................................................................................................................ 61 
8.1 
PREPARING THE STUDY AGENT: INFECTED MOSQUITO PREPARATION STAGE .......................................................... 61 
8.1.1 
SOURCE CASE IDENTIFICATION (P. VIVAX PATIENT(S)): ..................................................................................... 61 
8.1.2 
MOSQUITO PREPARATION AND MEMBRANE FEEDING: ..................................................................................... 62 
8.2 
ADMINISTERING THE STUDY AGENT TO THE VOLUNTEERS: .................................................................................. 62 
9. 
SAFETY............................................................................................................................................ 63 
9.1 
SAFETY EXPERIENCE FROM PREVIOUS CHMI STUDIES ........................................................................................ 63 
9.2 
WHAT DO WE EXPECT TO DISCOVER DURING THE CHALLENGE? ........................................................................... 65 
9.3 
RISKS ........................................................................................................................................................ 66 
9.3.1 
PHLEBOTOMY........................................................................................................................................... 66 
9.3.2 
BLOOD DONATION (UP TO 250 ML) ............................................................................................................. 66 
9.3.3 
MOSQUITO BITES ...................................................................................................................................... 67 
9.3.4 
PLASMODIUM VIVAX INFECTION .................................................................................................................. 67 

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9.3.5 
RISK OF RELAPSE OF MALARIA ..................................................................................................................... 67 
9.3.6 
CONTINGENCY PLAN IF SUBJECT GOING MISSING POST-CHALLENGE .................................................................... 68 
9.4 
SAFETY DETECTION, ASSESSMENT, DOCUMENTATION AND REPORTING ................................................................. 68 
9.4.1 
DEFINITIONS ............................................................................................................................................ 69 
9.4.2 
CLINICAL LABORATORY PARAMETERS AND OTHER ABNORMAL ASSESSMENTS QUALIFYING AS ADVERSE EVENTS OR 
SERIOUS ADVERSE EVENTS: ....................................................................................................................................... 70 
9.4.3 
REPORTING PROCEDURES ........................................................................................................................... 72 
9.5 
EVENTS OR OUTCOMES NOT QUALIFYING AS ADVERSE EVENTS OR SERIOUS ADVERSE EVENTS ................................... 72 
9.5.1 
PREGNANCY ............................................................................................................................................. 73 
9.6 
SAFETY MONITORING .................................................................................................................................. 74 
9.6.1 
MONITORING ........................................................................................................................................... 74 
9.6.2 
MEDICAL MONITOR GROUP ....................................................................................................................... 74 
9.6.3 
DATA AND SAFETY MONITORING BOARD ...................................................................................................... 74 
10. STATISTICS ...................................................................................................................................... 75 
11. DATA, DIRECT ACCESS TO SOURCE DATA/DOCUMENTS .................................................................... 75 
12. QUALITY CONTROL AND QUALITY ASSURANCE PROCEDURES ........................................................... 75 
12.1 
AUDIT & INSPECTION .................................................................................................................................. 75 
13. ETHICS ............................................................................................................................................ 76 
13.1 
PARTICIPANT CONFIDENTIALITY ..................................................................................................................... 76 
13.2 
BENEFIT .................................................................................................................................................... 77 
13.3 
DATA HANDLING AND RECORD KEEPING .......................................................................................................... 77 
14. INSURANCE ..................................................................................................................................... 77 
15. PUBLICATION POLICY ...................................................................................................................... 78 
16. COMPENSATION ............................................................................................................................. 78 
17. REFERENCES .................................................................................................................................... 80 
18. APPENDIX A:   MOSQUITO BITING PROCESSES DIAGRAM ................................................................. 83 

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19. APPENDIX B: TREATMENT ALGORITHM ............................................................................................ 84 
AMENDMENT HISTORY     
Amendment No. 
Protocol Version No. 
Date issued 
Author(s) of changes 
 
 
 
 
 
LIST OF ABREVIATIONS:  
AE 
Adverse event 
β-HCG 
Beta – Human Chorionic Gonadotropin 
BP 
Blood Pressure 
BT 
Body Temperature 
BUN 
Blood Urea Nitrogen 
CBC 
Complete Blood Count 
CHMI 
Controlled human malaria infection 
Cr 
Creatinine 
CRF 
Case Record Form  
CRO 
Contract Research Organization 
CSP 
Circumsporozoite 
CYP2D6 
Enzyme predicting primaquine metabolism (P450 enzyme) 
DBP 
Duffy Binding Protein 
DMFA 
Direct Membrane Feeding Assay 
DSMB 
Data Safety Monitoring Board 
FBS 
Fasting Blood Sugar 

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FTM 
Faculty of Tropical Medicine 
FTMCTU 
Clinical Therapeutic Unit (healthy volunteer ward), Faculty of Tropical 
Medicine 
GCP 
Good Clinical Practice 
GMP 
Good Manufacturing Practice 
HBsAg 
Hepatitis B surface antigen 
HCV 
Hepatitis C virus 
HDL 
High Density Lipoprotein 
HIV 
Human Immunodeficiency virus 
HTLV 
Human T cell Lymphotrophic virus 
ICF 
Informed Consent Form 
ICH 
International Conference on Harmonisation 
JE 
Japanese Encephalitis 
LDL 
Low Density Lipoprotein 
LFT 
Liver Function Test 
MFA 
Membrane Feeding Assay 
MORU 
Mahidol-Oxford Tropical Medicine Research Unit 
MVRU 
Mahidol Vivax Research Unit 
OxTREC 
The Oxford Tropical Research Ethics Committee 
PCR 
Polymerase Chain Reaction 
P. falciparum 
Plasmodium falciparum 
PI 
Principal Investigator 

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PQ 
Primaquine 
PR 
Pulse Rate 
P. vivax 
Plasmodium vivax 
PvCSP 
P. vivax circumsporozoite 
PSC 
Programme Steering Committee 
qPCR 
Quantitative Polymerase Chain Reaction 
RECs 
Research Ethics Committees 
RR 
Respiratory Rate 
RUNMC 
Radboud University Nijmegen Medical Centre 
SAE 
Serious Adverse Event 
SOP 
Standard Operating Procedure 
TMDR 
Tropical Medicine Diagnostic Reference Laboratory 
ULN 
Upper Limit of Normal 
USMMVP 
U.S. Military Malaria Vaccine Program 
WHO 
World Health Organization 
WI 
Work Instruction

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1. SYNOPSIS 
 
Study Title 
A clinical study to assess the feasibility of a controlled human Plasmodium vivax 
malaria infection model through experimental sporozoite infection in Thai 
Adults 
Short Study Title 
Vivax malaria human infection studies in Thailand 
Study site 
Clinical Therapeutic Unit (FTMCTU), Hospital for Tropical Diseases, Mahidol 
Vivax Research Unit, Medical Entomology Department and TMDR, Faculty of 
Tropical Medicine, Mahidol University, Bangkok Thailand. 
Protocol Number  
MAL19002 
Study Design 
Controlled human infection study. 
Overview 
This is a sporozoite-challenge clinical study with the primary objectives of 
assessing the feasibility and safety of controlled human sporozoite P. vivax 
malaria infection in upto six healthy volunteers, and developing a bank of P. 
vivax-infected blood for use as inocula in future controlled human blood stage 
P. vivax malaria infection studies. Secondary objectives are to assess the growth 
of and the immune response to P. vivax infection and assess the induction of 
sexual gametocytaemia post-CHMI via the natural route of malaria infection 
(mosquito bite). 
Study Participants 
Healthy adult volunteers, aged 20-55 years: malaria naïve and ABO blood group 
O. 
Sample size  
Up to 6  
Planned Study Period  
Approximately 1 year from the time of antimalarial treatment initiation. There 
will be a 6 months screening process prior to the malaria challenge and 
approximately 9-16 days pre-patent period.  
Objectives 
Primary objectives: 

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• 
To assess the feasibility and safety of controlled human P. vivax 
malaria infection in six healthy human volunteers, through 
experimental sporozoite infection (mosquito bite). 
• 
To obtain up to 250 mL of blood from each infected volunteer and 
freeze down the blood to create inocula for use in future P. vivax 
challenge studies.  
Secondary objectives: 
• 
To assess the immune response to primary P. vivax infection 
delivered by mosquito bite. 
• 
To assess gametocytaemia following primary P. vivax infection 
delivered by the mosquito bite. 
Study Endpoints 
Primary endpoints: 
• 
Feasibility and safety of P. vivax sporozoite human challenge, as 
measured by successful infection (development of detectable 
persistent parasitaemia +/- clinical symptoms) and AE(S) 
occurrences. 
• 
Collection and freezing down of up to 250 mL P. vivax-infected 
blood from each of the 6 volunteers. 
Secondary endpoint: 
 
• 
Cellular and humoral Immune response to primary P. vivax 
infection. 
• 
Gametocytaemia pre-treatment, as measured by qPCR. 
Medicinal Product  
None 
Medical monitor (chair) 
Dr. Lorenz Von Seidlein, MD  

 
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2. BACKGROUND, OVERVIEW AND RATIONALE 
2.1 
Plasmodium vivax malaria 
Plasmodium vivax (P. vivax) is one of the five Plasmodium species known to cause human malaria. It is 
geographically the most widespread, and accounts for most of the cases of non-P. falciparum malaria. 
Although generally less severe than falciparum malaria vivax malaria is associated with some mortality 
(particularly in children) and significant levels of morbidity, a disease burden which has been chronically 
under-appreciated [1]. P. vivax accounts for up to 50% of malaria cases in South and Southeast Asia, and 
between 71-81% of cases in South America with an estimated global number of between 106 and 313 million 
clinical cases per year. Following sporozoite inoculation by a mosquito clinical cases are not only due to the 
resulting primary infection but are also due to relapses from the hypnozoites in the liver, which occur weeks 
to years after the primary infection [2]. Whereas most of the vivax burden is in Asia and South America, 
there are between 6 - 15 million cases of vivax malaria in Africa per year [3].     
2.2 
The impact of Plasmodium vivax infection 
Plasmodium vivax infection has been shown to cause significant morbidity and attributable mortality, with 
reported complications including severe anaemia, acute respiratory distress syndrome, shock [4-6]. A 
reduction in fatal growth when mothers are infected with P. vivax, despite effective antimalarial therapy, 
has also been shown [7]. The full socio-economic impact of vivax infection is not known, but overall global 
cost due to lost productivity is estimated at US$ 1.4 to 4.0 billion per year [2]. Those affected by P. vivax 
malaria are typically poor with inadequate access to affordable healthcare and with little financial reserve, 
perpetuating the cycle of poverty [8]. 
Control of P. vivax is challenging. This is due to multiple factors including relapses, difficulty detecting 
asymptomatic infection, resistance to antimalarial medications, and a lack of understanding of parasite 
biology [9]. Recent calls for control and ‘eradication’ of malaria worldwide [10] have focused attention on 
this neglected disease and the need for development of an effective P. vivax vaccine to be used alongside 
current control methods. Consequently, the revised Malaria Vaccine Technology Roadmap to 2030 now 
recognises the importance of P. vivax and calls for a vaccine to achieve 75% efficacy over two years – equally 
weighted with P. falciparum in an era of renewed political will to move towards malaria elimination and 
eradication [11]. 
Unlike P. falciparum, to date there has been relatively little research into this malaria species. One of the 
limiting factors has been the inability to culture P. vivax in vitro over a prolonged period. Recently there has 
been an increase in vivax vaccine research, with several candidate vaccines being developed and taken 

 
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forward to clinical trial [12, 13]. However, none of these vaccines has progressed past a Phase IIa trial, and 
options for assessing the protective efficacy of new candidates are extremely limited. 
2.3 
Challenges in P. vivax drug and vaccine development 
The unique characteristics of P. vivax pose some specific challenges in studying its biology and designing 
strategies to control it [14]. For example, the lack of a method for continuous culture of P. vivax blood-
stages makes it difficult to perform in vitro growth inhibition assays to identify synergistic combinations of 
blood-stage antigens that can be targeted to inhibit P. vivax blood-stage growth with high efficiency. In the 
absence of a P. vivax blood-stage culture system, production of infected mosquitoes for sporozoite- or 
transmission-stage studies also requires access to P. vivax patients in endemic areas. Several groups have 
recently succeeded in establishing short-term P. vivax culture for invasion assays using enriched 
reticulocytes from cord blood [15], but such methods are still dependent on access to fresh P. vivax isolates 
from malaria patients, limiting the routine use of such assays to endemic regions. In order to study 
hepatocyte invasion stages of P. falciparum as well as P. vivax, attempts have been made to establish in 
vitro liver models [16, 17]. In addition, a reliable humanised mouse model for P. vivax liver stages is now 
available [18]. Both approaches will help to identify and test novel drugs and vaccines targeting hepatocyte 
stages. Studies on expression of parasite proteins in hypnozoites will determine if these latent stages can 
also be targeted with novel drugs and vaccines. 
2.4 
Vaccine development 
Given that a P. vivax vaccine will in most cases be used in low transmission settings, the ideal vaccine would 
have high efficacy (more than 90%) and block transmission. The development of such a vaccine will likely 
require a combination of multiple antigens that act synergistically to achieve high efficacy. For example, 
achieving high rates of blood-stage growth inhibition may require targeting a combination of key blood-
stage antigens involved in reticulocyte invasion. A combination of blood-stage antigens with liver-stage 
antigens may be needed to achieve high efficacy, and inclusion of antigens from sexual- and mosquito-
stages will be needed to inhibit transmission. It is therefore necessary to initiate efforts to combine antigens 
both within and across developmental stages to achieve synergy and attain the goal of developing a highly 
effective vaccine for P. vivax malaria.  The purpose of developing and deploying sporozoite- and blood-stage 
human challenge models is to accelerate and rationalise vivax vaccine development. Conducting Controlled 
human malaria infection (CHMI) studies in malaria-endemic countries using malaria-exposed volunteers 
provides a means of testing vaccine candidates in a study population genetically and immunologically similar 
to the target population for vaccine deployment. P. vivax vaccine candidates can be validated and down-
selected to ensure that only the most promising candidates proceed to time-consuming and expensive field 
trials against natural challenge. 

 
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Table 1: P. vivax vaccine candidates under development:  from ref 12. 
Vaccine candidate 
Development 
Phase 
Lifecycle 
stage 
Antigen 
Delivery system 
VMP001 
Phase I/IIa END Liver-stage 
PvCSP 
Rec. protein-AS01B 
CSV-S,S 
Pre-clinical 
Liver-stage 
PvCSP 
HBsAg fusion-AS01B 
PvCSP-LSP 
Phase I END 
Liver-stage 
PvCSP 
Synthetic peptides-Montanide ISA 
720 
ChAd63-PvTRAP/MVA-
PvTRAP 
Pre-clinical 
Liver-stage 
PvTRAP 
Prime-boost, viral vectors 
PvDBPII 
Phase I 
Blood-stage 
PvDBP 
Rec. protein-GLA-SE 
PvDBPII-DEKnull 
Pre-clinical 
Blood-stage 
PvDBP 
Rec. protein 
ChAd63-PvDBPII/MVA-
PvDBPII 
Phase I/IIa 
Blood-stage 
PvDBP 
Prime boost, viral vectors 
PvMSP119 
Pre-clinical 
Blood-stage 
PvMSP1 
Rec. protein-Montanide ISA720 
Pvs25H 
Phase Ia END 
Transmission-
stage 
Pvs25 
Rec. protein-Alhydrogel; 
Rec. protein-Montanide ISA 51 
Pvs28 
Pre-clinical 
Transmission-
stage 
Pvs28 
Rec. protein-adjuvant 
Pvs25-IMX313 
Pre-clinical 
Transmission-
stage 
Pvs25 
Rec. protein-adjuvant 
AnAPN1 
Pre-clinical 
Mosquito 
midgut Ag 
AnAPN1 
Rec. protein-adjuvant 
2.5 
Controlled human infection studies 
The controlled human infection or the microbial challenge studies of human volunteers is the deliberate 
infection of human volunteers with micro-organisms under highly controlled conditions. Human challenge 
models have contributed uniquely to the understanding of the pathogenesis, immune responses and the 
treatment and prevention of numerous microbial diseases including influenza, cholera, typhoid, hepatitis 
and malaria [25].  

 
MIST 1 protocol V 10.0 dated 14 February 2022                                               Page 20 of 85 
In vaccine and therapeutic drug development there are frequently multiple candidates, and it is often 
difficult to decide which should be taken forward to expensive clinical field testing. At the proof of concept 
stage of development controlled human challenge trials can be used to assess the efficacy of a novel 
therapeutic or the protective efficacy of a candidate vaccine in a small number of volunteers. This alternative 
research methodology is now more widely used to reduce the cost and time investment to accelerate the 
development of drugs and vaccines. A review by the UK Academy of Medical Sciences on microbial challenge 
studies recognised that such studies are desirable for providing proof of concept for prophylactic and 
therapeutic interventions and can significantly accelerate progress to Phase III studies [25]. 
2.6 
Controlled human malaria infection studies 
Controlled Human Malaria Infection (CHMI) can either be undertaken via 1.) Inoculation of sporozoites by 
mosquito bite or 2.) Direct injection of sporozoites or of plasmodium-infected blood. The inoculation of 
sporozoites by mosquito bite allows both liver and blood stage infection to develop, while inoculation with 
parasitized erythrocytes leads to blood stage infection only. After the inoculation, the subject is carefully 
monitored and the infection is truncated by anti-malarial drug treatment that is initiated promptly according 
to pre-defined study specific criteria, for instance the onset of microscopic patency and/or detection of a 
predefined parasitaemia by quantitative polymerase chain reaction (qPCR).  
2.7 
Controlled human malaria infection with Plasmodium falciparum 
Plasmodium falciparum (P. falciparum) is a microbe particularly well-suited to “challenge” studies. It has a 
relatively short asymptomatic period, a well-established diagnostic laboratory test (blood film microscopy), 
and rapid recovery with no long-term infectious state following appropriate and timely treatment. Studies 
involving controlled human malaria infection are a powerful tool for investigating malaria vaccine and 
prophylactic and therapeutic drug efficacy [26]. With an increasing number of candidate malaria vaccines 
being developed, the number of centres conducting controlled human falciparum malaria infection studies 
is expanding in developed countries, and they are now also being conducted in endemic areas of Africa [26].  
The first well-documented controlled human malaria infection study with laboratory-reared infectious 
mosquitoes was carried out in 1986 at the US Walter Reed Army Institute of Research (WRAIR), the US Naval 
Medical Research Institute (NMRI) and the US National Institutes of Health (NIH) [30]. The following year, 
the efficacies of the first recombinant protein and synthetic peptide P. falciparum vaccines were reported 
for experimentally infected volunteers [31, 32].  Controlled human malaria infection has now become 
established as a key tool to assess the efficacy of novel malaria vaccines and drugs; a total of 1,343 
volunteers were experimentally infected with P. falciparum between 1985 and 2009 [33], [26]. As controlled 
human malaria infection trials are carried out in a controlled environment, they allow detailed evaluation 
of parasite growth and immunological responses, providing key information for vaccine and drug 
development [26]. Currently controlled human challenge infections trials are routinely carried out at: the 

 
MIST 1 protocol V 10.0 dated 14 February 2022                                               Page 21 of 85 
US Military Malaria Vaccine Program (USMMVP); Maryland, USA; Radboud University Nijmegen Medical 
Centre (RUNMC), Netherlands; the University Of Oxford, UK; QIMR Berghofer, Brisbane Australia, and, more 
recently, Seattle Biomed, USA and the KEMRI-Wellcome Programme in Kilifi, Kenya. Other groups in Africa 
who have done falciparum challenge including IHI in Tanzania and Lamberene, in Gabon. 
2.8 
 Controlled human malaria infection with Plasmodium vivax 
In contrast to P. falciparum, modern controlled human malaria infection studies with P. vivax have been 
conducted in much less frequently, with only a small handful of studies reported over the past decade. This 
is largely due to the inability to culture P. vivax long-term in vitro. In only two of the studies published to 
date has efficacy of immunisation been assessed (Table 2) [34]. Unlike with P. falciparum, there is also a risk 
of clinical relapse weeks to years after sporozoite infection if hypnozoites are not cleared from the liver, as 
these have the potential to reactivate.  
Table 2:  Overview of published Plasmodium vivax challenge studies. 
Trial Site 
Number of 
volunteers 
Pre-patent 
period (days)a 
Number of 
infected 
mosquitoes OR 
inoculum 
Number of 
volunteers 
with patent 
parasitaemia 
References 
Sporozoite (mosquito-bite) challenge studies 
Cali, Columbia 
18 
9 – 13 
2 - 10 
17/18 
[19] 
Cali, Columbia 
17 Duffy 
positive 
5 Duffy 
negative 
9 – 16 
2 - 4 
17/17 (Duffy 
positive) 
0/5 (Duffy 
negative) 
[20] 
Cali, Columbia 
7 malaria-naïve 
9 semi-immune 
11 – 13 
2 - 4 
16/16 
[22] 
WRAIR, USA 
6 malaria-naïve 
6 malaria -
naive 
9-12 
12-14 
5 
5 
6/6 
6/6 
[57] and 
unpublished data 
(NCT00935623) 
WRAIR, USA 
27 vaccinees 
10 – 13 
10 – 11 
5 
33/33 
[24]   

 
MIST 1 protocol V 10.0 dated 14 February 2022                                               Page 22 of 85 
Trial Site 
Number of 
volunteers 
Pre-patent 
period (days)a 
Number of 
infected 
mosquitoes OR 
inoculum 
Number of 
volunteers 
with patent 
parasitaemia 
References 
6 infectivity 
controls 
 
Cali, Columbia 
12 Duffy 
positive 
vaccines 
2 Duffy positive 
controls 
5 Duffy 
negative 
controls 
12 – 13 
2 - 4 
7/12 vaccinees 
2/2 Duffy 
positive 
controls 
0/5 Duffy 
negative 
controls 
[23] 
Oxford university 
Oxford, UK 
( 2018) 
2 Blood group 
O Rh negative 
 
10-11 
5 
2/2 
[Personal contact    
(Draper S. and 
Minassian A)] 
Blood-stage challenge studies (IBSM) 
QIMRB, Australia 
(ACTRN1261200109
6842) 
2 
8-9 
13,000 genome 
equivalents 
2/2 
[21] 
QIMRB, Australia 
(ACTRN1261300100
8718) 
6 
8-9 
31,786 (± 11,947) 
as determined by 
qPCR (= 15 
5 viable P. vivax 
parasites) 
6/6 
[35] 
 
 QIMRB, Australia 
(ACTRN1261400093
0684) 
2 
2/2 positive 
day 5  
Inoculum –  
564 PRBC  
2/2 
[3]Collins et al in 
preparation * 

 
MIST 1 protocol V 10.0 dated 14 February 2022                                               Page 23 of 85 
Trial Site 
Number of 
volunteers 
Pre-patent 
period (days)a 
Number of 
infected 
mosquitoes OR 
inoculum 
Number of 
volunteers 
with patent 
parasitaemia 
References 
 QIMRB, Australia 
(ACTRN1261600017
4482) 
24 
21/24 
positive day4 
3/24 positive 
day5  
Inoculum –  
564 PRBC  
 24/24  
[3] Collins et al in 
preparation * 
 QIMRB, Australia 
NCT02573857 
8  
8/8 positive 
day 4  
Inoculum –  
564 PRBC  
8/8 
McCarthy et al 
(submitted 2019) 
 QIMRB, Australia 
ACTRN12617001502
325 
4  
All positive 
day 4  
Inoculum –  
564 PRBC  
4/4 
[59] 
Oxford, dose finding 
(inoculation) 
6  
All positive 
day 14-16 
1:5x2, 1:20x2, 
whole vialx2 
6/6 
[Personal contact    
(Draper S. and 
Minassian A)] 
Oxford, vaccine trial 
(ChAd63 PvDBP and 
MVA PvDBP) 
8 
Data not 
available 
Data not 
available 
8 
[Personal contact    
(Draper S. and 
Minassian A)] 
a The pre-patent period refers to the period before malaria diagnosis: by blood film (sporozoite) or qPCR 
(blood-stage).  
Despite a paucity of modern challenge trial experience, there is however an extensive history of deliberate 
infection with P. vivax; most notably in malariotherapy, which was carried out for the treatment of 
neurosyphilis almost a century ago. The Austrian psychiatrist Julius Wagner-Jauregg later received a Nobel 
Prize for his work with this treatment [36], and the practice was widely adopted as the only effective 
treatment available at the time. Malariotherapy provided a wealth of information about P. vivax infection, 
which has been reviewed previously [37]. Deliberate infection with P. vivax was also conducted in the USA 
from the 1940s to 1970s in prisoners involved in the Malaria Research Project at the Illinois State 
Penitentiary. These studies mainly examined compounds for their potential use as antimalarials [38]. Similar 
studies were also carried out at the United States Penitentiary, Atlanta. Key discoveries of the biology of P. 
vivax were made during this period including, for example, the association between Duffy negativity and 
resistance to P. vivax infection [39]. Following on from the studies of P. vivax infection conducted in Illinois, 

 
MIST 1 protocol V 10.0 dated 14 February 2022                                               Page 24 of 85 
controlled human challenge infections were carried out to see if prior exposure to irradiated mosquitoes 
could confer protection by immunization. Rieckmann et al. [40] reported no protection against controlled 
human challenge infections in three volunteers previously exposed to P. vivax-infected irradiated 
mosquitoes on four occasions at intervals of 2 – 4 weeks (total of <200 mosquitoes). 
Three P. vivax controlled human challenge infection studies assessing the ability to ‘immunise’ with X-
irradiated sporozoites also took place in Maryland, USA during the 1970s [58]. Following immunisation, 
challenge infection was initiated by periodic exposure to the bites of non-irradiated infected mosquitoes in 
three volunteers in separate experiments, and blood films were taken at least daily for all volunteers to 
assess outcome. These experiments in Maryland demonstrated that controlled human malaria infection 
studies with both P. falciparum and P. vivax could be successfully carried out, but the studies were very 
small. 
More recent P. vivax mosquito-bite controlled human challenge infections trials have taken place in Cali, 
Colombia and at the Walter Reed Army Institute of Research (WRAIR), Maryland, USA.  
The first of the Cali trials involved eighteen healthy volunteers exposed to the bites of 2-10 infected An. 
albimanus mosquitoes [19]. P. vivax infection was established in mosquito lots fed on blood from fifteen 
patients presenting to outpatient clinics at the Immunology Institute in Cali and Buenaventura with P. vivax 
infection. Four mosquito lots had to be discarded due to co-infection with P. falciparum, hepatitis B or 
hepatitis C in the donor blood. Seventeen of the eighteen volunteers developed P. vivax malaria, confirmed 
by blood smear, with a pre-patent period between 9 and 13 days. At diagnosis, volunteers were treated 
with standard P. vivax therapy consisting of chloroquine (600 mg initially, followed by 450 mg 24 and 48 
hours later) to clear blood-stage infection and primaquine (PQ) (30 mg/day for 14 days) to achieve radical 
cure. Levels of parasitaemia ranged from 75-420 parasites/µL; parasites were cleared within 48 hours of 
starting treatment in all infected volunteers. There were no serious adverse events (SAEs) in this trial, but 
seven volunteers required fluid therapy due to nausea and vomiting, and five developed blurred vision 
lasting 2-3 days after treatment initiation. Authors speculated that the volunteer who did not develop 
malaria had surreptitiously taken anti-malarial medication, but this was never confirmed. 
The second P. vivax mosquito-bite controlled human challenge infections trial was carried out by the same 
group in Colombia, aiming to demonstrate the reproducibility of this method of infection using three 
different An. albimanus mosquito lots fed on blood from three P. vivax-infected donors [20]. Seventeen 
individuals whose red blood cells were positive for the Duffy antigen/chemokine receptor (DARC, “Duffy 
positive”) and five Duffy negative controls were enrolled into the study. Volunteers were randomly assigned 
to three groups (with six Duffy positive individuals in two of the groups and five in the third group), and 
exposed to the bites of 2-4 infected mosquitoes. The Duffy negative controls were assigned across the three 

 
MIST 1 protocol V 10.0 dated 14 February 2022                                               Page 25 of 85 
groups, with two controls in the first two groups and one in the third. All Duffy positive volunteers (and 
none of the Duffy negative volunteers) developed blood-stage malaria. The pre-patent period ranged from 
9-16 days, and was different in the first group, with a median of 14 days, as opposed to a median of 10 days 
in the second two groups. 
A third P. vivax mosquito-bite controlled human challenge infections trial was carried out in Cali but this 
time among both ‘semi-immune’ (previously-exposed; n=9) and malaria-naïve adult volunteers (n=7) [22]. 
The pre-patent periods ranged between 11-13 days, with no significant difference between the malaria-
naïve and ‘semi-immune’ volunteers. Symptoms were significantly worse among the malaria-naïve subjects 
but there were no SAEs. One malaria-naïve volunteer presented three months after treatment with a P. 
vivax infection after visiting an endemic area. However, the investigators were unable to determine whether 
this episode of malaria was due to reinfection or relapse, as the volunteer had visited the same endemic 
area that the controlled human malaria infection study strain had come from. 
The most recent mosquito-bite controlled human challenge infections trial from the Cali group involved the 
use of radiation-attenuated sporozoites in Duffy positive and Duffy negative healthy adult volunteers, 
delivered by mosquito bite. Mosquitoes were infected as described above for the other trials conducted by 
this group. Sporozoites were attenuated by exposing the mosquitoes to 150 ± 10 cGy of gamma irradiation. 
Twelve Duffy positive volunteers and five Duffy negative controls completed the immunisation phase with 
exposure to P. vivax infected irradiated An. albimanus mosquitoes followed by mosquito-bite controlled 
human challenge infections. Two Duffy positive controls also completed the trial; these volunteers were 
exposed to non-irradiated, non-infected mosquitoes. Seven immunisations were carried out for each 
volunteer on weeks 0, 8, 12, 23, 48, 51 and 56 with a mean of 65 infectious bites for each immunisation. 
Two weeks after the final immunisation volunteers were treated with chloroquine and primaquine to clear 
any malaria infections that may have developed during the immunisation phase. Plasma levels of 
chloroquine and primaquine were checked prior to CHMI to ensure drug clearance. Challenge was carried 
out at week 64 using 2 - 4 P. vivax-infected mosquito bites, and volunteers were monitored daily with blood 
films from day 6. There were no reported SAEs related to immunisation, although one volunteer developed 
severe elevation of hepatic transaminases (>10 times the upper limit of normal [x ULN]) with associated 
abdominal pain and vomiting following mosquito-bite controlled human challenge infections, with no 
alternative cause found. These symptoms resolved spontaneously. The protective efficacy of the 
immunisation schedule was 42% (5/12 Duffy positive volunteers protected). In volunteers who developed 
malaria, the mean pre-patent period until blood film positivity was 12.8 days. Interestingly, all of the 
volunteers protected in this trial were female [23]. 
Only one other mosquito-bite controlled human challenge infections trial assessing a P. vivax vaccine has 
been published to date. The VMP001/ AS01B vaccine was tested in healthy malaria-naïve adults at the 

 
MIST 1 protocol V 10.0 dated 14 February 2022                                               Page 26 of 85 
WRAIR in the USA [24]. VMP001 is a soluble recombinant protein vaccine [41], encoding the P. vivax 
circumsporozoite protein (PvCSP) administered with the AS01B adjuvant (GlaxoSmithKline). The vaccine was 
administered to 30 volunteers in three cohorts (10 in each) at doses of 15 µg, 30 µg and 60 µg, given three 
times at a 4 week interval between the first and second dose; the third dose was given 8 (15 µg cohort), 6 
(30 µg cohort) or 4 (60 µg cohort) weeks after the second. Twenty-nine volunteers completed the 
vaccination phase, with twenty-seven proceeding to mosquito-bite controlled human challenge infections 
two weeks after final vaccination, along with 6 malaria-naïve controls. The mosquito-bite controlled human 
challenge infections with 5 P. vivax-infected An. dirus mosquitoes was undertaken. Laboratory-reared 
mosquitoes were fed on blood from a P. vivax-infected donor in a clinical laboratory in Mae Sot province in 
Thailand after screening by polymerase chain reaction (PCR) to ensure no co-infection with other 
Plasmodium species or blood-borne infections. Infected mosquitoes were then transported to WRAIR and 
maintained in their insectary until mosquito-bite controlled human challenge infections. Volunteers were 
treated following a diagnosis of vivax malaria by blood smear. The vaccine protective efficacy was 0 %; all 
volunteers had developed blood film-detectable parasitaemia by day 13. The median pre-patent period for 
all immunised volunteers was 11.9 days versus 10.7 days for infectivity controls. The challenge was well 
tolerated with no untoward reactogenicity following mosquito bites. No untoward SAEs were observed 
following the challenge and treatment phases. Volunteers were treated with standard chloroquine and 
primaquine therapy with rapid clearance of infection. However, two volunteers went on to have multiple 
relapses. One volunteer experienced two relapses (at weeks 8 and 18 after challenge), while the other 
experienced three (at weeks 11, 20 and 48 after challenge) [42]. By study completion the volunteers had 
been followed up for 5 years, and had not had any further relapses [24]. Exploratory genotyping for the 
cytochrome P450 (CYP) allele CYP2D6 was undertaken in 25 of the 33 volunteers. The volunteers with 
relapses were found to have either an intermediate-metaboliser phenotype or poor-metaboliser 
phenotype. These phenotypes were associated with significantly lower levels of primaquine clearance 24 
hours after dosing [42]. Primaquine is metabolised into redox-active metabolites by CYP2D6, and therefore 
individuals who are unable to metabolise the drug in sufficient quantities appear to be at risk of relapse 
from P. vivax.  
Because of these findings, we shall be assessing our healthy Thai volunteers for their ability to metabolise 
primaquine by genotyping for CYP2D6 at screening, in order to minimise the risk of subsequent relapse of 
infection. While there is not yet a fully validated correlation between CYP2D6 genotype and failure of 
primaquine radical cure, it is at present the best predictor of relapse that we have. We shall exclude any 
volunteer who is not a high metaboliser based on their CYP2D6 genotype. All volunteers will also be 
screened to ensure they have normal G6PD enzyme activity to prevent haemolytic anaemia during radical 
cure therapy with primaquine. 

 
MIST 1 protocol V 10.0 dated 14 February 2022                                               Page 27 of 85 
The most recent P. vivax mosquito-bite controlled human challenge infection trial was in 2 healthy malaria-
naïve volunteers carried out by The Malaria Vaccine Group in Oxford University; Oxford, UK in early 2018. 
Laboratory-reared mosquitoes were fed on blood from a P. vivax-infected donor in the Mahidol Vivax 
Research Unit (MVRU), Faculty of Tropical medicine, Bangkok, Thailand after screening of the patient’s 
blood by PCR to ensure no co-infection with other Plasmodium species or blood-borne infections. The 
infected mosquitoes were then transported to Imperial College, United Kingdom, where the mosquito-bite 
controlled human challenge infection trial with 5 P. vivax-infected An. dirus mosquitoes per volunteer was 
performed in the Imperial College insectary. The subjects were then transferred to the Centre for Clinical 
Vaccinology and Tropical Medicine, Oxford for clinical follow up. Volunteers were treated following a 
diagnosis of vivax malaria by blood smear and real time quantitative PCR. The pre-patent period for both 
participants was around 10-11 days. Both volunteers were blood group O rhesus negative. The blood 
donation was successfully performed in both subjects and no malaria relapse during first year follow up 
(Minassian A and Draper S, personal communication) (Table 2).  
2.9 
Background to first vivax CHMI study in Thailand (covered by current protocol) 
To date, most of the challenge models have been successfully performed in non-endemic settings such as 
in the US, UK and Australia. Up to date the challenge studies in UK in healthy vivax-naïve volunteers have 
not had any serious adverse event, malaria relapse, and lost follow-up (unpublished data obtained directly 
from lead investigator). However, the findings in high income settings may not be extrapolated to the target 
population for future vaccine deployment, which is more heterogeneous both in terms of vivax immunity 
and genetic background. The best volunteer population to test new vaccines is the eventual target 
population for vaccine deployment.  
As outlined above, the proposed P. vivax human challenge in Thailand would allow us to understand the 
immunological correlates of protection in an endemic setting, thereby informing the development of new 
vaccine candidates and rapid testing of the protective efficacy of candidate vaccines in the at-risk population 
in which they will be deployed. Advancing the development of such methods needs renewed emphasis on 
understanding the biology, pathogenesis and transmission of P. vivax. This study aims to assess the safety 
and feasibility of controlled human P. vivax malaria infection in Thailand. It also aims to build on the limited 
knowledge that exists on parasite growth dynamics, transmission and the human immune response to 
infection following infection by the natural route of delivery – mosquito bite. A major objective of the study 
is to provide a source of P. vivax infected blood to use in future vaccine efficacy trials of a sexual and 
asexual blood- and transmission-stage vaccines – making these studies more feasible in the future. We 
will be using An. dirus mosquitoes from our in-house sources  – reared in Thailand at the MVRU and 
Entomology Department, Mahidol University, Bangkok. MVRU is the source of the infected mosquitoes used 

 
MIST 1 protocol V 10.0 dated 14 February 2022                                               Page 28 of 85 
in previous CHMI study in Oxford described above. We shall be the first Asian centre to perform a controlled 
human malaria infection trial.  
Protocol to prepare P. vivax infected mosquitoes was approved by FTM-EC (Protocol # MUTM 2018-017). 
Patients with P. vivax for the infected mosquito preparation will be recruited at the malaria clinics in 
Thailand by MVRU team.   
For MIST 1 study, healthy vivax-naïve volunteers will be recruited at FTMCTU, Bangkok. 
2.10 Over view of the whole projects (Position of current study protocol within the overall 
Thailand vivax Controlled human challenge infection programme) 
This section aims to give the overview of all projects in this whole program. This will be a programme 
comprising multiple studies planning to conduct over the next 5 years in the Faculty of Tropical Medicine, 
Mahidol University, Thailand as follows: 
• 
Proof of feasibility and safety study and providing banked infected blood inocula for future blood 
stage human challenge studies.  
• 
Test the protective efficacy of pre-erythrocytic, blood stage and (ultimately) transmission blocking 
P. vivax vaccine candidates in the target population, in both vivax-naïve and semi-immune 
volunteers. 
• 
Characterise correlates of immune protection in the populations at risk, in vivax-naïve and semi-
immune volunteers. 
• 
Test the efficacy of novel drugs under development for anti-hypnozoite (anti-relapse) activity.  
 To achieve the major aims written above, the programme will compose of 5 or 6 subsequent studies. Each 
subsequent study will be informed by previous studies in the series (base on the results of previous study) 
and will be definitely covered by separate protocols with separate Ethics Committee submissions and 
approvals. The 5-year plan of controlled human malaria infection studies are as follows: 
• 
MIST 1 (covered by this current protocol). Initial mosquito infection (sporozoite) human challenge 
study (6 malaria-naïve healthy volunteers), MIST 1. This will be a proof of feasibility and safety study 
and providing banked infected blood inocula for future blood stage human challenge studies.  
• 
MIST 2. Blood stage inoculum human challenge in volunteers from an endemic area (20 semi-
immune volunteers) (using bank blood from MIST 1 to find the proper dose for nest vaccine testing 
study). This dose finding study aims to determine the most suitable inoculum ‘dose’ for human 
blood stage challenge in semi-immune subjects for later MIST trials of blood stage vaccine studies. 
The objective is to determine the most suitable inoculum ‘dose’ for human blood stage challenge in 
later CHMI trials of blood stage vaccine candidates.  

 
MIST 1 protocol V 10.0 dated 14 February 2022                                               Page 29 of 85 
• 
MIST 3. Mosquito (sporozoite) infection human challenge in volunteers from an endemic area. (20 
semi-immune volunteers). This will follow the study procedures of the first study (MIST 1), except 
that the volunteers will be recruited and screened from a malaria endemic area (Sai Yok, 
Kanchanaburi Province). The challenge (MIST 3) will take place in FTMCTU in Bangkok. These 
volunteers will be variably immune to malaria. The volunteers will receive full radical cure treatment 
regimen before challenge to clear any low level parasitaemia and hypnozoites from the liver at base 
line and then will be inoculated by infected mosquito bite. The radical cure will be given following 
the detection of parasitaemia. The immunological and parasitological data obtained will help define 
correlates of protection, and inform the design of future challenge studies for vaccine efficacy 
assessment in semi- immune volunteers.  
• 
MIST 4. Blood stage inoculum human challenge study to assess protective efficacy of a blood stage 
vaccine (50 volunteers, semi-immune). Study procedures will follow those of MIST 2 (dose selection 
study) above. This study (MIST 4) will be used to assess DBP-based or other blood stage vaccine. 
Volunteers will be randomized to vaccination or control groups, and inoculated using the blood 
banked from MIST 1 above in a dose determined by the results of MIST 2.  
• 
MIST 5. Mosquito (sporozoite) challenge studies to assess pre-erythrocytic vaccines (50 volunteers, 
semi-immune). These studies (MIST 5) will be used initially to test a CS-based or other pre-
erythrocytic vaccine. Study procedures will follow those in MIST 3, and radical cure will be given. 
Volunteers will be randomised to vaccination or control groups. 
• 
MIST 6 and 7 will be blood stage inoculum challenge or mosquito challenge studies to test further 
upcoming P. vivax vaccine candidates. The study procedures will follow MIST 4 or MIST 5 above, 
depending on type of vaccine to be tested. 
For the avoidance of doubt, this current protocol only covers MIST 1 ONLY, and all ethical submissions 
involving this protocol ONLY relate to this first study (MIST1) with the specified objectives described in the 
objectives section of this study in the following section.  
This P. vivax human challenge programme is a highly collaborative project involving the Faculty of Tropical 
Medicine (FTM), Mahidol Oxford Research Unit (MORU), Mahidol University, the Wellcome Kenya and 
Thailand Asia and Africa Programmes, in liaison with all the vaccine developers expected to have vaccine 
candidates ready for testing in the next five coming years. Multiple departments within FTM are involved, 
including the world leading Mahidol Vivax Research Unit (MVRU), Department of Entomology, Molecular 
Tropical Medicine and Genetics, The Tropical Medicine Diagnostic Reference Laboratory (TMDR), and the 
FTM Clinical Therapeutics Unit.  
The programme will be led, coordinated and capacity-built by an executive trial management team 
consisting of the programme co-PIs, site PIs, project/programme manager, and Controlled Human Malaria 

 
MIST 1 protocol V 10.0 dated 14 February 2022                                               Page 30 of 85 
Infection study experts. A Programme Steering Committee (PSC) led by an independent chair and the Dean 
of Faculty of Tropical Medicine will oversee the research. 
3. OBJECTIVES  
This protocol will cover only the initial mosquito infection human challenge study in up to 6 healthy malaria 
naïve volunteers (MIST 1). This will be a 1) Proof of feasibility study, 2) Providing banked P. vivax infected 
blood inocula for future blood stage human challenge studies, and 3) Providing data on the development 
of host immunity.  
The trial has the following objectives: 
 
3.1 
Primary objectives: 
• 
To assess the feasibility and safety of controlled human P. vivax malaria infection in six healthy 
human volunteers, through experimental sporozoite infection (mosquito bite). 
• 
To obtain up to 250 mL of blood from each infected volunteer and freeze down the blood to 
create inocula for use in future P. vivax challenge studies.  
3.2 
Secondary objectives: 
• 
To assess the immune response to primary P. vivax infection delivered by mosquito bite. 
• 
To assess gametocytaemia following primary P. vivax infection delivered by the mosquito bite. 
 
4. STUDY END POINTS 
4.1 
Primary endpoints: 
• 
Feasibility and safety of P. vivax sporozoite human challenge, as measured by successful 
infection (development of detectable persistent parasitaemia +/- clinical symptoms) and AE(S) 
occurrences. 
• 
Collection and freezing down of up to 250 mL P. vivax-infected blood from each of the 6 
volunteers. 
4.2 
Secondary endpoints: 
• 
Cellular and humoral Immune response to primary P. vivax infection. 
• 
Gametocytaemia pre-treatment, as measured by qPCR. 
 
5. STUDY DESIGN 

 
MIST 1 protocol V 10.0 dated 14 February 2022                                               Page 31 of 85 
5.1 
Summary of Trial Design 
This is a Plasmodium vivax mosquito-bite controlled human challenge infection trial in healthy 
malaria-naïve Thai adults. 
6. PARTICIPANT IDENTIFICATION AND RECRUITMENT 
6.1 
Trial Participants 
Up to six healthy, malaria-naïve Thai adults aged between 20 and 55 years with at least an 
undergraduate degree will be recruited at the FTMCTU at the Hospital for Tropical Diseases, Faculty 
of Tropical Medicine, Mahidol University, Bangkok, Thailand. 
6.2 
Inclusion criteria 
The volunteer must meet all of the following criteria to be eligible for the study: 
• 
Healthy adult aged 20 to 55 years with weight more than 50 kg. 
• 
Blood group O. 
• 
Red blood cells positive for the Duffy antigen/chemokine receptor (DARC). 
• 
Normal CYP2D6 genotype.  
• 
Normal blood levels of Glucose-6-phosphate dehydrogenase (G6PDH) by the WHO definition.  
•  COVID-19 vaccination at least two doses of COVID-19 vaccine(s) approved by WHO.   
• 
Agree to practice continuous effective contraception for the duration of study period until 3 
months post-challenge. 
• 
Agreement to refrain from blood donation during the course of the study and for 1 year after the 
end of their involvement in the study. 
• 
Willing to take a curative antimalarial regimen following challenge. 
• 
Willing to be admitted in the Hospital for Tropical Diseases for blood donation and clinical 
monitoring, until antimalarial treatment is completed and their symptoms are settling.  
• 
Willing to reside in Bangkok for the duration of the study, until all antimalarial treatment has been 
completed. 
• 
Reachable (24/7) by mobile phone during the period between challenge CHMI and completion of 
all antimalarial treatment. 
• 
Able to read and write in Thai and able to answer ALL questions on the informed consent 
questionnaire correctly. 
• 
Provided written informed consent to participate in the trial. 
• 
 Educational level: has at least an undergraduate degree. 
6.3 
Exclusion criteria 

 
MIST 1 protocol V 10.0 dated 14 February 2022                                               Page 32 of 85 
The volunteer MUST NOT enter the study if any of the following apply: 
• 
History of clinical malaria. 
• 
Positive malaria PCR OR malaria film OR malaria serology 
• 
  History of severe allergy to mosquito bite  
• 
Presence of any medical condition (either physical or psychological) which in the judgment of the 
investigator would place the participant at undue risk or interfere with the results of the study 
(e.g. serious underlying cardiac, renal, hepatic or neurological disease; severe malnutrition; 
congenital defects or febrile condition) 
• 
Presence of chronic disease or chronically use of medication.  
• 
Plan to travel outside of Bangkok within the period of challenge until 3 months after. 
• 
Use of systemic antibiotics with known antimalarial activity in the 30 days before challenge (e.g. 
trimethoprim-sulfamethoxazole, 
doxycycline, 
tetracycline, 
clindamycin, 
erythromycin, 
fluoroquinolones and azithromycin).  
• 
Use of immunoglobulins or blood products (e.g. blood transfusion) at any time in the 1 year 
preceding enrolment. 
• 
Venipuncture unlikely to allow blood donation according to the protocol as determined by the 
investigator. 
• 
Receipt of an investigational product or any vaccine in the 30 days preceding enrolment (D0), or 
planned receipt during the study period. 
• 
Prior receipt of an investigational vaccine likely to impact on interpretation of the trial data or the 
P. vivax parasite as assessed by the Investigator. 
• 
Any confirmed or suspected immunosuppressive or immunodeficient state, including HIV 
infection, asplenia, history of splenectomy, recurrent, severe infections, and chronic infection. 
• 
Immunosuppressant medication within the past 6 months preceding enrolment (D0) (inhaled and 
topical steroids are allowed). 
• 
History of allergic disease or reactions likely to be exacerbated by malaria infection. 
• 
Female participant who is pregnant, lactating or planning pregnancy during the course of the 
study.  
• 
Contraindications to the use of antimalarial treatment (e.g. chloroquine or primaquine or 
atovaquone / proguanil, DHA piperaquine). ** 
• 
Use of medications known to have a potentially clinically significant interaction with antimalarial 
drug that will be used in this study (chloroquine or primaquine or atovaquone / proguanil, DHA/ 
piperaquine).  
• 
Use of medications known to cause prolongation of the QT interval as state in the section of 
prohibited drugs that may have effect on prolongation of the QT interval.  

 
MIST 1 protocol V 10.0 dated 14 February 2022                                               Page 33 of 85 
• 
Known existing positive family history in both 1st AND 2nd degree relatives < 50 years old for cardiac 
disease. 
• 
Family history of congenital QT prolongation or sudden death. 
• 
Any clinical condition known to prolong the QT interval. 
• 
History of cardiac arrhythmia, including clinically relevant bradycardia. 
• 
Screening ECG demonstrates a QTc interval ≥ 450 ms  
• 
Suspected or known or history of alcohol abuse  
• 
Suspected or known or history of drug abuse.  
• 
Concurrently participating in another clinical study, at any time during the study period. 
• 
Haemoglobin < 13 g/dL in male, < 12g/dL in female (Thai Red Cross). 
• 
Mean corpuscular volume (MCV) < 80 fL/cell  
• 
  Finding on safety laboratory values as defined below: 
• 
 AST > 40 U/L for male, and > 32 U/L for female (upper normal range), or 
• 
ALT > 41 U/L for male, and > 33 U/L for female (upper normal range), or 
• 
Total Bilirubin > 1.2 mg/dL, (upper normal range), or   
• 
Creatinine (Cr) > 1.17 mg/dL for male, and > 0.95 mg/dL for female (upper normal range), 
or   
• 
Abnormalities corrected calcium and magnesium blood levels 
• 
Fasting blood sugar (FBS) > 100 mg/dL, or  
• 
Cholesterol LDL (low density lipoprotein) > 160 mg/dL or Triglyceride > 200 mg/dL  
• 
 Thalassaemia disease or haemoglobinopathies. 
• 
Positive for a blood borne or vector borne infectious disease (HIVI-II, HBV, HCV, Dengue, Zika, 
Chikungunya, Filariasis, JE, and malaria antigen, Anti HTLVI and Anti-HTLVII antibody, Syphilis test 
(TPHA) 
• 
Positive for COVID-19 testing as diagnosed by RT-PCR 
** Link of the lists of medications with QTc prolongation: 
https://crediblemeds.org/pdftemp/pdf/CombinedList.pdf  
7. STUDY PROCEDURES 
This is a sporozoite-challenge clinical study with the primary objectives of assessing the feasibility and 
safety of controlled human sporozoite P. vivax malaria infection in six healthy volunteers, and developing a 
bank of P. vivax-infected blood for use as inocula in future controlled human blood stage P. vivax malaria 
infection studies. Secondary objectives are to assess the growth of and the immune response to P. vivax 
infection, and assess the induction of sexual gametocytaemia post-CHMI via the natural route of malaria 
infection (mosquito bite). 

 
MIST 1 protocol V 10.0 dated 14 February 2022                                               Page 34 of 85 
Up to six healthy, malaria-naïve adults aged between 20 and 55 years with weight more than 50 kg will be 
recruited at the FTMCTU in the Hospital for Tropical Diseases, Faculty of Tropical Medicine, Mahidol 
University, Bangkok. Volunteers will be recruited and receive mosquito-bite sporozoite challenge in two 
batches. The first batch will consist of 2 volunteers, and the second batch of 4 volunteers.  A P. vivax 
mosquito-bite controlled human challenge infection (CHMI) by sporozoites will be delivered to the first 
batch of volunteers at the insectarium unit, Department of Entomology, Faculty of Tropical Medicine, 
Mahidol University, Bangkok. The procedures will be performed strictly following the approved work 
instruction (WI) and clinical monitoring will be performed during the post-challenge period at the FTMCTU 
as an inpatient until the antimalarial medications (chloroquine) are completed. Chloroquine and primaquine 
will be prescribed according to current Thai national guideline. The second batch of volunteers will receive 
mosquito bite CHMI after the careful evaluation of the safety profiles / outcomes of the first 2 volunteers 
(at 28 days after treatment initiation as the minimum time interval). 
During admission, the volunteers will have blood taken at regular intervals post-challenge to assess 
parasitemia, immune response following P. vivax infection, and they will be monitored closely until they 
meet the criteria for blood donation and treatment. Up to 250 mL of blood will be taken prior to treatment 
from the successfully infected volunteers. The volunteers will then be treated with a standard course of oral 
chloroquine and a 2-week course of directed observed therapy (DOT) of oral primaquine for radical cure of 
P. vivax hypnozoites.   
Volunteers will be followed up for 1 year after antimalarial treatment initiation. They will be asked to come 
back to the outpatient clinic on D 28, D 90, 180, and 1 year after treatment initiation. During this phase, the 
volunteers will be contacted fortnightly by email/ phone call / other social communications e.g message 
(SMS), WhatsApp, Line, to ensure they remain well and asymptomatic.  
Alternative medications including dihydroartemisinin + piperaquine, atovaquone + proguanil and 
intravenous artesunate may be prescribed if indicated as per the treatment algorithm in Appendix B. 
7.1 
Recruitment of volunteers 
Volunteers will be screened and recruited at the FTMCTU, Faculty of Tropical Medicine, Mahidol University 
following Recruitment Process for Healthy Volunteer SOP.  
In brief, the volunteers may be recruited by use of an advertisement approved by the ethics committee and 
distributed or posted in the following places: 
• 
On a MIST website operated by the  study group   
• 
In public places with the agreement of the owner / proprietor. 
• 
Via presentations (e.g. presentations at lectures or invited seminars).  

 
MIST 1 protocol V 10.0 dated 14 February 2022                                               Page 35 of 85 
We may contact individuals who have participated in previous clinical trials from FTMCTU’s databases. 
These volunteers will have expressed an interest in receiving information about all future studies in FTMCTU 
for which they may be eligible. 
7.2 
Informed consent 
The informed consent process: The investigator will explain the purpose of the study with the help of and 
consistent with the Participant Information Sheet (PIS) prior to any study related procedures being 
undertaken. The volunteer must personally sign and date the latest approved version of the informed 
consent form (ICF) before any study specific procedures are performed.  
The information sheet and informed consent form will be explained to the volunteers detailing no less than: 
the exact nature of the study; the implications and constraints of the protocol; the known side effects and 
any risks involved in taking part. The aims of the study and all procedures to be carried out will be explained. 
It will be emphasised that there is no direct benefit from participating the trial.  
• 
It will be clearly stated that participation is entirely voluntary and that refusing to participate will 
not involve any penalty or affect the volunteers’ right to receive standard medical care. 
• 
It will also be emphasized that if they do consent to participate and are enrolled, that they are free 
to withdraw from the study at any time, for any reason, without any penalty or prejudice to future 
care, and with no obligation to give the reason for withdrawal.  
The consent information will emphasize that blood samples (from blood donation) will be taken and stored 
indefinitely for use in other, ethically approved research. The future use of samples may result in the 
development of a commercially viable product to which the volunteers may not have any rights. The 
volunteer will have the opportunity to question the investigator, or other independent parties to decide 
whether or not they will participate in the study.  
If they do decide to participate, volunteers will be asked to complete a questionnaire testing their 
understanding of the trial before signing the consent. This helps to ensure that individuals understand the 
trial sufficiently to give informed consent. After the volunteer answers all questions in the questionnaire 
correctly, they will be asked to sign and date two copies of the consent form, one for them to take away 
and keep, and one to be stored in the investigator’s site file and retained at the study site. These forms will 
also be signed and dated by the investigator. Volunteers who fail to answer all questions correctly on their 
first attempt will be allowed to re-take the questionnaire following further discussion with the investigator. 
Only the subject who able to subsequently answer all questions in the questionnaire correctly will be asked 
to give the consent and will be screened for the trial. Only 2 attempts will be permitted in total. 
Hospital consent for HIV test: This will be obtained prior to counselling and HIV test using the standard HIV 
consent form of Hospital for Tropical Diseases. Pre and post counselling for HIV screening and reporting will 

 
MIST 1 protocol V 10.0 dated 14 February 2022                                               Page 36 of 85 
be conducted with the support of physician from the Hospital for Tropical Diseases. In case a volunteer is 
found to be HIV positive, follow up measures including but not limited to providing counselling and 
treatment according to standard hospital procedure will be arranged through a physician from the Hospital 
for Tropical Diseases. 
7.3 
Screening and eligibility assessment 
The screening will aim to recruit up to 6 healthy volunteers and at least 2 back-up healthy volunteers using 
the same screening procedures. 
The screening and eligibility assessment will be performed in the FTMCTU, Hospital for Tropical  Diseases, 
Mahidol University following hospital registration as an outpatient. This will ensure that volunteers will 
receive prompt medical care as patients if required. Registration through the hospital system can also help 
prevent the volunteer from participating in other clinical trials simultaneously. This issue will be discussed 
and emphasized with the volunteer prior to screening and also through the study period.  
Screening procedures are outlined in the schedule of procedures in Table 6.   
7.3.1 
Screening visit 1 
Informed consent will be obtained prior to performing any study activities, including screening tests. Once 
informed consent is given, a screening number will be assigned in sequential order. Screening numbers will 
be issued consecutively (SCR-MIST1-001, SCR-MIST1-002…and so on). The volunteer will be advised to fast 
at least 8-12 hours prior to the screening process. All volunteers will have their medical history taken, a 
physical examination, urinalysis, electrocardiogram (ECG), ABO and rhesus system blood grouping, 
haemoglobin, testing for G6PD genotyping, G6PD enzyme level, a  complete blood count (CBC) and blood 
disease, biochemistry tests, Duffy antigen/chemokine receptor (DARC), blood-borne diseases including 
Hepatitis B profile (HBsAg, anti-HBc, anti-HBs), anti-HCV, HIV antibodies, anti-HTLV-I and anti-HTLV-II, and  
syphilis test (TPHA) and vector-borne diseases including Dengue, Zika, Chikungunya, JE, and malaria 
diagnosis, a filariasis RDT. The serum pregnancy test (for females), CYP2D6 genotyping, and malaria serology 
(exposure to malaria) will be done during this stage.  
If an abnormal finding at screening is deemed to be clinically significant, the volunteer will be informed and 
appropriate medical care arranged with the permission of the volunteer.  
Exclusion of the volunteer from enrolling in the trial or withdrawal of a volunteer from the trial will be at 
the discretion of the Investigator. 
The maximum time between the screen visit 1 and the malaria challenge day is 180 days 
Additional screening for safety reassurance will be performed on day -7 and day -1 (7 and 1 days before the 
mosquito bite) as described in the following sections.  

 
MIST 1 protocol V 10.0 dated 14 February 2022                                               Page 37 of 85 
7.3.2 
Screening visit 2: 7 days before challenge (D-7) 
The volunteers will attend for a repeat blood test 7 days before challenge (D-7) for HIV I-II, HBV, HCV, 
Dengue, Zika, Chikungunya, JE, malaria diagnosis, serology for anti HTLV-I and anti HTLV-II antibody, syphilis 
test (TPHA), filariasis, malaria serology (exposure to malaria), urinalysis, and a urine pregnancy test (for 
females) according to Table 6.1 to ensure no new acquisition of infection prior to challenge. Nasopharyngeal 
swab will be collected for testing of COVID-19 by RT-PCR. 
Other screening activities will be undertaken according to Table 6. 
This is important to ensure they have not acquired any new infections listed in the exclusion criteria since 
their first screening as their blood donation will be used in the future to administer malaria challenges to 
healthy volunteers.  
These tests for vector borne and blood borne infections are in line with the standard guidelines for blood 
donor selection;  
• 
The guidelines of the Joint UK Blood Transfusion and Tissue Transplantation Services Professional 
Advisory Committee (https://www.transfusionguidelines.org/transfusion-handbook/3-providing-safe-
blood/3-2 tests-on-blood-donation) 
• 
Guidelines on Assessing Donor Suitability for Blood Donation of World Health Organization 2012; 
https://www.who.int/bloodsafety/publications/guide_selection_assessing_suitability.pdf) 
The guidelines of National Blood Center, Thai Red Cross; 
(https://english.redcross.or.th/content/page/949) 
• 
Expert advices 
The maximum time between the screening visit 2 and the challenge day is 14 days. 
7.3.3 
Screening visit 3/ a day before the challenge day (1 day before challenge, D-1) 
Blood will be taken for haematology (CBC), biochemistry testing, Epstein Barr virus (EBV), cytomegalovirus 
(CMV), malaria immunology, and serum pregnancy test for females, in accordance with Table 6.1 and the 
subject screening work flow shown in Figure 1. In addition, blood collection will be performed to test for 
CMV IgM/IgG antibody, EBV VCA (IgM)/IgG antibody, and anti EBNA and EA complex antibody. COVID-19 
serology Point-of-Care (POC) test will be done to look at participants’ immunity from the current/previous COVID-
19 infection (asymptomatic) and/or prior immunization. Nasopharyngeal swab will also be collected for 
COVID-19 testing by RT-PCR. 
Other screening activities will be undertaken according to Table 6.  

 
MIST 1 protocol V 10.0 dated 14 February 2022                                               Page 38 of 85 
Figure 1. Screening Work Flow  
Exclude: 
If meet one of these 
exclusion criteria  
• ICF 
• History taking and general physical 
examination, ECG, including compliance 
evaluation, ABO, Rh, Hb 
• G6PD  
• Urinalysis, serum pregnancy test (females) 
Exclude: 
• Non O blood group,  
• G6PD is not normal (as 
defined by the WHO 
definition) 
• Hb <12g/dl (female) or 
Hb <13g/dl (male) 
• Potential non 
compliance 
• Unhealthy as judged by 
MD.  
• Meet any exclusion 
criteria 
Screening visit 1 (ICF 
sign) (up to D -180) 
Volunteer 
Exclude: 
If meets any of these 
exclusion criteria 
Screening visit 2 (D-7) 
Repeat; qPCR for HIVI-II, HBV, HCV, Dengue, Zika, 
Chikungunya, JE, malaria diagnosis (blood film, 
qPCR), serology for Anti HTLV-I and Anti HTLV-II 
antibody, Syphilis test (TPHA), Filaria, urine 
pregnancy test, urinalysis, Malaria serology4 
(exposure to malaria), and COVID-19 testing by 
RT-PCR 
• CYP2D6 genotyping 
• Malaria serology3 (exposure to malaria) 
Exclude: 
If meet one of these 
exclusion criteria 
Screening visit 3 (D-1) 
• 
Serum pregnancy test in females  
• 
CMV, EBV (by RT-PCR and serology test) 
• 
Blood biochemistry 
• 
Haematology (CBC) 
• 
Malaria immunology 
• 
COVID-19 testing by RT-PCR and 
serology POC Test 
Exclude: 
If meet this exclusion 
criterion 
• Duffy blood group 
• Blood biochemistry 
• Haematology and blood disease    
• Malaria diagnosis (blood film, qPCR), HIV 
serology, HBs profile1, HCV antibody2, 
Dengue, Zika, Chikungunya, JE, anti HTLV-I 
and anti HTLV-II antibody, syphilis test 
(TPHA), and a filariasis RDT 

 
MIST 1 protocol V 10.0 dated 14 February 2022                                               Page 39 of 85 
1 HBsAg or isolate HBc antibody  
     2 HCV antibody 
    3 AlphaScreen assay will be used to determine the quantitation of malaria antigens-specific antibodies (up 
to 300 P. falciparum and 300 P. vivax antigens). A seropositivity cut-off will be set as half the lowest non-
negative value from of the assayed samples. #a positive malaria history refers to a volunteer who 
previously had malaria infection which enables to reactive to more than 10% malaria antigens in the 
library. 
  4 Multiplex Bead Based Immunoassay will be performed to determine the magnitude of antibodies against 
recent exposure of 5-8 blood-stage malaria antigens. A seropositivity cut-off will be calculated by the 
average of median fluorescence intensity (MFI) plus 2 standard deviation of negative control. 
7.4 
Interventions in volunteers 
This section describes the clinical procedures for evaluating study volunteers and management during and 
after challenge.  
7.4.1 
Inpatient phase until end of antimalarial treatment:  
This section involves the baseline assessment, enrolment, challenge by mosquito bite (mosquito bite), blood 
donation, and antimalarial treatment.  
7.4.1.1 Admission day (baseline assessment) (D-1) 
The volunteers will be admitted to Hospital for Tropical Diseases, Faculty of Tropical Medicine, Mahidol 
University as inpatients following hospital registration to ensure the maximum safety practice. The 
registration can help detect if the volunteer has participated in any other clinical trial during the study 
period. Any new medical issues or symptoms that may have arisen will be again assessed. The venipuncture 
for serum pregnancy, CMV, EBV, CBC, malaria immunology and blood biochemistry will be performed 
according to Table 6.1. The history taking and physical examination, COVID-19 testing, and other study 
activities will be undertaken according to Table 6. All inclusion and exclusion criteria will be checked. 
Full contact details for each volunteer will again be confirmed and documented, including home and work 
addresses, mobile phone number, home and work landline telephone numbers where available and next-
of-kin address and telephone numbers. Volunteers must also provide the investigators with the name and 
24-hour telephone number of a close friend, relative or housemate who will be kept informed of their 
whereabouts for the duration of the study. 
7.4.1.2 Challenge: infected mosquito bites on day 0 (D0)  
All inclusion and exclusion criteria will be checked again to ensure eligibility criteria have been met. Study 
activities will be undertaken according to Table 7. Any new medical issues or symptoms that have arisen will 
be assessed. Results of vector borne and blood borne infectious diseases (of D-7) and pregnancy test (of D-
1, if female) must be available and reviewed prior to challenge. If all inclusion criteria are fulfilled and none 

 
MIST 1 protocol V 10.0 dated 14 February 2022                                               Page 40 of 85 
of the exclusion criteria apply, then the volunteer will be enrolled into the full study (proceeding to the 
challenge phase). Enrolment number will be assigned to each volunteer in sequential number of the format 
Study Code, Site Number, Sequential Number e.g. MIST1-TH01-001, MIST1-TH01-002…… MIST1-TH01-006.  
The back-up volunteers must also be available during the period of the malaria challenge (during the 
mosquito bites), in case one of the volunteers in the batch withdraws at the last minute or any unexpected 
event occurs. This means that confirmation of whether or not a back-up volunteer will be needed for the 
challenge will not be made until the challenge (the whole mosquito biting process) has been successfully 
completed. 
Challenge will be administered according to the SOP for sporozoite challenge by mosquito bites at the 
designated insectarium unit, Department of Entomology, Faculty of Tropical Medicine, Mahidol University. 
Following successful completion volunteers will return to FTMCTU for clinical management. The procedures 
are described in section 8. Other study activities will be undertaken according to Table 7. Summary of the 
mosquito biting process as detailed in Appendix A. 
1. Prepare infected mosquitoes 
2. Briefing of volunteers before the malaria challenge. 
3. Perform malaria challenge process in a secure room in the insectary. 
3.1 Clean volunteer forearm following to the mosquito biting work instruction. 
3.2 Bite each volunteer by five infectious mosquitoes (by placing his or her forearm over the pot 
containing mosquitoes) for 5-10 minutes. 
4. Return volunteer to Hospital for Tropical Diseases for admission until disease management is completed 
7.4.1.3 Day 1 to Day 4 post challenge 
The liver-stage of malaria infection is asymptomatic and lasts approximately one week. Even so the 
volunteers will be daily reviewed in the morning on days D1 to D4 post-challenge by using the daily well-
being checklist form.  The once daily general physical examination will be done. Blood will be collected on 
Day 2 for assessing the immune response and CBC and Day 3 for liver function tests.  
In case volunteers have fever but malaria blood film, malaria qPCR, and testing for vector-borne diseases 
are both negative, COVID-19 testing will be performed. 
7.4.1.4 Days 5 post challenge to Day of blood donation (D5 – DBl donation)  
Volunteers will be clinically monitored twice daily from D5 (in the morning and evening) by clinical research 
team. The team will consist of the investigators who are physicians (with experience in acute medicine and 
infectious diseases and familiarity assessing patients with malaria) and nurses. 

 
MIST 1 protocol V 10.0 dated 14 February 2022                                               Page 41 of 85 
• 
Well-being checklist questioning, physical examination will be performed. Venipuncture will 
be performed according to Table 6.1. 
• 
In the clinical well-being checklist form, the volunteers will be questioned whether they have 
experienced any malaria-related symptoms (such as fever, chills, sweating, malaise). These 
symptoms will be listed as solicited AEs.  
• 
The severity of symptoms will be carefully assessed. 
• 
The qPCR and blood film for malaria and gametocyte qPCR will be monitored twice daily from 
D5 until blood donation. Then once daily until the volunteer has two consecutive negative blood 
film readings and on the day 7 of primaquine treatment (Dpq 7) and day 14 of primaquine 
treatment (Dpq 14). 
• 
If volunteer present with a clinical feature(s) of COVID-19 at the discretion of the trial physician, 
and malaria blood film and malaria qPCR are both negative, COVID-19 testing will be performed. 
• 
Blood for assessing the immune response on day 5 and day of blood donation.  
• 
 Blood collection will be performed to test for CMV IgM/IgG antibody, EBV VCA (IgM)/IgG 
antibody, and anti EBNA and EA complex antibody at 2-4 weeks after the inoculation. 
• 
COVID-19 serology Point-of-Care (POC) test will also be assessed at 14 days after the inoculation 
to look at participant’s immunity.  
• 
Urine pregnancy test before chloroquine treatment.   
Other activities and the details of blood taken during the study period are given in Table 6.1 and Table 7.  
At a time-point that will depend on the level of parasitaemia and/or the degree of the volunteers’ symptoms 
(as per algorithm, Table 3), we will take up to 250 mL of blood from them within 24 hours of the clinical OR 
laboratory criteria being met (as per Table 3). The blood donation will be performed following the Blood 
Donation SOP then processed and frozen down following the parasite banking SOP for future use in the 
future subsequence blood-stage controlled human challenge trials (MIST2 - MIST7). The study team will 
make sure that volunteer tolerate the blood donation procedure and that they are provided with adequate 
symptom relief.  
Blood donation will be done only in volunteers with a haemoglobin higher than 10 g/dL and who are clinically 
stable. 
The ranges of haemoglobin level for a blood donation are describe as follow; 
• Hb > 11 g/dL:  blood donation 250 mL  
• Hb > 10 g/dL to 11 g/dL: blood donation 200 mL  
• Hb ≤ 10 g/dL: no blood donation 
 

 
MIST 1 protocol V 10.0 dated 14 February 2022                                               Page 42 of 85 
Table 3. Blood donation criteria 
Blood Donation 
Criteria 
• Febrile illness and any parasitaemia, and volunteer considered 
clinically fit for blood donation 
OR 
• Parasitaemia > 50,000 parasite /mL by microscopy without 
signs and symptoms, and volunteer considered clinically fit for 
blood donation 
 
Blood donation must be complete within 24 hour of these 
criteria being meet. 
7.4.1.5 Antimalarial treatment 
A study physician  will then immediately prescribe antimalarial treatment, chloroquine and a 14-day course 
of primaquine, 30 mg once daily using the direct observed therapy (DOTs) to ensure 100% drug compliance. 
Any side effect from the antimalarials will be rapidly dealt within a controlled environment. They will 
continue to have blood films and qPCR once daily until clinically recovered AND two consecutive negative 
of malaria blood films (completion of the chloroquine treatment course). In case blood donation occurred 
before day 14, blood collection will also be performed to test for CMV IgM/IgG antibody, EBV VCA (IgM)/IgG 
antibody, and anti EBNA and EA complex antibody at 2-4 weeks after the inoculation. In addition, COVID-19 
serology Point-of-Care (POC) test will also be assessed at 14 days after the inoculation to look at participant’s 
immunity. 
The volunteers will be then discharged from the hospital. Before discharge from the hospital, COVID-19 
testing will be carried out to confirm that subjects did not acquire COVID-19 during their inpatient stay. After 
discharge, they will attend FTMCTU daily for directly observed treatments (DOTs) until they have completed 
the remaining dosages of 14-day course of the primaquine treatment (DendPQ). Diary card will be given to the 
volunteers on the discharge day and will be reviewed on every subsequent visit.  
If any volunteer reaches day 21 post-challenge without reaching the treatment criteria, they shall be started 
on 3-day course of antimalarial (chloroquine) and a 14-day course of primaquine, 30 mg once daily.  
If any contraindications to chloroquine are identified, an alternative antimalarial dihydroartemisinin + 
piperaquine or Malarone (atovaquone + proguanil) will be prescribed. If a volunteer is unable to tolerate an 
oral antimalarial, the appropriate parenteral antimalarial therapy will be prescribed.  
If a volunteer withdraws/is withdrawn from the study after challenge but before reaching the criteria for 
malaria treatment, then a complete, appropriate, curative course of antimalarial therapy must be 
completed. The importance of this will be emphasized to volunteers at screening. 

 
MIST 1 protocol V 10.0 dated 14 February 2022                                               Page 43 of 85 
Table 4: Antimalarial treatment criteria. 
Malaria treatment Start  
Criteria  
• Blood donation completed  
           OR 
• Symptoms highly suggestive of malaria infection and judged by 
investigator as requiring antimalarial treatment, in the absence of a 
positive blood smear (other causes of symptoms should be considered 
and if appropriate treated) (Appendix B. Treatment algorithm) 
            OR 
• Day 21 reached and not develop symptom and blood parasitemia 
according to the blood donation criteria 
 
Table 5: Chloroquine and Primaquine treatment dose 
Weight 
Day 1 
Day 2 
Day 3 
Day 4-14 
CQ 
Tab 
PQ 
mg 
CQ 
Tab 
PQ 
mg 
CQ 
Tab 
PQ 
mg 
PQ 
mg 
25-50 kg 
4 
10 
1 
10 
1 
10 
10 
Above 50 kg 
4 
30 
4 
30 
2 
30 
30 
• 
National Malaria Treatment Guideline 2015 (Thailand), CQ 150 mg base/tablet 
7.4.1.6 Supportive treatments and medications 
The malaria symptoms (e.g. fever, nausea, vomiting, and dehydration) will be relieved accordingly. 
Paracetamol (500 mg orally up to four times a day) and dimenhydrinate (50 mg orally) will be prescribed 
according to clinically needed. Intravenous fluid supplementation may be given following physician decision. 
All medications used in the trial will be recorded in medical record.  
If the volunteers meet the criteria for severe malaria (by WHO definition) or fail to improve within 48 hours 
of starting anti-malarial therapy, malaria experts in the Faculty of Tropical Medicine will be consulted. The 
volunteers will be managed according to Thai National Guideline as well as the Hospital for Tropical 
Diseases’ standing order/ procedures/ guideline for severe malaria. 

 
MIST 1 protocol V 10.0 dated 14 February 2022                                               Page 44 of 85 
The algorithm to be used for P. vivax malaria treatment is attached in the protocol as Appendix B. The 
Investigators are able to treat any volunteer for malaria regardless of the blood film microscopy or qPCR 
result if they are clinically concerned or a volunteer wishes to withdraw from the study.  
7.4.2 
Follow up assessment phase (after completion of antimalarial treatment) 
After discharge from the hospital and completion of 14 days of primaquine daily DOT (Dend PQ), and if the 
volunteer remains asymptomatic or has mild, self-resolving symptoms, the follow-up assessment will be 
performed at FTMCTU as an out-patient on day 28 (DRx28), day 90 (DRx90), day 90 (DRx90), and 1 year (DRx 1yr) 
post-treatment initiation.  
On the primaquine end date (Dend PQ) the volunteers will receive full counselling on the remaining possibility 
of relapse and the signs and symptoms that they have to be able to observe early and report in the diary 
card and seek medical advice.  A diary card to self-document any abnormal symptoms will be given to them, 
and the contact channel with the study team will be again emphasized.     
All volunteers will be followed up according to the schedule in Table 7.  
Follow up visit after complete the radical cure are detailed as following.  
7.4.2.1 Day 28 post antimalarial treatment initiation visit (D Rx 28) 
The volunteers will be asked to visit FTMCTU as an outpatient using the hospital registration system. Diary 
cards will be reviewed and collected, a history taken using the clinical well-being check list, a physical 
examination will be performed and AEs assessed. Venipuncture will also be performed to detect malaria 
parasites by blood film and qPCR, malaria gametocyte qPCR, malaria immune response, CBC, and 
biochemistry according to Table 6.1. Other study activities will be undertaken according to Table 7. 
7.4.2.2 Between Day 28 and Day 90 post antimalarial treatment initiation (D Rx29 - D Rx89) 
The volunteers will be contacted fortnightly (biweekly) by email / phone call / other social communications 
e.g. message (SMS), WhatsApp, Line, to ensure they remain well and asymptomatic. They will not be limited 
to contact only with the study team, but the volunteer will have to understand that they must stay in 
Bangkok until day 90 after challenge and always immediately contact the study team as soon as they observe 
any abnormal symptoms or would like to contact the team for any other reason.  
7.4.2.3 Day 90 post antimalarial treatment initiation (D Rx 90) 
The volunteers will be asked to visit FTMCTU as an outpatient using the hospital registration system. History 
taken using the clinical well-being check list, physical examination will be performed and AEs will be 
assessed. Diary cards will be reviewed and collected. According to Table 6.1, venipuncture will be performed 
to detect malaria antigen by blood film and qPCR, malaria gametocyte qPCR, malaria immune response, 
CBC, biochemistry, and blood collection for future cross matching* with the volunteers who will receive the 
blood inoculum in the future MIST studies. Other study activities will be undertaken according to Table 7.  

 
MIST 1 protocol V 10.0 dated 14 February 2022                                               Page 45 of 85 
* Blood sample for cross matching may be collected any time ≥ 90 days after antimalarial                   
treatment initiation (time of blood donation)  
7.4.2.4 Between Day 90 and Day 1 year post antimalarial treatment initiation (DRx 91 to DRx 364) 
The volunteers will be contacted fortnightly by email/ phone call / other social communications e.g message 
(SMS), WhatsApp, Line to ensure they remain well and asymptomatic.  
7.4.2.5 Day 180 post antimalarial treatment initiation (D Rx 180) 
The volunteers will be asked to visit FTMCTU as an outpatient using the hospital registration system. 
According to Table 6.1, venipuncture will be performed to test for the serologic markers of hepatitis B virus 
(HBsAg, Anti-HBc, Anti-HBs), hepatitis C virus (Anti-HCV), HTLV I and HTLV II (anti HTLV I and anti HTLV II 
antibody), HIV (HIV antibodies), and syphilis (TPHA).  
** Blood sample may be collected any time ≥ 180 days after antimalarial treatment initiation (time of blood 
donation)  
7.4.2.6 1 year post antimalarial treatment initiation 
The volunteers will be asked to visit FTMCTU as an outpatient. Venipuncture will be performed to detect 
malaria antigen by blood film and qPCR, malaria gametocyte qPCR, malaria immune response, and CBC 
according to Table 6.1. Other study activity will be undertaken according to Table 7. 
 
Figure 2. Study diagram
 
 
 
 

 
MIST 1 protocol V 10.0 dated 14 February 2022                                               Page 46 of 85 
Table 6: Schedule of clinical reviews for volunteers pre-challenge (Timeline/Window are in relation to the 
day of challenge). 
Screening phase 
Event 
Screening 1  
Screening 2 
Screening 3 
Attendance number 
1 
2 
3 
Timeline (days) 
Up to D-180 
D-7 
D-1 
Window (days - days) 
(D-180) - (D-7) 
(D-14) - (D-7) 
D-1 
Inclusion / Exclusion 
criteria 
X 
X 
X 
Informed Consent 
Questionnaire 
X 
 
 
Informed consent 
X 
 
 
Medical History 
X 
X 
X 
Physical Examination  
X 
X 
X 
Body weight and 
heightf 
X 
 
X 
Urinalysis 
X 
X 
 
Electrocardiogram 
X 
 
 
Vital signs a  
X 
X 
 
Filariasis RDT 
X 
X 
 
G6PD 
X 
 
 
Haemoglobin 
X 
 
 
Blood typing ABO, 
Rhesus 
X 
 
 
Duffy antigen 
X 
 
 

 
MIST 1 protocol V 10.0 dated 14 February 2022                                               Page 47 of 85 
Screening phase 
Event 
Screening 1  
Screening 2 
Screening 3 
Attendance number 
1 
2 
3 
Timeline (days) 
Up to D-180 
D-7 
D-1 
Window (days - days) 
(D-180) - (D-7) 
(D-14) - (D-7) 
D-1 
CBC and 
haematological 
diseases (e.g. 
haemoglobinopathy, 
thalassaemia)  
X 
 
 
 
Blood biochemistry  
X b 
 
X c 
Infectious diseases 
(HIVI-II, HBV, HCV, 
Dengue, Zika, 
Chikungunya, JE, 
malaria diagnosis, 
Anti HTLV I and Anti 
HTLV II antibody, 
Syphilis test (TPHA) 
X d 
X e 
 
β-HCG testing  
(women only) 
X 
(Serum pregnancy test) 
X 
(Urine pregnancy test) 
X 
(Serum pregnancy test) 
Epstein-Barr virus 
(EBV), 
Cytomegalovirus 
(CMV) 
(by PCR and serology 
testing) 
 
 
X 
Haematology (CBC) 
 
 
X 
CYP2D6 genotype 
X 
 
 

 
MIST 1 protocol V 10.0 dated 14 February 2022                                               Page 48 of 85 
Screening phase 
Event 
Screening 1  
Screening 2 
Screening 3 
Attendance number 
1 
2 
3 
Timeline (days) 
Up to D-180 
D-7 
D-1 
Window (days - days) 
(D-180) - (D-7) 
(D-14) - (D-7) 
D-1 
Malaria serology  
X 
X 
 
Malaria Immunology 
 
 
X 
COVID-19 testing 
(nasopharyngeal 
swab [RT-PCR]) 
 
X  
(RT-PCR) 
X  
(RT-PCR) 
COVID-19 testing 
(nasopharyngeal 
swab (serology POC 
test) 
 
 
X  
(POC test) 
a Vital signs include blood pressure, pulse, respiratory rate and temperature. 
b Biochemistry including electrolytes (sodium, potassium, calcium, magnesium, chloride, bicarbonate), blood 
urea nitrogen (BUN), creatinine (Cr), liver function tests (LFTs), fasting blood sugar (FBS), and lipid profile 
(cholesterol, triglyceride, HDL, LDL) 
c Biochemistry including electrolytes (sodium, potassium, calcium, magnesium, chloride, bicarbonate), blood 
urea nitrogen (BUN), creatinine (Cr), and liver function tests (LFTs) 
d HIV, HBV, and HCV will be tested for antibodies. HBV serological profile includes HBsAg, anti-HBc, and anti-
HBs.  
e  HIVI-II, HBV, and HCV will be tested by qPCR. 
f Height will be measured only at Screening 1

 
MIST 1 protocol V 10.0 dated 14 February 2022                                               Page 49 of 85 
Table 6.1: Blood collection during study period 
Phase 
Clot blood 
EDTA blood 
NaF 
CPD 
Total 
blood 
volume 
(ml) 
Total 
accumulat
ion blood 
volume 
(ml) 
Volume 
Test 
Volume 
Test 
Volume 
Test 
Volume 
Test 
Screening 1 
7 
Blood typing  
13.5 
Duffy antigen 
2 
Fasting 
blood 
sugar 
  
  
22.5 
22.5 
Blood 
biochemistry 
Thalassemia 
  
  
  
  
Serum HCG 
CBC 
  
  
  
  
HIV  Ab 
G6PD 
  
  
  
  
HBs profile, anti-
HCV 
CYP2D6 
  
  
  
  
Syphillis (TPHA) 
HTLVI-II 
  
  
  
  
  
  
Filaria 
  
  
  
  
  
  
Malaria  
  
  
  
  
  
  
malaria exposure (serology) 
  
  
  
  
  
  
JE 
  
  
  
  
  
  
DEN 
  
  
  
  
  
  
CHIK 
  
  
  
  
  
  
Zika 
  
  
  
  
Screening 2 
1 
Syphillis (TPHA) 
13.5 
malaria  
  
  
  
  
14.5 
37 
  
  
Malaria exposure (serology 
  
  
  
  
 
  
  
JE 
  
  
  
  
  
  
DEN 
  
  
  
  
  
  
CHIK 
  
  
  
  

 
MIST 1 protocol V 10.0 dated 14 February 2022                                               Page 50 of 85 
Phase 
Clot blood 
EDTA blood 
NaF 
CPD 
Total 
blood 
volume 
(ml) 
Total 
accumulat
ion blood 
volume 
(ml) 
Volume 
Test 
Volume 
Test 
Volume 
Test 
Volume 
Test 
  
  
Zika 
  
  
  
  
  
  
Filaria 
  
  
  
  
  
  
HCV Ag 
  
  
  
  
  
  
HBV Ag 
  
  
  
  
  
  
HTLVI-II 
  
  
  
  
  
  
HIV  Ag 
  
  
  
  
Screening 3 
9 
Blood 
biochemistry 
16 
malaria immunology 
  
  
  
  
25 
62 
Serum HCG 
CBC 
  
  
  
  
  
 CMB, EBV 
serology 
CMV 
  
  
  
  
  
  
EBV 
  
  
  
  
Admission 
D0-D4 
3 
Liver 
function 
test 
12 
Malaria immunology 
  
  
  
  
15 
77 
  
  
CBC 
  
  
  
  
Admission 
D5-blood 
donation 
7 
Liver 
function 
test 
64 
Malaria blood film, qPCR, 
gametocyte qPCR, 
hemoglobin 
  
  
Up to 
250 
Blood 
donation 
321 
398 
  
 CMB, EBV 
serology 
Immunology 
  
  
  
  
  
  
CBC 
  
  
  
  

 
MIST 1 protocol V 10.0 dated 14 February 2022                                               Page 51 of 85 
Phase 
Clot blood 
EDTA blood 
NaF 
CPD 
Total 
blood 
volume 
(ml) 
Total 
accumulat
ion blood 
volume 
(ml) 
Volume 
Test 
Volume 
Test 
Volume 
Test 
Volume 
Test 
Treatment 
phase 
3 
Liver function 
test  
22 
Malaria blood film, qPCR, 
gametocyte qPCR, 
hemoglobin 
  
  
  
  
25 
423 
  
  
Malaria immunology 
  
  
  
  
CBC 
 
 
 
 
 
 
DRx 28 
5 
Blood 
biochemistry 
14 
CBC 
  
  
  
  
19 
442 
  
  
Malaria blood film, qPCR, 
gametocyte qPCR 
  
  
  
  
  
  
Malaria immunology 
  
  
  
  
DRx 90 
5 
Blood 
biochemistry 
14 
CBC 
  
  
  
  
69 
511 
  
  
Malaria blood film, qPCR, 
gametocyte qPCR 
  
  
  
  
  
  
Malaria immunology 
  
  
  
  
50 
Blood collection for cross 
matching* 

 
MIST 1 protocol V 10.0 dated 14 February 2022                                               Page 52 of 85 
Phase 
Clot blood 
EDTA blood 
NaF 
CPD 
Total 
blood 
volume 
(ml) 
Total 
accumulat
ion blood 
volume 
(ml) 
Volume 
Test 
Volume 
Test 
Volume 
Test 
Volume 
Test 
DRx 180** 
 4 
Syphillis 
HIV 
HBs profile 
anti-HCV 
1 
HTLVI-II 
 
 
 
 
 
5 
516 
DRx 1 Yr 
  
  
14 
Malaria blood film, qPCR, 
gametocyte qPCR 
  
  
 
 
14 
530 
  
  
CBC 
  
  
  
  
  
  
Malaria immunology 
  
  
  
  
* Blood sample for cross matching may be collected any time ≥ 90 days after antimalarial treatment initiation (time of blood donation)  
** Blood sample may be collected any time ≥ 180 days after antimalarial treatment initiation (time of blood donation)  

 
MIST 1 protocol V 10.0 dated 14 February 2022                                                                                                                                                                   Page 53 of 85 
Table 7: Schedule of Clinic Attendances Post-challenge  
Event 
Admission Phase 
OPD visit 
for PQ DOT 
Follow up Phase 
Timeline  
D0 
D1-D4 
D5 - DBl donation DStart CQ/PQ - 
Dd/c 
D1st OPD  -  
DEnd PQ 
DRx28 
Between 
DRx28 &  
DRx90 
DRx90 
DRx91 to 
1 yr. 
DRx180 
DRx1 yr. 
Window (days) 
0 
0 
0 
 
0 
±9 
 
±14 
 
±14 
±14 
Inclusion / Exclusion criteria 
X 
 
 
 
 
 
 
 
 
 
 
Medical History (well-being 
check list)a 
X 
X 
(Once 
daily) 
X 
(Twice daily) 
X 
(Once daily) 
X 
X 
 
X 
 
 
X 
Body weight  
X 
 
X 
(Blood 
donation) 
 
 
X 
 
X 
 
 
X 
Physical examination a 
X 
X 
X 
X 
X 
X 
 
X 
 
 
X 
Urine pregnancy test (women 
only)  
 
 
X            
(before CQ 
Tx) 
 
 
 
 
 
 
 
 
Vital signs a 
X 
X 
X 
X 
X 
X 
 
X 
 
 
X 
Local & Systemic Events 
 (AE/SAE) b 
X 
X 
X 
X 
X 
X 
 
X  
 
 
X 

 
MIST 1 protocol V 10.0 dated 14 February 2022                                                                                                                                                                   Page 54 of 85 
Event 
Admission Phase 
OPD visit 
for PQ DOT 
Follow up Phase 
Timeline  
D0 
D1-D4 
D5 - DBl donation DStart CQ/PQ - 
Dd/c 
D1st OPD  -  
DEnd PQ 
DRx28 
Between 
DRx28 &  
DRx90 
DRx90 
DRx91 to 
1 yr. 
DRx180 
DRx1 yr. 
Window (days) 
0 
0 
0 
 
0 
±9 
 
±14 
 
±14 
±14 
Mosquito bite malaria 
sporozoite challenge 
X 
 
 
 
 
 
 
 
 
 
 
Treatment for Malaria c 
 
 
 
X 
X 
 
 
 
 
 
 
Diary card 
 
 
 
 
X 
X 
 
X 
 
Malaria Blood Film and qPCR 
and Gametocyte PCR 
 
 
X 
(Twice daily)  
X 
(Once daily) 
X 
(Once daily 
on DPQ 7 and 
DPQ 14)      
X 
 
X 
  
 
X 
Haemoglobin  
 
 
X  
X 
X 
(DEnd PQ)  
 
 
 
 
 
 
Haematology (CBC) 
 
X 
(D2) 
 
X 
(D5, D Bl 
donation) 
 
X 
(DRx7) 
X 
 
X 
 
 
X 
Biochemistry d 
 
 
 
 
X 
 
X 
 
 
 
Liver function Tests 
 
X 
X  
 
X  
 
 
 
 
 
 

 
MIST 1 protocol V 10.0 dated 14 February 2022                                                                                                                                                                   Page 55 of 85 
Event 
Admission Phase 
OPD visit 
for PQ DOT 
Follow up Phase 
Timeline  
D0 
D1-D4 
D5 - DBl donation DStart CQ/PQ - 
Dd/c 
D1st OPD  -  
DEnd PQ 
DRx28 
Between 
DRx28 &  
DRx90 
DRx90 
DRx91 to 
1 yr. 
DRx180 
DRx1 yr. 
Window (days) 
0 
0 
0 
 
0 
±9 
 
±14 
 
±14 
±14 
(D3) 
(Present with 
parasitaemia) 
(DEnd PQ) 
Malaria Immunology  
 
X 
(D2) 
X 
(D5, DBl 
donation) 
 
X 
(DRx7) 
X 
 
X 
 
 
X 
Blood donation  
 
 
X 
 
 
 
 
 
 
 
 
Contact volunteers fortnightly  
 
 
 
 
 
 
X  
 
X   
 
 
COVID-19 testing (RT-PCR by 
nasopharyngeal swab) 
 
X e   
X f   
X  
(Dd/c) 
 
 
 
 
 
 
 
COVID-19 testing 
(serology POC test) 
 
 
X h 
 
 
 
 
 
 
 
Blood collection for cross 
matching 
 
 
 
 
 
 
 
x  
 
 
 
Infectious diseases (HBV, HCV, 
anti HTLV I and anti HTLV II 
antibody, HIV, and Syphilis test 
(TPHA)g 
 
 
 
 
 
 
 
 
 
X 
 

 
MIST 1 protocol V 10.0 dated 14 February 2022                                                                                                                                                                   Page 56 of 85 
Event 
Admission Phase 
OPD visit 
for PQ DOT 
Follow up Phase 
Timeline  
D0 
D1-D4 
D5 - DBl donation DStart CQ/PQ - 
Dd/c 
D1st OPD  -  
DEnd PQ 
DRx28 
Between 
DRx28 &  
DRx90 
DRx90 
DRx91 to 
1 yr. 
DRx180 
DRx1 yr. 
Window (days) 
0 
0 
0 
 
0 
±9 
 
±14 
 
±14 
±14 
EBV and CMV serology 
 
 
X i 
 
 
 
 
 
 
 
 
a  Vital signs include blood pressure, pulse, respiratory rate and body temperature. 
b  AE/SAEs  will be collected until 1 year. 
c  This treatment period could occur on any day between D6 and D21, blood draws will continue as per the Table for the relevant study day, up until the time-point of blood 
donation or Treatment. The treatments will be done on D21 if no criteria meet.  
d    Biochemistry including electrolytes (sodium, potassium, chloride, bicarbonate), blood urea nitrogen (BUN), creatinine (Cr), liver function tests (LFTs) 
e   In case volunteers have fever but malaria blood film, qPCR, and testing for vector-borne diseases are negative, COVID-19 RT-PCR will be tested. 
f  In case volunteers have fever but malaria blood film and qPCR are both negative, COVID-19 RT-PCR will be tested. 
g   HBV, HCV, and HIV will be tested for antibodies. HBV serological profile includes HBsAg, anti-HBc, and anti-HBs 
h   COVID-19 serology POC test will be done at 14 days after the inoculation. 
i    EBV and CMV serology will be done at 2-4 weeks after the inoculation. EBV and CMV including EBV VCA (IgM)/IgG antibody, anti EBNA and EA complex antibody, and CMV IgM/IgG antibody

 
 
 
MIST 1 protocol V 10.0 dated 14 February 2022                                                   Page 57 of 85 
7.5 
Details of study clinical procedures/laboratory tests 
Procedures will be performed at the time points indicated in the schedule of procedures (Tables 6-7). 
Additional procedures or laboratory tests may be performed, at the discretion of the investigators if clinically 
necessary. All clinical and laboratory systems will be inspected by a Contract Research Organization (CRO) 
appointed by the Wellcome Trust (first inspection in March 2019, second inspection prior to study initiation), 
and study procedures monitored by the MORU Clinical Trials Support Group.  
7.5.1 
Clinical procedures 
7.5.1.1 Body Weight and Height 
Body Weight and Height will be measured at the time points indicated in the schedule of procedures (Tables 
6 & 7). 
7.5.1.2 Vital signs 
Pulse rate (PR), blood pressure (BP), respiratory rate (RR) and body temperature (BT) will be measured at the 
time points indicated in the schedule of procedures (Tables 6 & 7). 
7.5.1.3 Urinalysis 
Urine will be tested for the presence of clinically significant proteinuria, glucosuria or haematuria (out of 
normal range) at the screening visit.  
7.5.1.4 Electrocardiogram 
An electrocardiogram will be performed at the screening visit.  
7.5.2 
Laboratory procedures 
The Tropical Medicine Diagnostic Reference Laboratory (TMDR) will be the central reception point for all blood 
samples collected from FTMCTU, and will follow a sample management system designed by the MORU Sample 
Management Centre. TMDR will aliquot diagnostic and research samples and be responsible for transfer to 
designated laboratories for testing and retrieve the result generated from the following laboratories  
▪ Diagnostic Laboratory Unit, Hospital for Tropical Disease, Faculty of Tropical Medicine, Mahidol 
University 
1. Haematology: complete blood count (CBC) and blood grouping (A/B/O, Rhesus status)   
2. Biochemistry: consisting of electrolytes, blood urea nitrogen (BUN), creatinine (Cr), liver function 
tests (LFTs), fasting blood sugar (FBS) and lipid profile (cholesterol, triglyceride, HDL, LDL).  
3. Serum beta-human chorionic gonadotrophin (serum β-HCG)  
 
4. Diagnostic serology; HBs profile (HBsAg, anti-HBc, anti-HBs), HCV antibodies, and HIV antibodies. 
5. Urinalysis 
6. Urine pregnancy test 

 
 
 
MIST 1 protocol V 10.0 dated 14 February 2022                                                   Page 58 of 85 
7. CMV, EBV serology 
 
▪ Tropical Medicine Diagnostic Reference Laboratory (TMDR) 
1. Hemoglobin 
2. Serology for HTLV I, HTLV II  
3. Dengue PCR 
4. Filariasis by rapid antigen test  
▪ Malaria Laboratory, Mahidol-Oxford Tropical Medicine Research Unit 
1. Malaria microscopic examination 
▪ Microbiology and Immunology Department, Faculty of Tropical Medicine, Mahidol University 
1. qPCR for Chikungunya  
2. qPCR for Zika virus  
▪ Molecular Tropical Medicine & Genetics Department, Faculty of Tropical Medicine, Mahidol University 
1. qPCR for Malaria  
▪ ATGenes 
1. G6PD genotyping 
2. Duffy antigen  
3. Haematological diseases: haemoglobinopathy analysis, CBC, haemoglobin electrophoresis and 
molecular studies for alpha and beta thalassaemia trait 
4. CYP2D6 genotype testing and reporting of expected metaboliser phenotype. 
▪ Virology Department, Ramathibodi Hospital, Mahidol University 
1. qPCR for HIV1 virus, HIV 2 virus 
2. Hepatitis B PCR 
3. qPCR for hepatitis C virus 
4. qPCR for Japanese encephalitis virus. 
5. Cytomegalovirus 
6. Epstein-Barr virus 
▪ Serology Department, Ramathibodi Hospital, Mahidol University 
1. Syphilis serology  
▪ Division of Hematology and Oncology, Siriraj Hospital 
1. G6PD enzyme level 

 
 
 
MIST 1 protocol V 10.0 dated 14 February 2022                                                   Page 59 of 85 
▪ Mahidol Vivax Research Unit (MVRU), Faculty of Tropical Medicine, Mahidol University 
1. Screening for background exposure to malaria (screening 2): Multiplex Bead Based Immunoassay will 
be performed to determine the magnitude of antibodies against recent exposure of 5-8 blood-stage 
P. vivax antigens. A seropositivity cut-off will be calculated by the average of median fluorescence 
intensity (MFI) plus 2 standard deviations of negative control.   
2. Gametocyte qPCR 
3. Immunology: Immunological responses to P. vivax infection.  
4. Cryopreservation of P. vivax infected blood (parasite banking) 
Samples may also be sent to collaborating laboratories within and outside the Thailand for immunomonitoring 
and/or harmonisation of key immunological assays. There are two collaborating laboratories;  
1) Ehime University, Matsuyama, Japan, and 2) KEMRI, Kenya 
The AlphaScreen assay will be used to quantify malaria antigens-specific antibodies (up to 300 P. falciparum 
antigens and 300 P. vivax antigens). A seropositive cut-off will be set as half the lowest non-negative value of 
the assayed samples. Positive malaria history refers to a volunteer who previously had a malaria infection 
leading to antibodies in their serum which reactive to more than 10% of the malaria antigens in the library. 
This may include human DNA and RNA analysis. 
With the volunteers’ informed consent, any leftover cells and serum [not including up to 250 mL blood 
inoculum] will be stored for 10 years in the FTM/MORU Sample Management System for future immunological 
and genetic analysis of malaria-specific responses. 
7.5.3 
Blood donation, processing and storage 
Two hundred fifty cubic centimeters (cc) of blood will be taken with a CPD whole blood collection kit (Imuflex 
whole blood filter PL saving with, Terumo). The blood will be leukocyte depleted using the inline 
leukodepletion filter that is a component of the blood collection pack. The blood will be transported back to 
the facility at Hospital for Tropical Diseases where it will be processed and frozen according to laboratory 
procedures in the spirit of Current Good Manufacturing Practice (cGMP). Immediately on arrival, the red cells 
will be separated from the plasma by centrifugation, and the blood then cryopreserved using a method 
described elsewhere [21]. Briefly, a ratio of 2 volumes of GMP quality Glycerolyte 57 to 1 volume of infected 
cell pellet will be used. The first 20% of the Glycerolyte 57 volume will be added dropwise with gentle agitation, 
and the suspension will be incubated for 5 minutes at room temperature before the remaining Glycerolyte 57 
is added. The RBC-Glycerolyte mixture will be stored as 1.5 ml aliquots in 2 ml cryovial tube then frozen under 
controlled conditions at a temperature of -80°C for overnight. Aliquots are transferred and stored in a 
dedicated liquid nitrogen tank in a secure environment. This freezing process will be performed according to 
the blood donation and sample processing work instruction (WI) and Laboratory Standard Operating 
Procedure (SOPs) under controlled conditions in the laboratory, similar to the procedures used to freeze and 

 
 
 
MIST 1 protocol V 10.0 dated 14 February 2022                                                   Page 60 of 85 
thaw the blood being used for CHMI trials in Oxford [54]. Aliquots of blood will be tested for bacterial 
contamination with a validated blood culture technique, mycoplasma, endotoxin as well as sterility and other 
key QC parameters according to a defined testing plan. CMV, EBV, Blood-borne (HIV, HBV, HCV, HTLV I-II, 
Syphilis) and vector-borne (DEN, CHIK, JE, and Zika) infectious diseases will be tested to ensure no new 
acquired infection during the admission phase. Genetic analysis of the parasite will be performed for in depth 
characterization of the banked parasite. There is good evidence that P. vivax withstands the freezing and 
thawing process and can subsequently be successfully used in short-term culture and invasion assays [55]. 
7.6 
Definition of start and end of trial 
The start of the trial is defined as the date of the first screening visit for first volunteer. The end of trial is the 
date of the last follow-up (1 year) of the last volunteer. 
7.7 
WITHDRAWAL OF VOLUNTEERS 
In accordance with the principles of the current revision of the Declaration of Helsinki (updated 2013) and any 
other applicable regulations, a volunteer has the right to withdraw from the study at any time and for any 
reason, and is not obliged to give his or her reasons for doing so. In addition the volunteer may withdraw/be 
withdrawn from further study procedures at any time in the interests of the volunteer’s health and well-being, 
or for any of the following reasons: 
• 
Administrative decision by the Investigator. 
• 
Ineligibility (either arising during the study or retrospectively, having been overlooked at screening). 
• 
Volunteer non-compliance with study requirements. 
• 
An AE which requires discontinuation of the study involvement or which results in inability to continue 
to comply with study procedures. 
• 
Current COVID-19 infection during a participant’s admission in the hospital 
The medical monitors may recommend withdrawal of volunteers. The reason for withdrawal from further 
study procedures will be recorded in the case record form (CRF). If a volunteer withdraws after having 
completed a course of antimalarials, as much long-term safety data collection as possible, including 
procedures, such as safety bloods, will continue to be collected, with the agreement of the volunteer. For all 
AEs, appropriate follow-up visits or medical care will be arranged, with the agreement of the volunteer, until 
the AE has resolved, stabilised or a non-trial related causality has been assigned. 
If a volunteer withdraws from the study after challenge but before reaching the criterion for malaria diagnosis, 
a complete, appropriate, curative course of antimalarial therapy must be completed by standard chloroquine 
treatment and a 2-week course of 30 mg/day of primaquine. The importance of this will be emphasized to 
volunteers at screening. If a volunteer refuses to take antimalarial therapy after malaria diagnosis, a rapid 
assessment of mental state and capacity will be undertaken, with the involvement of psychiatric and infectious 

 
 
 
MIST 1 protocol V 10.0 dated 14 February 2022                                                   Page 61 of 85 
diseases specialists. If necessary and if in accordance with the law in Thailand the volunteer may be detained 
for appropriate medical management. 
If a volunteer withdraws from the study, blood samples collected before their withdrawal from the trial will 
be used/stored unless the volunteer specifically requests otherwise. Similarly, all data collected up to the point 
of withdrawal will be stored, unless they specifically request for it to be destroyed. Volunteers are free to 
request that their blood samples be destroyed anytime during or after the study. However, up to 250 mL of 
blood that they donate will not be destroyed after they have donated it, as it will be impossible to repeat this 
from a “back-up” volunteer half way through the study. However, the sample will be “delinked” so that there 
can be no tracing of it back to the volunteer who donated it. This shall all be made clear in the consent form. 
In all cases of volunteer withdrawal, excepting those of complete consent withdrawal, long-term safety data 
collection will continue if volunteers have already undergone challenge process. Withdrawn volunteers before 
blood donation will be replaced. 
8. Description and administration of the study agents (which is the P. vivax 
infected mosquitoes) 
 
In this study, the study agent that we apply / administer to the volunteer is not a drug (since it is not drug 
trial). It is “P. vivax infected-mosquitoes” and the dosage given is “5 infected mosquitoes per one volunteer”. 
This section composes of 2 parts; 1) How to prepare the study agent (infected mosquitoes) (See section 9.1.)  
and 2) How to administer the study agent to the volunteers (how to perform mosquito biting) (See section 
9.2.) 
8.1 
Preparing the study agent: Infected mosquito preparation stage 
The process to producing the “P. vivax infected mosquito” are a) Source case identification (P. vivax patient(s)), 
b) mosquito preparation, and c) membrane feeding: 
8.1.1 
Source case identification (P. vivax patient(s)):  
P. vivax patients are recruited from a clinic or hospital in endemic areas in Thailand where FTM has a study 
site. The molecular analysis is performed by the FTM laboratory in Bangkok to exclude mixed malaria species 
infection. Testing of donor blood for blood-borne and endemic mosquito-borne (vector borne) infections in 
the source patient as per the following details; 
• 
Screening for blood-borne infection: HIV-1 and -2, HTLV I-II infection, Hepatitis B and C (HBV and HCV), 
and syphilis 
• 
Screening for vector-borne infections endemic in Thailand that could potentially be transmitted from 
the patient via the Anopheles mosquito: Dengue virus, Zika virus, Chikungunya virus, Japanese B 
encephalitis virus and Filariasis 

 
 
 
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• 
Screening for mixed malaria infections: molecular speciation for P. vivax, P. falciparum, P. ovalae , P. 
malariae , and P knowlesi 
 
8.1.2 
Mosquito preparation and membrane feeding:  
The Mahidol Vivax Research Unit (MVRU) and the Department of Entomology, Faculty of Tropical Medicine, 
Mahidol University, Bangkok, will provide the Anopheles mosquitoes for the challenge. They are reared in the 
laboratory and fed on rigorously screened human blood (purchased from the Red Cross) to maintain the colony 
and induce egg-laying. The prepared mosquitoes are infected via a membrane feeder (membrane feeding 
assay (MFA)) containing infected blood from a P. vivax patient (as explained in #1). Up to 3,000 mosquitoes 
are used per patient’s blood feed. 
Prior to the mosquitoes biting volunteers, the presence of P. vivax infection is checked:  
a) 7-9 days post blood-feeding by counting oocysts visualised on dissection of the midgut; and  
b) 14 days post blood-feeding by grading the number of sporozoites visualised on dissection of salivary 
gland.  
The mosquitoes are only used for challenge if the infection rate is >75% and grading of sporozoite  > +3. (This 
mosquito preparation protocol was already approved by FTMEC (No# MUTM 2018-017). 
8.2 
Administering the study agent to the volunteers:  
• 
Mosquito challenge of volunteers at the insectarium Unit, Department of Entomology 
Once the mosquitoes have been fed with P. vivax infected blood (by membrane feeding assay (MFA)) they 
need to be used in a sporozoite challenge (biting the study subject) between 14-21 days later to ensure 
maintained infectivity (it usually takes 14 days for the sporozoites to reach the salivary glands).  
The challenge steps: Challenge (mosquito biting) of volunteers by using these infected mosquitoes will be 
carried out in the insectarium unit within the Faculty of Tropical Medicine, Mahidol University, Bangkok.  
The volunteer(s) will be hospitalized a night before the mosquito biting day (D0) to ensure their safety / 
illegibility and to avoid the traffic burden. 
Mosquitoes are transported directly from MVRU in a temperature-controlled box at 26 +/- 2oC and 
immediately transferred to a secure room within the insectary of the Entomology Department. Each pot of 
mosquitoes is kept in a strong transparent plastic storage box. All manipulation of mosquitoes is performed 
within a secure room. The challenge is performed in a part of the secure insectary that is isolated by 
appropriate and approved mechanisms.   

 
 
 
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Prior to challenge, all volunteers are briefed. The arms will be cleaned according to the mosquito biting work 
instruction. Use by the volunteer of lotion or perfume is forbidden on the day of mosquito challenge. 
For challenging, 5 mosquitoes will be aspirated into a small box.  Each volunteer will be exposed to the bites 
of five infectious mosquitoes by placing their forearms over the mosquito cup for 5-10 minutes at a time.  
Engorged mosquito(es), as indicated by the presence of a blood meal in the abdomen), is/are individually 
taken out of the pot to be dissected and assessed for sporozoite load (grading the load of sporozoite by using 
graded 0 to +4), ONLY a gland rating of +2 or more, representing 10 or more observed sporozoites, qualifies 
as being infectious). If, by this method the volunteer is found to have been inoculated by less than five infected 
mosquitoes, further mosquitoes are allowed to feed on the volunteer until a total of 5 appropriately infected 
mosquitoes have fed. The bite-challenge procedure continues until the volunteer has been bitten by 5 
infectious mosquitoes (Appendix A). This mosquito biting process will be performed strictly following the 
Mosquito Bite Malaria Sporozoite Challenge work instruction.  
The rationale for 5 infectious mosquito bites is based on the extensive previous experience with P. falciparum, 
where sporozoites inoculated by <5 mosquitoes have led to an irregular infection in malaria-naive human 
volunteers. However, this does not appear to be the case for P. vivax: 100% of the 45 volunteers (controls and 
vaccines) bitten by five P. vivax-infected mosquitoes in the WRAIR US study developed patent parasitaemia, 
and also of the 53 non-vaccinated volunteers (either malaria-naïve or semi-immune) bitten by between 2 and 
4 infected mosquitoes in the Cali studies, 52 developed patent parasitaemia (about 100-1,000 sporozoites are 
inoculated per bite). However, to ensure reliable infection in our small study of just 6 volunteers, we have 
chosen to proceed with 5 successful infectious mosquito bites. 
9. SAFETY  
9.1 
Safety experience from previous CHMI studies   
Following a collaborative consensus process involving investigators from the USMMVP, Sanaria, University of 
Maryland, University of Oxford, RUNMC, the Seattle Biomedical Research Institute and the KEMRI-Wellcome 
Kilifi Research Programme, a consensus document; “Standardization of Design and Conduct of P. falciparum 
Sporozoite Challenge Trials” was developed, and provides a comprehensive guide to the appropriate conduct 
of controlled human malaria infection studies [33]. Although there remain minor differences between centres 
in follow-up procedures in controlled human malaria infection trial conduct, there is consensus on the 
following key points. 
• 
All volunteers should have a medical assessment no longer than 48 hours before challenge, including 
an interim medical history, directed physical examination, pregnancy test for female volunteers. 

 
 
 
MIST 1 protocol V 10.0 dated 14 February 2022                                                   Page 64 of 85 
• 
Follow-up visits should be scheduled at least once daily, but may increase in frequency to twice daily, 
starting at day 5-7 post-sporozoite challenge. At all visits volunteers should be questioned about the 
occurrence of adverse events (AEs) and use of medication. 
• 
Grading and reporting of adverse events should be performed using international and local guidelines. 
It should be noted that the occurrence of a low frequency of grade 3 severe adverse events, of short 
duration, and with no long-term sequelae, is not unexpected in control human challenge studies. A 
minority of those challenged are known to experience grade 3 systemic adverse events and this fact 
should be included in the informed consent form. 
• 
Vital signs should be recorded at least once daily and at any subsequent visits for medical attention. 
Directed physical examination should be performed when necessary. 
• 
It is critical that every volunteer must receive every dose of antimalarial therapy. In some settings fully 
directly observed treatment will be essential. Where directly observed treatment is not used, 
investigators must follow volunteers closely to ensure compliance with the treatment regimen. 
• 
After challenge, all volunteers should be followed until they have completely finished antimalarial 
treatment. 
• 
Volunteers should be evaluated at least two weeks after finishing treatment. 
• 
A local safety monitor and an independent safety monitoring committee should be established to act 
as independent experts in evaluating adverse events. The safety monitor or monitoring committee 
may advise the investigators on initiating anti-malarial treatment for a specific volunteer or volunteer 
group. While safety monitoring committees are not a requirement for Phase I trials, they should be 
considered a requirement for challenge trials which have an efficacy component and which have major 
potential safety concerns. 
Volunteer safety is of paramount importance, and we will follow these guidelines. The following measures are 
in place to safeguard volunteer safety: 
• 
Volunteers will only be enrolled in the study if investigator judges this is appropriate. 
• 
Volunteers’ understanding of the trial information will be tested by means of a questionnaire at 
screening. This provides further confidence that fully informed consent has been obtained. 
• 
Before challenge, full contact details for each volunteer will be documented, including home address 
and mobile telephone numbers. Mobile telephone numbers will be verified prior to challenge to 
ensure the volunteers are easily contactable. Home and work landline telephone numbers where 
available and next-of-kin address and telephone numbers will also be documented. Volunteers must 
also provide the Investigators with the name and 24 hour telephone number of a close friend (s), 
relative (s) or housemate (s) who live nearby and who will be kept informed of their whereabouts for 
the duration of the study. 

 
 
 
MIST 1 protocol V 10.0 dated 14 February 2022                                                   Page 65 of 85 
• 
The volunteers will be hospitalized throughout the challenge processes until clinically recovered AND 
the completion of the antimalarial course (chloroquine) AND until two consecutive negative blood 
films. The 14 days of primaquine for radical cure will be performed under direct observed therapy 
(DOT).  
• 
Volunteers will be able to contact a medically qualified member of the study team 24 hours a day 
throughout the study period. 
• Volunteers will be required to contact the study team and their GP if they feel feverish or unwell in 
the 1 years following the challenge. 
• The 5 years insurance for malaria infection will be provided. 
9.2 
   What do we expect to discover during the challenge? 
This part of the protocol aims to clearly review the events related to experience from the previous control 
human challenge studies in order to foresee the possible event(s) after the challenge that we may have to 
prepare to encounter within this study (almost all of the data comes from P. falciparum controlled human 
challenge studies). 
• 
Nearly all unvaccinated volunteers in challenge studies develop symptoms of clinical malaria infection 
within 2 weeks following the challenge (means nearly all success in the induce the infection by this 
intervention) 
• 
Approximately 1/5 of volunteers in P. falciparum CHMI studies temporarily develop symptoms graded 
as severe (symptoms that prevent daily activities, most of them were fever and dehydration), but severe or 
life-threatening malaria has never occurred [43]. Vivax malaria is less dangerous than falciparum malaria, 
though severity of initial symptoms may be similar. 
• 
Routine laboratory checks generally show a moderate decrease in leukocyte and platelet numbers 
during infection, with no change in haemoglobin concentration [44].  
• 
Bleeding or thrombogenic complications have never been described [43, 44].  
• 
Abnormalities of liver enzymes have been observed, but these abnormalities have rarely resulted in 
clinical manifestations (just one volunteer with raised ALT associated with abdominal pain and vomiting in the 
recent RCT from Cali [23]) and they resolved after a few days. 
As of 2011, human malaria challenge infections have been conducted with P. falciparum in over 1,300 
volunteers [43-45]. Recently, safety concerns were raised in a young volunteer who suffered a cardiac event 
shortly after treatment for diagnosed malaria. This was diagnosed as probable myopericarditis, although 
ischaemia could not be ruled out. Although a definite relationship between the cardiac event, which resolved 
fully and rapidly, and the experimental malaria infection was not established [46] (and this was also seen with 
P. falciparum, not P. vivax), it has been generally agreed that volunteers with an increased risk of cardiac 

 
 
 
MIST 1 protocol V 10.0 dated 14 February 2022                                                   Page 66 of 85 
disease should be excluded from such trials [33]. A further case of myopericarditis has since been identified in 
a recent challenge study, also at the Nijmegen, Netherlands, centre, but in this case the individual was also 
diagnosed with an intercurrent rhinovirus infection so that the relation to malaria infection is again uncertain. 
There was a brief episode of clinical chest pain and the volunteer made a full recovery [47]. 
9.3 
Risks 
9.3.1 
Phlebotomy 
The maximum volume of blood drawn over the study period should not compromise these otherwise healthy 
volunteers. There may be minor bruising, local tenderness or pre-syncopal symptoms associated with 
venipuncture, which will not be documented as AEs if they occur. 
Due to the 250 mL blood donation, each volunteers will have a total (maximum) of 530 mL taken over the 
study period (~3 months from admission phase to 1 year). This is approximately in the range of the amount of 
a regular blood donation (350-500 mL) every 3 months. In addition, a recent multicentre study by NHS Blood 
and Transplant (in Oxford and Cambridge) compared outcomes in 50,000 regular blood donors who were 
randomised to different intervals between blood donations (as regularly as giving 470 mL every 8 weeks in 
one group). They found no differences in tolerance/ outcomes over a 3-year follow-up between the different 
groups (verbal communication, data as yet unpublished). Donating 816 mL over 4 months amounts to 124 mL 
less than that donated by the 8-week interval group in the NHSBT study, so we are confident that this volume 
should not compromise our volunteers in any way. However, we do acknowledge that these volunteers will 
not be true “healthy volunteers” at the time of blood donation as they will be infected with malaria and may 
be symptomatic from this. For this reason, they shall be closely monitored in a controlled environment at the 
time of donation, with access to symptomatic relief, including fluid therapy, if required. Reassuringly, a similar 
amount of blood has been taken from research volunteers with P. vivax infection in 3 previous studies, and 
this has resulted in no untoward adverse events over and above those attributable to the P. vivax infection 
itself [21, 49, Minassian A and Draper S, personal communication]. The same or larger amount (500 mL) has 
also been taken from patients with P. falciparum malaria [50, 51]. 
Participants’ blood samples will be collected by well - trained medical personnel with aseptic technique to 
prevent infection or complications. 
9.3.2 
   Blood donation (up to 250 mL) 
Close clinical monitoring to ensure that volunteer tolerates the blood donation. This procedure will be carried 
out during the working hour.  If the volunteer’s haemoglobin falls below 10 g/dL with clinical stable regardless 
of their level of parasitaemia, then the blood donation shall be withheld and they shall be treated with 
antimalarials immediately. 

 
 
 
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9.3.3 
    Mosquito bites 
Mosquito bites may cause local inflammatory reactions with redness, itching, swelling, scaling and/or 
tenderness. Topical anti-histamine cream for use twice daily for 3 days post-mosquito bite will be dispensed 
to the volunteers on the day of challenge. Serious allergic reactions including anaphylaxis have not been seen 
in challenge studies to date, but may occur and for this reason volunteers will be inoculated in an area where 
Advanced Life Support trained physicians and a defibrillator are immediately available. The volunteers will be 
screen and excluded if suspected as severe allergy to the insect bite or mosquito bite. Data regarding itching 
and use of antihistamine cream will be collected on diary cards but, as this is an expected part of the challenge 
procedure, it will not be classed as an adverse event in the immediate post-challenge period. 
9.3.4 
    Plasmodium vivax infection 
Base on previous studies, volunteers are likely to develop symptomatic malaria infection following challenge 
[48]. Volunteers will be followed up closely post-challenge and only enrolled in the study if they are deemed 
reliable and capable of complying with the intensive follow-up schedule.  
As described above, the volunteers will be hospitalized in the Hospital for Tropical Diseases, Faculty of Tropical 
Medicine, Mahidol University for close clinical monitoring, treatment (antimalarials) and symptomatic relief 
(fluids, analgesia and/or anti-emesis) until parasite becomes negative. The treatments algorithm as well as the 
hospital guideline will be fully reviewed and strictly followed. This study initiation will be officially acknowledge 
to the hospital to ensure all the facility are in place. The expert of the Faculty of Tropical Medicine will be 
officially acknowledged prior to the study. The medical monitors will be reachable for the whole study 
processes.  
A very small proportion of volunteers in previous P. vivax blood-stage and sporozoite-stage challenge studies 
have temporarily required intravenous fluid therapy for nausea and vomiting prior to treatment [23, 49].  
It is relevant to note that safety and parasitological data from 128 malaria-naïve volunteers participating in 
challenge studies in Oxford and the Netherlands were analysed and compared to a report from the US Military 
Malaria Vaccine Program. The authors found that cohorts with a longer prepatent period or a higher peak 
parasitaemia did not consistently show a higher frequency of adverse events [52]. However, these data were 
on P. falciparum challenge, and there is a lot less experience with P. vivax challenge. 
9.3.5 
     Risk of relapse of malaria 
Infection with P. vivax malaria carries with it a significant risk of relapsing disease if sub optimally treated. This 
can occur because parasites (hypnozoites) can stay dormant in the liver and then reactivate into the blood 
causing clinical disease weeks, months or even years later. This risk is minimised and mitigated by: 
a.) giving volunteers a 14-day course of primaquine treatment after / together with their initial 3-days of 
schizonticidal antimalarials, as this drug specifically eradicates any remaining dormant parasites from the liver.  

 
 
 
MIST 1 protocol V 10.0 dated 14 February 2022                                                   Page 68 of 85 
b.) screening volunteers for their ability to metabolise primaquine efficiently, by checking their blood for 
CYP2D6 genotypes. Only volunteers who have genotypes associated with “high-metaboliser” status will be 
enrolled into the study.  
c.) following up volunteers after treatment by email and / or telephone every two weeks for a year in order to 
capture any symptoms suggestive of relapse and any medical attention sought. They shall also be encouraged 
to inform their local doctor and the study investigators as soon as possible should any symptoms occur. If they 
do relapse they shall be referred to hospital for treatment immediately. The study group will provide  
treatment for 5 years of P. vivax malaria relapse. 
9.3.6 
    COVID-19 
The risk mitigation steps will be taken for the face to face research during COVID-19 pandemic which follow 
the local guidance (Hospital for Tropical Diseases). 
9.3.7 
Contingency plan if subject going missing post-challenge  
The volunteers will be inpatients for entirety of the challenge phase, from the day before mosquito challenge 
until the completion of schizonticidal antimalarial treatment. They will then receive daily directly observed 
primaquine treatment for 14 days. It is unlikely that a volunteer will become absent during hospitalization 
(before schizonticidal treatment is completed). The chance of a subject missing radical treatment with 
primaquine daily DOT should be very low following the counseling  carried out during the screening process. 
If the volunteer does fail to attend for a scheduled clinical visit after discharge from the hospital (during the 
primaquine DOT phase), and if the volunteer is un-contactable by telephone or instant messaging, the 
following stakeholders will be informed: 
· 
All Investigators. 
· 
The volunteer’s nominated contact and next of kin. 
· 
The trial sponsor. 
· 
The medical monitor group. 
· 
The ethical committee(s). 
· 
The local police department. 
Active efforts will be made to locate the volunteer while all parties will aim to preserve the volunteer’s 
confidentiality. Volunteers will be informed of this during screening. 
9.4 
Safety detection, assessment, documentation and reporting 
The investigators and designated site staff is/are responsible for the detection, assessment, documentation 
and reporting of events meeting the criteria and definition of an AE or SAE as provided in this protocol. Each 

 
 
 
MIST 1 protocol V 10.0 dated 14 February 2022                                                   Page 69 of 85 
volunteer will be instructed to contact the investigator immediately should the volunteer manifest any signs 
or symptoms they perceive. 
9.4.1 
Definitions 
9.4.1.1 Adverse Event (AE): 
An AE is any untoward medical occurrences in a volunteer, which may occur during or after administration of 
a study intervention (in this case, vivax parasite challenge) and does not necessarily, have a causal relationship 
with the intervention. An AE can therefore be any unfavorable and unintended sign (including an abnormal 
laboratory finding), symptom or disease temporally associated with the study intervention, whether or not 
considered related to the study intervention. Any events occurring between screening and D-1 will be 
considered as baseline/, pre-existing conditions. This information will be recorded in the medical records.  
9.4.1.2 Serious Adverse Event (SAE): 
An SAE is an AE that results in any of the following outcomes, whether or not considered related to the study 
intervention. 
• Death (i.e. results in death from any cause at any time). 
• Life-threatening event (i.e. the volunteer was, in the view of the Investigator, at immediate risk of 
death from the event that occurred). This does not include an AE that, if it occurred in a more serious 
form, might have caused death. 
• Persistent or significant disability or incapacity (i.e. substantial disruption of one’s ability to carry out 
normal life functions). 
• Transfer of inpatient care to the intensive care unit, if the Investigators assess that a higher level of 
intervention and intense monitoring is required to manage symptoms following controlled human 
malaria infection, blood donation, or drugs (over and above what can be provided by the Investigators 
in research bay). Hospitalization (including inpatient or outpatient hospitalization for an elective 
procedure) for a pre-existing condition that has not worsened unexpectedly does not constitute a SAE. 
• An important medical event (that may not cause death, be life threatening, or require hospitalisation) 
that may, based upon appropriate medical judgment, jeopardise the volunteer and/or require medical 
or surgical intervention to prevent one of the outcomes listed above. Relapse of malaria would be 
classified as an SAE. 
• Congenital anomaly or birth defect. 

 
 
 
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9.4.2 
Clinical laboratory parameters and other abnormal assessments qualifying as adverse events or 
serious adverse events:  
In absence of diagnosis, clinically abnormal laboratory findings (e.g. clinical chemistry, haematology, and 
urinalysis) or other abnormal assessments that are judged by the investigator to be clinically significant will be 
recorded as AE or SAE if they meet the definition of an AE or SAE. Clinically significant abnormal laboratory 
findings or other abnormal assessments that are present at baseline and significantly worsen following the 
start of the study will also be reported as AEs or SAEs. 
The investigator will exercise his or her medical and scientific judgment in deciding whether an abnormal 
laboratory finding or other abnormal assessment is clinically significant. 
If a test is deemed clinically significant, it may be repeated, to ensure it is not a single occurrence. If a test 
remains clinically significant, the volunteer will be informed and appropriate medical care arranged as 
appropriate with the permission of the volunteer. Decisions to exclude the volunteer from enrolling in the trial 
or to withdraw a volunteer from the trial will be at the discretion of the Investigator.  
In addition, COVID-19 infection in volunteers acquired during in-patient stay challenge will be 
considered an SAE and COVID-19 treatment will be provided to the volunteers free of charge. If volunteers 
have to stay in the hospital for SAE, admission compensation at 2,000 THB per night will be provided to the 
volunteers. 
9.4.2.1 Causality assessment 
The investigator is obligated to assess the relationship between study procedure and/or antimalarial 
medications and the occurrence of each AE/SAE. The investigator will use clinical judgment to determine the 
relationship. Alternative plausible causes, such as natural history of the underlying diseases, concomitant 
therapy, other risk factors, and the temporal relationship of the event to the study procedure and antimalarial 
medications will be considered and investigated. The relationship of the adverse event with the study 
procedures will be categorized as unrelated, unlikely to be related, possibly related, probably related or 
definitely related (Table 8). An intervention-related AE refers to an AE for which there is a possible, probable 
or definite relationship to the study intervention. The delegated clinician will use clinical judgment to 
determine the relationship.  
Table 8: Guidelines for assessing the relationship of study intervention to an AE 
0 
Unrelated 
No temporal relationship to study intervention and 
Alternate aetiology (clinical state, environmental or other 
interventions);and 
Does not follow known pattern of response to CHMI, blood donation 
or drug. 

 
 
 
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1 
Unlikely to be related 
Unlikely temporal relationship to study intervention and 
Alternate etiology 
likely 
(clinical state, environmental 
or other 
interventions) and 
Does not follow known typical or plausible pattern of response to 
challenge, blood donation or drug. 
2 
Possibly related 
Reasonable temporal relationship to study intervention; or 
Event not readily produced by clinical state, environmental or other 
interventions; or 
Similar pattern of response to that seen with previous challenge, blood 
donation or similar drug. 
 
3 
Probably related 
Reasonable temporal relationship to study intervention; and 
Event not readily produced by clinical state, environment, or other 
interventions or 
Known pattern of response seen with previous challenge, blood 
donation or drug. 
4 
Definitely related 
Reasonable temporal relationship to study intervention; and 
Event not readily produced by clinical state, environment, or other 
interventions; and 
Known pattern of response seen with previous challenge, blood 
donation or drug. 
 
9.4.2.2 Assessment of intensity 
Each adverse event will be graded by the investigator and designated study staff according to Common 
Terminology Criteria for Adverse Events (CTCAE) Version 5.0.   
In the rare case that an adverse event is not graded in the CTCAE, then that event should be graded as follows: 
Grade 1: 
Mild AE 
Grade 2: 
Moderate AE 
Grade 3: 
Severe AE 
Grade 4: 
Life-threatening or disabling AE  
Grade 5:  
Death related to AE 
All AE/SAE will be collected throughout the first 3 month after challenge or until a satisfactory resolution 
occurs. Malaria relapse will be collected throughout the entire 1 year period. All AEs that result in a volunteer’s 
withdrawal from the study will be followed up until a satisfactory resolution occurs, or until a non-study 
related causality is assigned (if the volunteer consents to this). In cases where the volunteer has to do AE self-
detection and self- assessment, the simple grading for the self-assessment on the diary card will be used (Table 
9)  

 
 
 
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The simple grading for use for the self- assessment for severity as in Table 9 
Table 9: Severity grading criteria for the self-assessment for AEs. 
GRADE 0 
None 
GRADE 1 
Mild: Transient or mild discomfort (<48hours);no medical intervention/therapy 
required 
GRADE 2 
Moderate: Mild to moderate limitation in activity – some assistance may be needed; no 
or minimal medical intervention/therapy required 
GRADE 3 
Severe: Marked limitation in activity, some assistance usually required; may require 
medical intervention/therapy 
9.4.2.3 Assessment of outcomes 
The investigator will assess the outcome of all AEs (including SAEs) recorded during the study as: 
• 
Recovered/resolved. 
• 
Recovering/resolving. 
• 
Not recovered/not resolved. 
• 
Recovered with sequelae/resolved with sequelae. 
• 
Fatal (SAEs only). 
During the inpatient phase, the adverse events (including all SAEs) will be assessed by the investigator and the 
delegated study staff.  
While the volunteer is followed as an outpatient, a diary card will be provided, and the volunteer will be 
instructed how to self-assess the cause and severity of AEs. This self-assessment symptom diary will be 
checked, discussed, collected and recorded at each clinic visit. 
9.4.3 
    Reporting procedures 
For SAEs 
In order to comply with current regulations on serious adverse event reporting to Ethics and regulatory 
authorities (if applicable), the event will be documented accurately and notification deadlines respected.  
SAEs will be reported to the medical monitor group immediately (within 24 hours) of the Investigators’ being 
aware of their occurrence. 
SAEs will also be reported to ethics committees, the Data and Safety Monitoring Board (DSMB), and the 
regulatory authority (if applicable), in accordance with reporting requirements and according to required 
timelines.  
9.5 
     Events or outcomes not qualifying as adverse events or serious adverse events  

 
 
 
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9.5.1 
Pregnancy 
Volunteers are informed that for the safety of volunteer and their child, pregnancy is prohibited until 3 months 
after the challenge. Female study volunteers are asked to use appropriate contraceptive methods to prevent 
pregnancy while they participate in the study for 3 months after the mosquito challenge.  
Appropriate contraceptive methods include: 
- Established use of oral, injected or implanted hormonal contraceptives 
- Intrauterine Device or Intrauterine System 
- Barrier methods (condoms or diaphragm with additional spermicide) 
- Male sterilisation and female 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 volunteer. Periodic 
abstinence (e.g., calendar, ovulation, symptothermal, post-ovulation methods) and withdrawal are not 
acceptable methods of contraception. 
Female volunteers will be tested for pregnancy immediately prior to challenge and during follow up as 
indicated in the schedule of procedures (Table 6). 
Should a volunteer become pregnant during the trial, she will be treated with antimalarials immediately and 
will be withdrawn from the study. We will not routinely perform venepuncture on such volunteers, other than 
blood films to check that the parasitaemia has been cleared by the antimalarial treatment. With the 
volunteer’s permission she shall be followed up until pregnancy outcome. The management of any volunteer 
found to be pregnant at any time after challenge up to the point of malaria treatment will be discussed with 
the on-call infectious diseases consultants at Faculty of Tropical Medicine and the Mahidol-Oxford Tropical 
Medicine Research Unit, including advice on antimalarial drug choice. 
Should a volunteer become pregnant after receiving antimalarial treatment (but prior to the end of the study), 
they shall be discontinued from the study as soon as we have confirmed that their parasitaemia has cleared. 
The volunteer will still be followed up until 1 year after challenge.  
During pregnancy the following should always be considered as an SAE: 
• 
Spontaneous abortion, (spontaneous pregnancy loss before/at 22 weeks of gestation) 
• 
Ectopic and molar pregnancy 
• 
Stillbirth (intrauterine death of fetus after 22 weeks of gestation). 
• 
Any early neonatal death (i.e. death of a live born infant occurring within the first 7 days of life).  

 
 
 
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• 
Any congenital anomaly or birth defect identified in the offspring of a study subject (either during 
pregnancy, at birth or later) regardless of whether the fetus is delivered dead or alive. This includes 
anomalies identified by prenatal ultrasound, amniocentesis or examination of the products of 
conception after elective or spontaneous abortion. 
However, pregnancy occurring in participants within 3 months of challenge will be considered a 
SAE.  
Furthermore, any SAE occurring as a result of a post-study pregnancy AND considered by the investigator to 
be reasonably related to the study procedures will be reported. While the investigator is not obligated to 
actively seek this information from former study volunteers, he/she may learn of a pregnancy through 
spontaneous reporting. 
9.6 
Safety monitoring 
9.6.1 
Monitoring  
Monitoring will be performed using established Monitoring plan. Independent monitoring will be performed 
by Clinical Trials Supporting Group (CTSG), MORU. Following written standard operating procedures, the 
monitors will verify that the clinical trial is conducted and data are generated, documented and reported in 
compliance with the protocol and Good Clinical Practice (GCP). The investigators will provide direct access to 
all trial related source data/documents and reports for the purpose of auditing by the Sponsor and inspection 
by local and regulatory authorities. 
9.6.2 
Medical Monitor Group 
A Medical Monitor group, representing the Sponsor, will be appointed for oversight of safety in this clinical 
study. The Medical Monitor group will be responsible for safety assessments. The monitor will review the 
study prior to initiation and will be available to advise the Investigators on study-related medical issues and to 
act as a representative for the welfare of the subjects. The medical monitor does not have direct involvement 
in the conduct of the study. All serious adverse events will be reported to the medical monitor within 24 hours 
of becoming aware of the event. The medical monitor group is responsible for the review of the safety data 
and communicate with the PI and/or the DSMB, as appropriate.  
The medical monitor group will be chaired by Dr. Lorenz Von Seidlein M.D., and the members of the medical 
monitor group are 
1. Dr. Elizabeth Ashley, MD 
2. Dr. Rupam Tripura, MD 
9.6.3 
Data and Safety Monitoring Board 
DSMB will be independent of the Clinical Trials and Research Governance, Oxford University (Sponsor) and 
MIST programme team.  The DSMB will review the study prior to initiation; review the interim safety data 

 
 
 
MIST 1 protocol V 10.0 dated 14 February 2022                                                   Page 75 of 85 
reports; and review all SAEs according to DSMB charter. The Board may convene additional reviews if deemed 
necessary, on review of the safety data, as sent, periodically by the medical monitor. All SAEs will be reported 
by the site principal investigator to the DSMB at the same time as they are submitted to the ethics committees. 
The PI(s) will notify the Board and obtain a recommendation concerning continuation, modification, or 
termination of the study. The site principal investigator will submit written DSMB summary reports with 
recommendations to the ethics committees(s). The roles and responsibilities of the DSMB will be formalised 
in a charter agreed with the members of the DSMB. 
10.  STATISTICS 
The safety of the CHMI will be assessed by descriptive analysis of the frequency, incidence and nature of 
adverse events and serious adverse events arising during the study. Since this is a feasibility study conducted 
in six volunteers, only a brief statistical analysis plan is applicable.  
Sample size calculation 
The number of volunteers undergoing malaria challenge in this CHMI study will be up to 6, deemed sufficient 
to meet the primary objective of assessing the safety and feasibility of controlled human P. vivax malaria 
infection in Thailand. It is the first study in Asia, and if successful, larger similar studies will be performed per 
the study package described in the background, overview and rationale of the study section (section 2).  
11. DATA, DIRECT ACCESS TO SOURCE DATA/DOCUMENTS 
Source documents are original documents, data, and records from which volunteers’ CRF data are obtained. 
These include, but are not limited to, hospital records (from which medical history and previous and 
concurrent medication may be summarized into the CRF), clinical and office charts, registration logbook, 
laboratory and pharmacy records, diaries, radiographs, referral notes and correspondence. All documents will 
be stored safely in confidential conditions. On all study-specific documents, other than the signed consent, the 
volunteer will be referred to by the study volunteer number/code, not by name. Direct access to all trial related 
source data/documents will be granted to authorized representatives from the sponsor, Research Ethics 
Committees (RECs), and the regulatory authorities to permit trial-related monitoring, audits and inspections. 
12. QUALITY CONTROL AND QUALITY ASSURANCE PROCEDURES 
The study will be conducted in accordance with the current approved protocol, ICH GCP, relevant regulations 
and standard operating procedures. Data will be evaluated for compliance with the protocol and accuracy in 
relation to source documents. Following written standard operating procedures, the monitors will verify that 
the clinical study is conducted and data are generated, documented and reported in compliance with the 
protocol.  
12.1 Audit & Inspection 

 
 
 
MIST 1 protocol V 10.0 dated 14 February 2022                                                   Page 76 of 85 
The Quality Assurance will perform system based internal audits to check that trials are being conducted, data 
recorded, analysed and accurately reported according to study protocols, departmental SOPs and in 
compliance with ICH GCP. The audit schedule includes laboratory activities. The internal audits will supplement 
the monitoring process and will review processes not covered by the monitor. 
The Sponsor, trial sites, ethical committee(s), and authorised individuals may carry out audit to ensure 
compliance with the protocol, GCP and appropriate regulations. GCP inspections may also be undertaken by 
the regulatory authority to ensure compliance with protocol and national regulations. The sponsor will assist 
in any inspections. 
13. ETHICS 
Ethical approval will be sought prior to commencing the study through the relevant Research Ethics 
Committees. Indemnity for the trial will be provided by the University of Oxford. SAEs will be reported to the 
medical monitor group, DSMB, and ethics committees. GCP certificate will be obtained by all 
staff/investigators prior to commencing the studies. 
The Investigators will ensure that this study is conducted in accordance with the principles of the Declaration 
of Helsinki 2013. The trial will adhere to the ICH GCP. 
The protocol, informed consent form, participant information sheet, and other written participant 
information/materials and Advertisement will be submitted to appropriate Research Ethics Committees 
(RECs), and regulatory authorities (if applicable) for written approval. The Principal Investigator (PI) will submit 
and, where necessary, obtain approval from the above parties for all amendments to the original approved 
documents. The Investigator will notify deviations from the protocol or SAEs occurring at the site to REC(s) of 
these if necessary in accordance with procedures. The principal investigator shall submit a report once a year 
throughout the study, or on request, to the ethic committees. In addition, an End of Study notification and 
final report will be submitted to the ethic committees. 
13.1 Participant confidentiality 
All data used for analysis will be de-identified; personal identifying information such as names and telephone 
numbers will not be used for analysis. Files containing identifiable information will be stored separate from 
other study data, in secure locations. Only the Sponsor’s representative, Investigators, the clinical monitor, 
the ethical committee(s) and the regulatory authorities will have access to these records. Photographs may be 
taken to be shown to other professional staff, used for educational purposes, or included in a scientific 
publication. Photographs will not include the volunteer’s face and if required the volunteer’s written informed 
consent will be sought before photographs are taken. The photographs will be stored as confidential records, 
as above. 

 
 
 
MIST 1 protocol V 10.0 dated 14 February 2022                                                   Page 77 of 85 
Volunteers’ data and results from blood analyses stored in our database may be shared with other researchers 
to use in the future. However, the other researchers will not be given any information that could identify the 
subject. 
13.2 Benefit 
The expected benefit is to provide information about the feasibility and safety of controlled human P. vivax 
malaria infection studies for future testing of pre-erythrocytic candidate vaccines, about the growth of and 
the immune response to P. vivax infection, and to create a bank of P. vivax-infected blood for future testing 
of candidate blood stage vaccines. All of these will benefit patients with malaria infections in the future, and 
potentially lead to development of a vaccine to prevent infections altogether. However there are no direct 
benefits to the participants taking part in this study. 
13.3 Data handling and record keeping 
The investigators will maintain and retain appropriate medical and research records and essential documents 
for this trial in compliance with ICH E6 (R2) GCP and regulatory and institutional requirements for safety and 
protection of confidentiality of volunteers. The principal Investigators are responsible for data management 
and for delegating the receiving, entering, cleaning, querying, analysing and storing all data that accrues from 
the study. The study staff will enter the data into the volunteers’ CRFs, which will be in a paper and/or 
electronic format. This includes safety data, laboratory data (both clinical and immunological) and outcome 
data. Data will be managed and stored in MACRO® database, a GCP-compliant electronic data capture system. 
A study data management plan will outline detailed procedures for data capture, storage, curation and 
preservation.  
The study protocol, documentation, data and all other information generated will be held in strict confidence. 
Only authorized, trained study staff will have access to study records. The investigators will permit authorized 
representatives of the sponsor, ethical committee(s), regulatory authorities (if applicable), authorized 
representative of sponsor, and the monitors to examine (and when required by applicable law, to copy) clinical 
records for the purposes of quality assurance reviews, audits and evaluation of the study safety and progress. 
No information concerning the study or the data will be released to unauthorised third parties, without prior 
written approval of the Sponsor.  
Medical’s record will be kept at study site for approximately 15 years after study completion. Data will be fully 
anonymised and stored indefinitely in the secure Database. 
14. INSURANCE 

 
 
 
MIST 1 protocol V 10.0 dated 14 February 2022                                                   Page 78 of 85 
The University of Oxford has a specialist insurance policy in place - Newline Underwriting Management Ltd, at 
Lloyd’s of London – which would operate in the event of any volunteer suffering harm as a result of their 
involvement in the research. 
15.  PUBLICATION POLICY 
All Investigators will be involved in reviewing drafts of the manuscripts, abstracts, press releases and any other 
publications arising from the study. Authorship will be determined in accordance with the International 
Committee of Medical Journal Editors (ICMJE) guidelines and other contributors will be acknowledged. 
16.  COMPENSATION 
Volunteers will be compensated for their time and for the inconvenience caused by procedures as below. This 
amount of compensation is calculated based on the cost of living in Bangkok for time and traveling. According 
to the consensus recommended by the controlled human challenge study group meeting in 2015, the 
compensation specifically on the risk was also advised (Bambery B, Selgelid M, Weijer C, et al., Ethical criteria 
for human challenge studies in infectious disease. Public Health Ethics. 2016. 9; 92-103.)  
 
Table 10: Estimated compensation amounts. 
Activity 
Compensation (THB) 
Number of visits 
Total 
1. Screening visit 1 (D -180) 
2,000 
1 
2,000 
2. Screening visit 2 on D-7 
1,500 
1 
1,500 
3. Admission per night 
2,000 
Around x 18 nights 
36,000 (estimated) 
4. Inconvenience of blood 
donation: 
5,000 
1 
5,000 
5. Primaquine DOT per visit 
(out-patient) 
1,000 
11 
11,000 
6. D Rx28 
1,500 
1 
1,500 
7. D Rx90 
1,500 
1 
1,500 
8. DRx180 
1,500 
1 
1,500 
9. 1 yr. 
1,500 
1 
1,500 
 

 
 
 
MIST 1 protocol V 10.0 dated 14 February 2022                                                   Page 79 of 85 
Time in Trial 
(approx.) 
Maximum no. of 
visits 
Maximum volume of 
blood taken1 (mL) 
Total compensation amount (THB) 
1 year and 6 
months 
18 
530 
61,500 
Remark:  
1. Maximum volume of blood taken is 530 ml (estimated blood donation is D12-D14). In case volunteer 
does not reach the treatment criteria, microscopy and malaria qPCR and Gametocyte qPCR will be 
performed twice daily until D21, so the maximum volume of blood taken will be 260 mL. 
In case volunteers has to come for extra visit(s), they will be compensated for their time and for the 
inconvenience for 1,000 THB per day. 

 
 
 
MIST 1 protocol V 10.0 dated 14 February 2022                                                   Page 80 of 85 
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MIST 1 protocol V 10.0 dated 14 February 2022                                             Page 83 of 85 
18. Appendix A:   MOSQUITO BITING PROCESSES DIAGRAM  
 
 
 
 
 
Subject will be briefed before 
the challenge. 
The challenge process will be 
performed in the secure room 
in the insectary. 
Forearm will be cleaned. 
Subject will be bitten by five 
infectious mosquitoes per 
volunteer (by placing their 
forearms 
over 
the 
pot 
containing mosquitoes) for 5-
10 minutes. 
 
Infected mosquitoes 
Subject will be hospitalized 
at Bangkok Hospital for 
Tropical Disease until disease 
management is completed. 
 
 

 
MIST 1 protocol V 10.0 dated 14 February 2022                                                                                                                                                                Page 84 of 85 
 
19. APPENDIX B: TREATMENT ALGORITHM 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
*Mild clinical features, any one of the following and no moderate or severe features described below: 
• 
Intermittent fever ( < 38 C, in 4-6 hour)  
• 
Nausea  <2 vomiting episodes in 24 hours 
• 
Discomfort 
• 
Mild dehydration 
**Moderate clinical features, any one of the following and no severe features described below: 
• 
Sustained high fever ( > 38 C,  in 4 to 6 hour)  
• 
Severe vomiting (> 3 times in 24 hours),  
• 
Moderate dehydration 
***Severe clinical features, any one of the following:  
• 
Severe prostration  
• 
Unstable vital signs (BP less than 80/50 mmHg, resting pulse > 120, weak pulse, RR > 25 BPM ) 
• 
Alteration of consciousness 

 
MIST 1 protocol V 10.0 dated 14 February 2022                                                                                                                                                                Page 85 of 85 
• 
Severe dehydration 
• 
Acute dyspnea 
• 
Oliguria in previous 24 hours 
• 
Clinical deteri 
****Antimalarial treatments will be prescribed either on D21 or earlier 
following the physician judgements based on clinical and laboratory data.  
 
#Blood donation will be done according to physician judgement based on 
parasitaemia.