A Phase II Clinical Study to Assess the Safety, Immunogenicity, and Efficacy of Blood-stage Plasmodium Vivax Malaria Vaccine Candidate PvRII/Matrix-M in Healthy Thai Adults Living in Thailand
Phase: PHASE2
Conditions: Plasmodium Vivax Infection, Malaria Vaccine
Sponsor: University of Oxford

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PART A: PROJECT INFORMATION
A1. TITLE OF THE STUDY IN THAI AND ENGLISH
English: A Phase II Clinical Study to Assess the Safety, Immunogenicity, and Efficacy of Blood-
stage Plasmodium Vivax Malaria Vaccine Candidate PvDBPII/Matrix-MTM in Healthy Thai Adults
Living in Thailand, controlled human malaria infection (CHMI)
Study Code: MAL22004
Trial Registry Name: ClinicalTrials.gov; ClinicalTrials.gov ID: NCT05380388
A2. RESEARCH PROPOSAL VERSION DATE:
V.3.0, dated 14 December 2022
Type of submission
Initial review (first time submission)
Revision according to TMEC suggestions No…2..
Amendment No. ….…
A3. PRINCIPAL INVESTIGATOR NAME: Professor Nicholas Day and Research Professor
Dr. Jetsumon Sattabongkot Prachumsri

Faculty Staff (Go to A4.1)

Student, ID………………..… (Please check the following, and go to A4.2)

Research for Thesis

Research for Thematic Paper
M.Sc. (Trop. Med.)
M.C.T.M.
Ph.D. (Trop. Med.)
M.C.T.M. (T.P.)
Ph.D. (Clin. Trop. Med.)
Other, specify ……………

Other Mahidol Oxford Tropical Medicine Research Unit (MORU) Staff
(Please specify, and go to A4.1)

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A4. LIST NAME, AFFILIATION AND CONTACT DETAILS OF ALL INVESTIGATORS
A4.1 For Faculty staff and other
Name
Position
Contact address
E-mail address
Professor Nicholas
Day
Co-
Principal
Investigator
Mahidol Oxford Tropical
Medicine Research Unit
(MORU)
Faculty of Tropical Medicine
Mahidol University
420/6 Rajvithi Road
Bangkok 10400 Thailand
nickd@tropmedres.ac
Research Professor
Dr. Jetsumon
Sattabongkot
Prachumsri
Co-
Principal
Investigator
Mahidol Vivax Research
Unit (MVRU)
Faculty of Tropical Medicine
Mahidol University
420/6 Rajvithi Road
Bangkok 10400 Thailand
jetsumon.pra@mahidol.ac.t
h
Assoc. Professor
Kittiyod Poovorawan
Accountabl
e
Investigator
(FTM Thai
Staff) and
Site-
Principal
Investigator
Department of Clinical
Tropical Medicine
Mahidol Oxford Tropical
Medicine Research Unit
(MORU)
Faculty of Tropical Medicine
Mahidol University
420/6 Rajvithi Road
Bangkok 10400 Thailand
kittiyod.poo@mahidol.ac.th
Professor Sasithon
Pukrittayakamee
Co-
investigator
Mahidol Oxford Tropical
Medicine Research Unit
(MORU)
Faculty of Tropical Medicine
Mahidol University
420/6 Rajvithi Road
Bangkok 10400 Thailand
yon@tropmedres.ac
Assist. Professor
Podjanee Jittamala
Co-
investigator
Department of Tropical
Hygiene
Mahidol Oxford Tropical
Medicine Research Unit
(MORU)
Faculty of Tropical Medicine
Mahidol University
420/6 Rajvithi Road
Bangkok 10400 Thailand
podjanee@tropmedres.ac

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Name
Position
Contact address
E-mail address
Assist. Professor
Borimas
Hanboonkunupakarn
Co-
investigator
Department of Clinical
Tropical Medicine
Mahidol Oxford Tropical
Medicine Research Unit
(MORU)
Faculty of Tropical Medicine
Mahidol University
420/6 Rajvithi Road
Bangkok 10400 Thailand
borimas@tropmedres.ac
Professor Sir
Nicholas White
Co-
investigator
Mahidol Oxford Tropical
Medicine Research Unit
(MORU) Faculty of Tropical
Medicine
Mahidol University
420/6 Rajvithi Road
Bangkok 10400 Thailand
nickwdt@tropmedres.ac
Professor Arjen
Dondorp
Co-
investigator
Mahidol Oxford Tropical
Medicine Research Unit
(MORU) Faculty of Tropical
Medicine
Mahidol University
420/6 Rajvithi Road
Bangkok 10400 Thailand
arjen@tropmedres.ac
Dr. Angela M
Minassian
Co-
investigator
Centre for Clinical
Vaccinology and Tropical
Medicine,
Department of Biochemistry,
University of Oxford,
Churchill Hospital, Old
Road, Headington,
Oxford, OX3 7LE
angela.minassian@bioch.ox
.ac.uk
Professor Philip
Bejon
Co-
investigator
Kemri-Wellcome Trust
research program
Kilifi, Kenya
pbejon@kemri-
wellcome.org
Professor Simon J
Draper
Co-
investigator
Department of Biochemistry,
University of Oxford,
Dorothy Crowfoot Hodgkin
Building, South Parks Road,
Oxford, OX1 3QU, United
Kingdom
Simon.draper@bioch.ox.ac.
uk
Professor Mallika
Imwong
Co-
investigator
Molecular Tropical
Medicine and Genetics
Faculty of Tropical Medicine
mallika.imw@mahidol.ac.th

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Name
Position
Contact address
E-mail address
Mahidol University
420/6 Rajvithi Road
Bangkok 10400 Thailand
Assist. Prof. Wang
Nguitragool
Co-
investigator
Molecular Tropical
Medicine and Genetics
Faculty of Tropical Medicine
Mahidol University
420/6 Rajvithi Road
Bangkok 10400 Thailand
Wang.ngu@mahidol.edu
Professor Kesinee
Chotivanich
Co-
investigator
Mahidol Oxford Tropical
Medicine Research Unit
(MORU) Faculty of Tropical
Medicine
Mahidol University
420/6 Rajvithi Road
Bangkok 10400 Thailand
nok@tropmedres.ac
Dr. Wanlapa
Roobsoong
Co-
investigator
Mahidol Vivax Research
Unit (MVRU)
Faculty of Tropical Medicine
Mahidol University
420/6 Rajvithi Road
Bangkok 10400 Thailand
wanlapa.ros@mahidol.edu
Assist. Prof. Patchara
Sriwichai
Co-
investigator
The Department of Medical
Entomology
Faculty of Tropical Medicine
Mahidol University
420/6 Rajvithi Road
Bangkok 10400 Thailand
patchara.sri@mahidol.ac.th
Assoc. Professor
Pornsawan
Leaungwutiwong
Co-
investigator
Tropical Medicine
Diagnostic Reference
laboratory
Department of Microbiology
and Immunology
Faculty of Tropical Medicine
Mahidol University
420/6 Rajvithi Road
Bangkok 10400 Thailand
pornsawan.lea@mahidol.ac.
th
A4.2 For students: Not applicable
Name
Position
Contact address
E-mail address
Principal Investigator

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Accountable Investigator
(Student/Advisor)
Advisor
Co-advisor
Co-advisor
A5. RESPONSIBILITY OF PI
Whole study
Partial responsibility
A6. NUMBER OF YOUR ONGOING RESEARCH PROJECT(S)
1) Professor Nicholas Day: 5 studies
2) Research Professor Dr. Jetsumon Sattabongkot Prachumsri: 6 studies
A7. CONTACT PERSON
1. Professor Nicholas Day (Co-Principal Investigator)
Mahidol Oxford Tropical Medicine Research Unit (MORU)
Faculty of Tropical Medicine
Mahidol University
420/6 Rajvithi Road
Bangkok 10400 Thailand
Email: nickd@tropmedres.ac
2. Research Professor Dr. Jetsumon Sattabongkot Prachumsri (Co-Principal Investigator)
Mahidol Vivax Research Unit (MVRU)
Faculty of Tropical Medicine
Mahidol University
420/6 Rajvithi Road
Bangkok 10400 Thailand
Tel. 02 306 9187
Email: jetsumon.pra@mahidol.ac.th
Mobile phone: 08 1611 4241
3. Assoc. Professor Kittiyod Poovorawan (Accountable Investigator and Site-Principal
Investigator)
Department of Clinical Tropical Medicine
Faculty of Tropical Medicine

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Intervention
Mahidol University
420/6 Rajvithi Road
Bangkok 10400 Thailand
Email: kittiyod.poo@mahidol.ac.th
Mobile phone: (083) 149-6864
A8. NATURE OF THE STUDY- STUDY INVOLVING SPECIMEN COLLECTION
Clinical trial phase II -…/
Bioequivalence/ pharmacokinetic drug study
Prospective epidemiological research
Laboratory study
A9. IS THE PROJECTS A SINGLE CENTER OR MULTI-CENTER?
Single center
Multicenter (within Thailand)
Please specify the study sites ……………………………………………........
Multicenter (International)
Please specify the study sites …………………………………........................
A10. PROJECT SUMMARY IN THAI AND ENGLISH
A10.1 Full protocol summary in English (not more than 1,000 words per language)
This project is the third part of a 5-year research programme entitled "Malaria Infection Studies in
Thailand (MIST)", and is known as MIST3. MIST3's primary objectives are to assess the safety
and efficacy of the PvDBPII/Matrix-MTM vaccine candidate in healthy adult Thai participants and
to establish whether the PvRII/Matrix-MTM vaccine can demonstrate a reduced parasite
multiplication rate in vaccinated participants compared to a control group (placebo vaccine) in a
blood-stage controlled human malaria infection model. This study will recruit up to 46 eligible
healthy participants aged 20-55 in Thailand at the Faculty of Tropical Medicine, Mahidol
University. Twenty-three participants will receive three doses of the PvDBPII/Matrix-MTM
candidate vaccine, and 23 participants will receive three doses of the placebo vaccine (normal
saline). Safety and immunogenicity will be evaluated after each dose as per protocol.

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Approximately 3-4 weeks after receiving the third vaccination, all participants will undergo blood-
stage CHMI with Plasmodium vivax. The participants will be monitored closely as in-patients
during inoculation and after the parasite was detected or clinically indicated in the Hospital for
Tropical Diseases and treated according to the Research Proposal Submission Form.
A10.2 Part of study conducted by PI
Whole Study
Part of the Study
A11.
SOURCE(S) OF FUNDING/ SPONSOR(S) AND BUDGET (INFORMATION
REQUIRED FOR REVIEW AND CONSIDERATION)
Funded by:
Wellcome Trust
Budget amount:
6,000,000 THB
Expecting fund from: …………………
Budget amount: …………………………………...………….…………….
A12. DECLARE CONFLICT OF INTEREST
The Co-Principal Investigators and the Investigators declare they have no conflict of interest.
PART B: DETAILS OF THE STUDY (Describe only the responsibilities of the PI)
B1. BACKGROUND AND RATIONALE
The P. vivax malaria problem
Plasmodium vivax (P. vivax) is one of five Plasmodium species that cause human malaria and
accounts for the most cases of non- falciparum malaria worldwide. It is now the most widespread
human malarial infection in endemic areas outside Africa. The main reasons for the wide
geographical distribution of P. vivax are likely to be related to the following aspects of P. vivax
biology: (1) its ability to relapse from the dormant liver stage (hypnozoite), (2) its high
transmission potential due to early production of gametocytes, high infectivity to mosquitoes and
shorter development cycle in the vector - host compared to other plasmodia (1, 2).
P. vivax has been considered as causing ‘benign’ malaria, but recent large case series have
demonstrated that P. vivax infection is associated with significant morbidity and mortality (3).
Clinical cases are not only due to primary infection following the infected mosquito bite but also

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relapses from the hypnozoite stage, which occur weeks to years after primary infection (4).
Standard schizonticidal regimens are not effective against the hypnozoite. Radical cure of the
hypnozoite stage requires therapy with one of the 8-aminoquinolines, primaquine and tafenoquine.
However, both medications carry a significant risk of severe haemolytic anaemia in individuals
with glucose-6-phosphate dehydrogenase deficiency (G6PD deficiency) (5). There is no cheap,
reliable point-of-care G6PD test, so many P. vivax patients do not currently receive adequate anti-
hypnozoite treatment. More recently, the importance of the cytochrome P450 enzyme CYP2D6 in
the metabolism of primaquine to the active metabolite has been recognized (6, 7). A common
polymorphism in the CYP2D6 gene for the cytochrome P450 enzyme results in poor conversion of
primaquine to the active form, resulting in treatment failures. It is estimated that these two factors
combined may make nearly 40% of the population at risk of P. vivax infection either ineligible for
or unresponsive to primaquine therapy (5).
Recent calls for control and ‘eradication’ of malaria worldwide 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 recognizes the importance of P. vivax and calls for development of 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 (8, 9). More recently,
research into vivax malaria has increased with candidate vaccines being developed and taken
forward to clinical trial (10-12)
Challenges and directions in P. vivax vaccine development.
P. vivax malaria vaccine approach
Most of the P. vivax malaria vaccine candidates that are currently under development target
individual stages of the P. vivax parasite’s life cycle. Several highly abundant parasite proteins
have been identified as targets of natural immunity and in the recent years the list of possible
candidates has expanded.
Subunit vaccines for P. vivax have been developed based on these candidate antigens. There are
three major approaches for the development of malaria vaccines that correspond to the three stages
of the parasite’s life cycle as follow:

Pre-erythocytic antigens; vaccine using these antigens aim to prevent initial infection by
targeting the infective stage (sporozoites) that were introduced by the mosquito bite.
Alternatively, these vaccines can target antigens expressed by liver stage parasites to
prevent the emergence of merozoites into the blood stream e.g. P. vivax circumsporozoite
protein (PVCSP), and P. vivax thrombospondin related adhesive protein (PvTRAP).

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
Blood stage antigens; since all of the symptoms of malaria occur during this stage, the
majority of vaccines targeting antigens expressed during the blood stage are designed
primarily to prevent disease or morbidity and mortality associated with the disease
when parasites are in red blood cell (RBC). One approach is to target merozoite antigens
to prevent red blood cell invasion and reduce the density and prevalence of parasites in the
infected host RBC e.g. receptor-binding region II of P. vivax Duffy binding protein
(PvDBPII), P. vivax merozoite surface proteins (MSP 1), P. vivax apical merozoite antigen
1 (PvAMA1), and P. vivax reticulocyte binding protein (PvRBP).

Transmission blocking antigens; the transmission blocking vaccines target antigens
expressed during parasite lifecycle stages in the mosquito (e.g. gametocyte or oocyst
antigens). Transmission blocking vaccines aim to block malaria transmission from
mosquitoes to humans by preventing the malaria parasite from developing in the
mosquito. Although these vaccines would not directly prevent infection or disease, they
would assist elimination efforts through preventing the onward transmission of
infections. Candidates include parasite proteins expressed on surface of zygote (Pvs25) and
ookinete (Pvs28), and alanyl aminopeptidase 1 (AnAPN1), a highly conserved midgut
surface antigen of Anopheles mosquitoes that is essential for ookinete invasion and
development). (Table 1, Figure 1)

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Figure 1. Malaria vaccine leading candidate antigens
Ref. Barry and Arnott et al. Frontiers in Immunology, 2014 (359)

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Table 1. P. vivax malaria vaccine candidates under development (10)
Vaccine candidate
Development
Ph
Lifecycle
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-
Pre-clinical
Liver-stage
PvTRAP
Prime-boost, viral vectors
PvDBPII-DEKnull
Pre-clinical
Blood-stage
PvDBP
Rec. protein
PvDBPII
Phase I
Blood-stage
PvDBP
Rec. protein-GLA-SE
PvDBPII
Phase I/IIa
Blood-stage
PvDBP
Rec. protein- Matrix-M1TM
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

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Blood stage P. vivax controlled human malaria infection experience
There have been 8 studies of blood-stage P. vivax human challenge. Five studies were conducted at
the Queensland Institute of Medical Research (QIMR), Brisbane, Australia, and 3 studies have
been recently conducted by the University of Oxford, UK (13).
In 2018, Oxford University undertook the first proof-of-concept studies in Europe of P. vivax
human challenge. First, two healthy UK adult volunteers were safely infected with a clonal strain
of P. vivax by the bite of infected mosquitoes transported from Thailand (VAC068,
ClinicalTrials.gov: NCT03377296) (13). 250 mL blood was obtained from both volunteers at a
prespecified parasitaemia/clinical threshold. Following stringent safety screening, the parasite
stabilate from one of these donors (PvW1) was thawed and used to inoculate 6 healthy malaria-
naive United Kingdom adults by blood-stage CHMI, at 3 different dilutions (whole vial or a neat
dose, 1:5 dilution and 1:20 dilution) (VAC069A, NCT03797989). Parasitaemia developed in all
volunteers, who were diagnosed within 12.5-16.5 days of infection and had detectable
gametocytaemia by qPCR within 1-3 days of peak parasitaemia. They were successfully drug
treated with artemether/lumefantrine or atavoquone/proguanil.
The majority of volunteers (4/6) experienced either no or mild AEs, whilst 2/6 volunteers reported
malaria related clinical signs and symptoms. The majority of the AE symptoms resolved within a
few days after treatment initiation. Of the laboratory AEs recorded, transient lymphocytopenia and
thrombocytopenia were recorded in 4/6 and 2/6 volunteers, respectively. A transient grade 1-2
transaminitis was also seen in 4/6 volunteers at 6 days post treatment. ALT levels fully resolved to
pre-challenge levels with no associated abnormalities in other indices of liver function and no
associated clinical symptoms..
Based on these infectivity data, and accounting for possible variation in the number of pRBC
between thawed cryopreserved vials, an inoculum dilution of 1:10 was selected for future P. vivax
blood-stage human challenge studies. To-date, the PvW1 inoculum has been administered to a total
of 19 malaria-naïve UK volunteers as a primary challenge, with 12 and 2 volunteers going on to
receive a secondary and tertiary homologous repeat blood-stage challenge, respectively (VAC069
B-D re-infection studies) (12). In Thailand, the blood-stage challenge to identify optimal dilution is
on going and the result should be available in the first quarter of 2023.
PvDBPII/Matrix-MTM vaccine development
PvDBPII antigen in clinical studies
The clinical manifestations of malaria are caused by the blood stage of the malaria parasite's life
cycle, which involves repeated cycles of merozoite invasion, intracellular replication, and egress.
The invasion of human red blood cells by P. vivax is mediated by interaction of the P. vivax Duffy-

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binding protein (PvDBP) with its receptor, the Duffy antigen receptor for chemokines (DARCs)
(14-17). Duffy-negative individuals are largely resistant to P. vivax infection (14). The functional
receptor-binding domain of PvDBP, known as region II (PvDBPII), binds DARC to mediate
invasion by P. vivax merozoites (17). Although PvDBPII is polymorphic, the DARC binding
residues appear to be highly conserved across P. vivax strains (10). This finding implies that
PvDBPII may contain cross-reactive epitopes that trigger strain-transcending inhibitory antibodies.
Individuals living in endemic areas have been shown to generate high-titre binding-inhibitory
antibodies against PvDBPII that effectively block DARC binding by various PvDBPII alleles.
Importantly, the development of such binding-inhibitory antibodies is correlated with resistance to
P. vivax infection (18). These findings support the development of a subunit vaccine based on
PvDBPII to protect against P. vivax malaria, both immunologically and mechanistically (10).
For production of recombinant PvDBPII, codon optimised synthetic gene encoding PvDBPII
(M.W ~39kDa) was cloned in pET28a (+) vector and transformed into E. coli strain BLR(DE3)
pLysS. PvDBPII is expressed as inclusion bodies. Recombinant PvDBPII is denatured, refolded,
purified to homogeneity, filter sterilized and stored at -70 °C or below.
Matrix-MTM in clinical studies (also known as Matrix-M1TM)
GLA-SE, the adjuvant used in the PvDBPII vaccine trial described above, has shown comparable
immunogenicity to Alum in human clinical trials, and the antibody titres generated may not be
sufficient to protect against malaria, where very high antibody titres are required. When the P.
falciparum malaria VLP, R21, was tested in combination with Matrix-M1TM and compared head-
to-head against R21 in combination with GSK’s potent adjuvant AS01B, the immunogenicity in
humans was the same (ClinicalTrials.gov identifier: NCT02572388), confirming the potency of
Matrix-M1TM. We would naturally choose the best adjuvant for this proof-of-concept study, and so
have elected to combine PvDBPRII with Matrix-M1TM VAC079 Clinical Trial Protocol, V 6.0, 4
th March 2021, University of Oxford Page 30 of 108 Matrix-M1TM (MM) is a 40nm-sized complex
containing the adjuvant-active saponins obtained by chromatographic fractionation of extracts of
the bark of the Quillaja saponaria tree, phospholipid and cholesterol. Quillaja saponins are
triterpene glycosides, with molecular weights of the different saponins ranging from 1,800 – 2,000
Da. In water, saponin in concentrations of 200-500 ppm exist as monomers; at higher
concentrations they aggregate as micelles, with a molecular weight of approximately 100,000 Da.
Saponins are surface-active compounds with a variety of applications including in agriculture,
feed, food and beverage, mining, and veterinary vaccines, and are currently being developed in
human vaccine clinical trials addressing multiple target antigens. In aqueous solution, saponins are
excellent adjuvants and are used in commercial veterinary vaccines, e.g. vaccines against foot-and-
mouth disease, bovine mastitis, feline leukemia and equine influenza. An HPLC-purified fraction
from the same saponin source, called QS21, is a component of the AS01 adjuvant used in the pre-
erythrocytic vaccine RTS,S/AS01. Matrix-MTM adjuvant, in 1 of 2 formulations (named Matrix-

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M1TM or Matrix-M2TM), has been administered to 37734 individuals, of which 37197 have received
Matrix-M1TM, in a total of 29 clinical trials in the US, UK, Europe, Australia and Africa. These
trials include assessment of recombinant malaria candidate vaccines, the most advanced of which
is the malaria/ HBsAg fusion moiety R21, adjuvanted with Matrix-MTM, within six Phase I and
Phase I/II trials conducted in UK, East Africa and West Africa. The profile of adverse events
following vaccination with PvDBPII-Matrix M1 may be partially predicted from previous studies
assessing protein vaccines using the Matrix-MTM adjuvant. Local adverse events are likely to
include injection site pain, erythema, swelling, itching and warmth. Expected systemic adverse
events would include headache, fatigue, myalgia, arthralgia, malaise, feverishness, fever and
nausea. The majority of these symptoms are of mild intensity, and they are transient.
Safety and immunogenicity of the PvDBPII (Salvador I strain) vaccine formulated in
glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE) adjuvant was assessed in a single centre,
phase I, randomized, controlled, dose-escalating, single-blind study in India in 2016
(CTRI/2016/09/007289) [29]. Three doses of the PvDBPII/GLA-SE vaccine were administered by
the intramuscular route at 4 week intervals. The vaccine was administered to 27 healthy malaria-
naïve volunteers at doses of 10 μg (n=9), 25 μg (n=9) and 50 μg (n=9). Another 9 volunteers 
received GeneVac-B (hepatitis B vaccine from Serum Institute of India) as a control arm of the
study. The adjuvant used was glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE), a
synthetic Toll-like receptor 4 agonist (a proprietary adjuvant from Infectious Disease Research
Institute, Seattle, USA).
Immunogenicity data demonstrated that all three doses of PvDBPII (10, 25 and 50 μg) elicited 
antigen-specific and receptor blocking serum antibody responses. The 50 μg PvDBPII/GLA-SE 
vaccine dose elicited the highest antibody response against PvDBPII and the most persistent
binding-inhibitory antibodies against PvDBPII. Both Day 84 and Day 180 sera inhibited binding
not only of the homologous PvDBPII Sal I allele but also of three other PvDBPII alleles (P, O and
AH), which are commonly found in Papua New Guinea.
The vaccine candidate, PvDBPII/GLA-SE, which was tested in healthy Indian male adults, was
safe and well tolerated. No significant immediate reactogenicity was observed within the first hour
post immunization at all three dose levels (10, 25 and 50 μg PvDBPII formulated with GLA-SE [5 
μg of GLA]). No SAE was observed and no subject withdrew or was withdrawn from the study on 
account of safety. A single mild local solicited AE (pain at injection site) after the first dose of 10
μg PvDBPII was reported. No systemic solicited AEs were observed. There were similar 
incidences and intensity of unsolicited AEs among the subjects who received PvDBPII/GLA-SE
and those who received GeneVac-B (control hepatitis B vaccine). No clinically significant
difference was observed in haematology, biochemistry and urinalysis parameters post vaccination.
Vital signs showed no significant changes throughout the study. No abnormal findings were
observed during the post-study physical examination. There was no difference in the number of

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subjects who had biological values outside the normal ranges for different haematological and
biochemical parameters in the PvDBPII/GLA-SE and control vaccine group.
Recombinant PvDBPII formulated in Matrix-M adjuvant was assessed in an open-label Phase I/IIa
clinical trial (VAC079) in healthy adults living in the UK for safety, immunogenicity and efficacy
(Clinicaltrials.gov identifier: NCT04201431) (Ref). The PvDBPII/Matrix M vaccine was
administered intramuscularly to 16 healthy malaria-naïve volunteers at a dose of 50 μg PvDBPII 
protein mixed with 50 μg Matrix-M adjuvant (PvDBPII/Matrix-M). PvDBPII/Matrix-M was 
administered monthly (0, 1, 2 months) or in a delayed dosing regimen (0, 1, 14 months). The
delayed regimen was due to trial halts during the Covid-19 pandemic. Volunteers underwent
heterologous controlled human malaria infection (CHMI) with blood-stage P. vivax parasites
(using the PvW1 inoculum) (13) at 2-4 weeks following their last vaccination, alongside
unvaccinated controls. PvDBPII/Matrix-M was found to be safe and well tolerated with no safety
concerns in malaria naïve adults. Following vaccinations with PvDBPII/M-M, all solicited AEs
were mild to moderate in severity, with subsequent doses associated with increased frequency and
severity of solicited AEs as compared to the first dose. The most common solicited AEs reported
were injection site pain and headache. Incidents of transient lymphopenia of grade 2 severity were
observed following vaccinations with PvDBPII/M-M. Unsolicited AEs, deemed at least possibly
related to PvDBPII/M-M vaccinations, were of mild to moderate severity and self-limiting.
When given in a delayed dosing regimen (0, 1 month and 14 months), PvDBPII/Matrix-M elicited
high antibody responses against PvDBPII and binding inhibition titers. Following heterologous
controlled human malaria infection (CHMI) with blood-stage P. vivax parasites, PvDBPII/Matrix-
M given in the delayed boosting regimen demonstrated significant reduction (>50%) in the mean
parasite multiplication rate in the vaccinated group as compared to unvaccinated controls.
However, administration of PvDBPII/Matrix-M in a 0, 1 month and 2 months regimen elicited 10
fold lower antibody and binding inhibition titers and did not have any impact on parasite
multiplication rate following CHMI with blood-stage P. vivax parasites.
Why controlled human challenge trial?
Conventional vaccine development is complex. The average development timeline is between 8
and 18.5 years, estimated costs are $200 million to $900 million, and the probability of success of
less than 10% (19, 20). Vaccine clinical trials aim to answer questions related to the immune
response, immune correlates of protection, protective efficacy, and target populations. It is
expected that most vaccines will fail because of inadequate protective efficacy. Increasing
recognition of this critical bottleneck in conventional vaccine development has led to demand for a
proof-of-concept clinical trial: the controlled human challenge trial.
In July 2014, the World Health Organization (WHO) held a consultation on Clinical Evaluation of
Vaccines: regulatory expectations and controlled human challenge trials were considered important

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for facilitating vaccine development (21). Controlled human challenge trials are thus a prime
example of ways in which clinical development of vaccines can be accelerated by facilitating early
rejection of poor vaccine candidates. Controlled human challenge trials in malaria initially raised
ethical debate, but have now gained acceptance through demonstrating their potential and the fact
that they have been safely performed in >3,000 volunteers all over the world (22).
The development of a safe and reproducible P. vivax-controlled challenge model in humans could
facilitate clinical development of P. vivax vaccines, through selecting efficacious candidates for
further clinical testing in more expensive and logistically challenging studies in the field.
P. vivax human challenged study in Thailand
This study is part of the long-term project "Malaria Infection Study in Thailand (MIST)" funded by
Wellcome Trust to develop the feasibility of P. vivax controlled human challenge trial in an
endemic area. The following section provides an overview of the projects in this programme,
which comprises multiple studies to be conducted over the next 5 years in the Faculty of Tropical
Medicine, Mahidol University, Thailand.
Most vivax malaria challenge studies performed so far have been in non-endemic settings such as
in the US, UK, and Australia. However, the findings in those settings may not be extrapolatable to
the target population for future vaccine deployment, which is may differ both in terms of immunity
to P. vivax and genetic background. The best volunteer population in which to test new vaccines is
the eventual target population for vaccine deployment. To date, the MIST1 and MIST2 are the
only CHMI trials performed in a P. vivax endemic population in Asia.
The MIST1 study aimed to assess the safety and feasibility of controlled human P. vivax malaria
infection following infection by the natural route of delivery – mosquito bite and provided a source
of P. vivax infected blood to use in future vaccine efficacy trials of a blood- and transmission-stage
vaccines. Up to date, there are two set of inocula obtained from MIST study.
MIST2 study was a blood stage inoculum dose finding study using the inocula produced from
MIST1) to identify the dose of the inocula to be used in future studies. In addition, MIST2 study
will serve as the basis for a challenge model for future P. vivax candidate vaccine efficacy studies.
P. vivax human challenge studies conducted in Thailand would allow us to understand the
immunological correlates of protection in an endemic setting, thereby informing the development
of new P. vivax 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.

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The overall objectives of the MIST programme
Proof of feasibility and safety study and providing P. vivax banked infected blood
inocula for future P. vivax 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.
Characterise correlates of P. vivax immune protection in the target population.
Test the efficacy of new drug candidates under development.
To achieve these major aims, the programme will compose of 4 to 7 studies. Each subsequent
study will be informed by the previous studies in the series and each study will be covered by
separate protocols with separate Ethics Committee submissions and approvals.
The MIST3 study (covered by this current protocol):
This MIST3 study is a Phase II clinical trial to primarily assess the safety of the PvDBPII/Matrix-
MTM vaccine in healthy volunteers and to establish whether the PvDBPII/Matrix-MTM vaccine can
demonstrate a reduced parasite multiplication rate in vaccinated participants compared to control
group (no malaria vaccine) using banked blood from MIST1 at an optimum inoculum dose as
determined by MIST2.
B2. STUDY OBJECTIVES AND ENDPOINTS
Primary Objectives:
1. To assess the safety and tolerability of the PvDBPII/Matrix-MTM vaccine.
2. To establish whether the PvDBPII/Matrix-MTM vaccine can demonstrate a reduced parasite
multiplication rate in vaccinated participants compared to controls (placebo vaccine) in a P.
vivax blood-stage controlled human malaria infection model

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Primary Endpoints:
1. Safety: Data on specific endpoints for safety and reactogenicity will be collected actively
and passively. The following parameters will be assessed:
a.
Occurrence of solicited local reactogenicity signs and symptoms in the 7 days
following each vaccination
b. Occurrence of solicited systemic reactogenicity signs and symptoms in the 7 days
following the vaccination
c.
Occurrence of unsolicited adverse events in the 28 days following each vaccination
d. Change from baseline for safety laboratory measures in the 28 days following
vaccination
e.
Occurrence of serious adverse events during the whole study duration and
feasibility of primary P. vivax blood-stage CHMI, as measured by (S)AE
occurrences and successful infection (development of detectable persistent
parasitaemia by thick blood film +/- clinical symptoms)
AE data will be collected at each clinic visit. It will be collected from diary cards, clinical
review, clinical examination (including observations) and laboratory results.
This AE data will be tabulated and frequency, duration and severity of AEs compared to the
AE data collected for the infectivity control group undergoing parallel CHMI recruited.
Hematological and biochemical laboratory values will be presented according to local grading
scales and tabulated by group, for comparison with the laboratory AE data collected for
infectivity control groups undergoing CHMI in parallel.
SAEs and withdrawal due to AE(s)/SAE(s) will be described in detail.
The vaccinated participants will be followed for approximately 1 year following their first
vaccination
2. Efficacy Endpoints:
Quantitative PCR-derived parasite multiplication rate (PMR) will be the primary efficacy
endpoint and a comparison of the endpoint between the participants vaccinated with
PvDBPII/Matrix-MTM and control group will constitute the primary analysis for efficacy.
The geometric means of three replicate PCR results obtained for each individual at each
timepoint will be used for model-fitting. Negative individual replicates will be handled as
specified in the MIST3 Statistical Analysis Plan. The PMR will be calculated using a linear
model fitted to log10-transformed qPCR data. The secondary efficacy endpoint will be log10

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cumulative parasitaemia by qPCR (area under the curve) for each individual up to the first day
on which a participant is treated.
Secondary objectives:
1. To assess the humoral and cellular immunogenicity of the PvDBPII-Matrix-MTM vaccine
candidate.
2. To assess immunological readouts for association with a reduced parasite multiplication
rate
3. To assess transmission of the parasite from each infected participant to Anopheles
mosquitoes
Secondary Endpoints:
1. Immunological Endpoint: PvDBPII-specific immunogenicity will be assessed by a variety
of immunological assays, with comparison before and after vaccination. The main outcome
measures will be humoral and B cell responses to the P. vivax Duffy-binding protein region
II (PvDBPII) – total IgG, isotypes and avidity; T cell responses to PvDBPII by flow
cytometry assays; in vitro functional PvDBPII inhibitory binding assays and in vitro
functional PvDBPII growth inhibition assays. Other established and exploratory
immunology assays may be carried out, including through collaboration with other
specialist laboratories, which may be outside of Thailand. This would involve transfer of
anonymized serum/plasma samples. Consent for this will be requested from participants.
Other analyses may be detailed in the MIST 3 laboratory plan. Some assays may be
duplicated at different sites. Some of these will involve analysis of frozen samples, and
others analysis of fresh samples. Any other immunological analyses performed will be
reported as not pre-specified in the trial protocol.
2. PMR and anti-PvDBPII antibody responses.
3. Percentage of mosquitoes infected and the average oocyst number per mosquito after
blood feeding.
B3. STUDY SITE(S)
Clinical Therapeutics Unit (FTM CTU, recruiting site), Hospital for Tropical Diseases, Mahidol
Vivax Research Unit (MVRU), Medical Entomology Department, Tropical Medicine Diagnostic
Reference Laboratory (TMDR), and Malaria Laboratory, Faculty of Tropical Medicine, Mahidol
University, Bangkok, Thailand

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B4. STUDY TIMELINES:
1. There will be up to 3 months screening process.
2. The duration that individual participants will be in the study is 1 year and 7 months.
3. After completion of the last participant’s last visit, there will be an estimated 3 months for
database lock and an estimated 3 months to analyse the data.
4. The total duration of this study is approximately 2 year and 3 months starting after EC
approvals.
B5. STUDY POPULATION AND SAMPLE
B5.1 Target population
46 healthy Thai adults, aged from 20 to 55 years will be randomized in a 1:1 ratio to the study
malaria vaccine or to a ‘sham’ vaccine normal saline control group (23 participants per study
group). All participants who received 3 doses of the malaria vaccine and those in the control
group will be challenged at the Hospital for Tropical Diseases, Faculty of Tropical Medicine,
Mahidol University, Bangkok, Thailand.
B5.2 Subject selection criteria
B5.2.1 Subject/inclusion criteria
The participant must meet all the following criteria to be eligible for the study:
1. Healthy Thai adults aged 20 to 55 years
2. Minimum educational level of high school or equivalent
3. Red blood cells positive for the Duffy antigen/chemokine receptor (DARC)
4. Women only: Must practice continuous effective contraception for the duration of the
study period until 3 months post-challenge.
5. Agreement to refrain from blood donation during the study and for 1 year after the
initiation of antimalarial treatment.
6. Willing to be admitted to the Hospital for Tropical Diseases for clinical monitoring as
required by the protocol until antimalarial treatment is completed and their symptoms
are settling, willing to take a curative antimalarial treatment following CHMI, and
willing to reside in Bangkok and its vicinity for 2 months after malarial treatment
initiation.
7. Able to read and write in Thai.
8. Provide written informed consent to participate in the trial
9. Answer all questions on the informed consent quiz correctly

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10. Completed COVID-19 vaccination with 2 doses of any WHO-approved vaccine
B5.2.2 Subject/exclusion criteria
The participant MUST NOT enter the study if any of the following apply:
1. Positive malaria qPCR OR malaria film prior to vaccination and challenge
2. Presence of any medical condition (either physical or psychological) that, in the judgment
of the investigator, would place the participant at undue risk (including the history of
clinically significant contact dermatitis) or interfere with the results of the study (e.g.,
underlying cardiac, renal, hepatic or neurological disease; severe malnutrition; congenital
defects or febrile condition)
3. Presence of chronic disease or chronic use of medication
4. Prior receipt of other investigational vaccine which is likely to impact the interpretation of
the trial data as assessed by the Investigator.
5. Any confirmed or suspected immunosuppressive or immunodeficient state, including HIV
infection, asplenia, history of splenectomy, recurrent severe infections, and chronic
infection
6. Immunosuppressant medication within the past 6 months preceding enrolment (D0) or plan
to use during the study (inhaled and topical steroids are allowed)
7. History of allergic disease or reactions likely to be exacerbated by malaria infection
8. Female participant who is pregnant as evidenced by positive beta-human chorionic
gonadotropin (β-HCG) test, or who is lactating or planning pregnancy during the course of 
the study.
9. Contraindications to the use of antimalarial treatment (e.g., chloroquine,
atovaquone/proguanil, or dihydroartemisinin/piperaquine)
10. Use of medications known to have potentially clinically significant interaction with the
antimalarial drugs that will be used in this study (chloroquine, atovaquone/proguanil, or
dihydroartemisinin/piperaquine)
11. History of cardiac arrhythmia, including clinically relevant bradycardia or Known existing
positive family history in both 1st AND 2nd-degree relatives < 50 years old for cardiac
disease
12. Family history of congenital QT prolongation or sudden death
13. Any clinical condition, including using medications known to prolong the QT interval or
screening electrocardiogram (ECG), demonstrates a QTc interval ≥ 450 ms. 
14. Suspected or known history of alcohol abuse or history of drug abuse.
15. Concurrently participating in another clinical study, at any time during the study period
16. Positive hepatitis B surface antigen or seropositive for hepatitis C virus, or HIV
17. Finding on safety laboratory values as defined below:

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
Abnormal ALT [>upper normal range]

Abnormal serum creatinine [>upper normal range]

Clinically significant abnormalities in corrected calcium and magnesium blood
levels

Haemoglobin < 11 g/dL
18. Blood group Rhesus negative
19. Blood incompatibility to the inoculum
20. History of allergic disease or reactions likely to be exacerbated by any component of the
vaccine
21. Any history of anaphylaxis in reaction to vaccinations
Vaccination and re-vaccination exclusion criteria
1. Acute disease at the time of vaccination. (acute disease is defined as the presence of a
moderate or severe illness with or without fever).
The following adverse events associated with vaccine immunisation constitute absolute
contraindications to further vaccine administration. If any of these events occur during the
study, the participant must be withdrawn and followed until the resolution of the event, as with
any adverse event:
1. Anaphylactic reaction following administration of the vaccine
2. Pregnancy
Exclusion criteria on the day of CHMI
The following constitute absolute contraindications to CHMI:
1. Acute disease, defined as a moderate or severe illness with or without fever
2. Pregnancy
3. 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)
B5.3 Sample size calculation
This is a randomized, double-blind, single site, Phase II, blood-stage P. vivax malaria vaccine
trial to assess the safety, immunogenicity and efficacy of the PvDBPII/Matrix-MTM vaccine
candidate against blood-stage challenge for the first time in Thailand, utilising a produced
cryopreserved inoculum source. Sample sizes for phase II clinical trials are usually small

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because safety information continues to be collected in phase II, hence few individuals are
expected to be subjected to intervention due to uncertainties in the safety profiles of the
interventions. In this study, the sample size determination is based on the efficacy endpoint. A
two-sided, two-sample t-test with group sample sizes of 18 participants in the PvDBPII/Matrix-
MTM vaccine group and 18 participants in the control group achieves at least 80% power to
detect at least a 2-fold reduction in the mean parasite multiplication rate (PMR) in the
PvDBPII/Matrix-MTM vaccine group compared to the control group. The coefficient of
variation on the original scale is 0.8. The significance level (alpha) is 0.050. Thus, assuming a
20% dropout rate, the number of participants in each group undergoing malaria vaccination and
or in the control group will be 23, making a total of 46 participants in the study. The sample
size calculations were performed in PASS 2020 using the “Tests for Fold Change of Two
Means (Log-Normal Data)” procedure.
B5.4 Recruitment Methods
Volunteers will be screened and recruited at the Faculty of Tropical Medicine’s Clinical
Therapeutics Unit (FTMCTU), Faculty of Tropical Medicine, Mahidol University in Bangkok
following Recruitment Process for Healthy Volunteer Standard Operating Procedure (SOP).
In brief, the volunteers may be recruited by use of recruitment materials approved by the ethics
committee and distributed or posted in the following places:

On the 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)
In addition, we may contact individuals who have participated in previous clinical trials from
the 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.
B5.5 Withdrawal/discontinuation criteria
In accordance with the principles of the current revision of the Declaration of Helsinki
(updated 2013) and any other applicable regulations, a participant 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 participant may withdraw/be withdrawn from further study
procedures at any time in the interests of the participant’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).

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
Participant 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.
The medical monitors may recommend withdrawal of participants.
The reason for withdrawal from further study procedures will be recorded in the Case Record
Form (CRF). If a participant 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 participant.
If a participant 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 with standard chloroquine treatment. The importance of this will be emphasized to
participants at screening. If a participant 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 diseases specialists. If necessary and if in accordance
with the law in Thailand, the participant may be detained for appropriate medical management.
If a participant withdraws from the study, blood samples collected before their withdrawal
from the trial will be used/stored unless the participant 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. Participants are free to request that their blood
samples be destroyed anytime during or after the study.
In all cases of participant withdrawal, except those of complete consent withdrawal, safety data
collection will continue if participants have already undergone challenge process.
Whenever possible attempt will be made to replace study participant(s) who withdrawn from
the study.
B5.6 Multi-site research
-
Not applicable
B6. RESEARCH METHODOLOGY
B6.1 Details of study design
Summary of trial design

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Phase II Randomized double-blind controlled study with CHMI, designed to assess the safety,
immunogenicity, and protective efficacy of PvDBPII/Matrix-MTM vaccine using RTPCR as the
primary diagnostic.
Overview
This is a Phase II randomized double-blinded controlled single-centre Plasmodium vivax
blood-stage CHMI trial to assess the safety, immunogenicity and efficacy of the candidate
malaria vaccine PvDBPII/Matrix-MTM. Healthy, Thai adults aged from 20 to 55 years will be
recruited and randomized at the Faculty of Tropical Medicine, Mahidol University in Bangkok.
Study group: Up to 23 healthy adults aged from 20 to 55 years will be recruited. These
participants will receive three doses of the PvDBPII/Matrix-MTM vaccine (each dose consisting
of 50 µg of vaccine and 50 µg of Matrix M) intramuscularly at 0, 1 and 7 months.
Between three to four weeks post-boost (3rd vaccination), all participants who receive 3 doses
of PvDBPII/Matrix-M vaccine will undergo P. vivax blood-stage CHMI, induced by injection
of P. vivax-infected erythrocytes.
Control group: Up to 23 healthy adults aged from 20 to 55 years will be recruited.
These participants will receive three doses of placebo vaccine, at a volume identical to that of
the PvDBPII/Matrix-MTM vaccine, intramuscularly at 0, 1 and 7 months.
Between three to four weeks post-boost (3rd vaccination), all participants who receive 3 doses
of placebo vaccine will undergo P. vivax blood-stage CHMI, induced by injection of P. vivax-
infected erythrocytes.
All participants will have blood taken at regular intervals following vaccination and in the post-
CHMI period to assess the immune response to vaccination and subsequent challenge, as well
as parasite growth dynamics and gametocytaemia.
Close monitoring will continue until participants meet criteria for antimalarial treatment or
until 21 days after challenge, when treatment will be started empirically.
Therapy will be with a standard course of chloroquine where not contraindicated. As infection
will be induced via intravenous injection of blood-stage parasites, there will be no liver-stage
infection and no hypnozoite formation, thereby eliminating the need for radical cure with
primaquine therapy. Follow-up at study site will be up to 1 year after antimalarial treatment
initiated.
Investigational Products

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PvDBPII Vaccine
PvDBPII was manufactured under Good Manufacturing Practice (GMP) conditions by
Syngene International, Bangalore, India, and the drug product was vialed and released by
Zydus Cadila, Ahmedabad, India. PvDBPII is supplied as a liquid at a concentration of 200
μg/mL in sterile aliquots in 0.5 mL clear glass vials. The vial is stored at -70oC or below.
Recombinant PvDBPII protein will be formulated at bedside with adjuvant Matrix-MTM just
prior to administration in the trial. Further details relating to batch release and manufacturing
can be found in the PvDBPII IMPD.
Storage of PvDBPII Vaccine
All vaccines will be stored between –70°C and –90°C. All movements of the study vaccines
will be documented. Vaccine accountability, storage, shipment and handling will be in
accordance local SOPs.
Matrix M1TM
Matrix M1 is manufactured under Good Manufacturing Practice conditions by Novavax AB
(Uppsala, Sweden). Matrix-M1TM will be supplied to the clinical site by Novavax AB and the
adjuvant will be labelled for investigational use only. Matrix-M1TM is formulated at a
concentration of 0.375mg/ml in PBS. The drug product is filled into sterile 3mL glass vials.
Matrix-M1TM (85 parts Matrix-A and 15 parts of Matrix-C) is obtained by simply mixing
Matrix-A  and Matrix-C, followed by dilution in PBS, filtration though filter 0.22 μm and 
filling into vials in a volume of 2 ml. Matrix-M1TM is a colourless slightly-opalescent non-
viscous liquid. Final batch certification and associated labelling takes place at the CTU,
Faculty of Tropical Medicine. Further details relating to batch release and manufacturing can
be found in the Matrix M1 IMPD.
Storage of Matrix-M TM
Matrix-M1TM is stored refrigerated at 2 to 8°C and protected from light. All movements of the
adjuvant will be documented. Accountability, storage, shipment and handling of Matrix-M1TM
will be in accordance with relevant local SOPs and forms.
Administration of Vaccine
Both vaccines (malaria and sham) will be administered intramuscularly in the deltoid muscle of
the arm, according to SOP Vaccination. The syringes containing the vaccine or 0.9% saline
will be prepared by the study pharmacist and labelled with the participant’s study numbers in
accordance with the randomization list. The vaccinating investigator will wear gloves and eye
protection. During administration of the vaccines, Advanced Life Support drugs and

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resuscitation equipment will be immediately available for the management of anaphylaxis. On
vaccination day, the study vaccine and adjuvant will be allowed to thaw to room temperature
and will be administered within 1 hour. For each immunization, one vial of PvDBPII will be
used per participant. One vial of adjuvant will be used for a maximum of two immunizations.
A mixture of PvDBPII at a dose of 50 μg, with 50μg of Matrix-M1TM will be administered to
all participants assigned to the vaccine group. Matrix-M1TM and PvDBPII will be mixed at the
bedside immediately prior to administration according to vaccine administration SOP. Any
vaccine not administered within the time frame will be destroyed.
PvDBPII antigen
(200 micrograms per mL)
Matrix-MTM
(375 micrograms per mL)
Volume immunized
0.39 mL
0.21 mL
0.38 mL
i.
0.39 mL of the thawed antigen will be mixed with 0.21 mL of Matrix MTM adjuvant.
ii.
Mix by ten gentle manual inversions to prepare the formulation.
iii.
From this reconstituted solution, 0.38 mL will be withdrawn for intramuscular
administration to the participants.
The control ‘sham’ vaccine syringes will contain 0.38 mL of 0.9% saline.
Vaccine calculation
Vaccine (PvDBPII) 0.5 mL
per vial
Matrix-MTM 0.75 mL per
vial
1 dose
1 vial
1 vial
3 doses
3 vials
3 vials
3 doses x 23 participants
69 vials
69 vials
B6.2 Details of procedures for specimen/data collection
B6.2.1 Screening and eligibility assessment
The screening will aim to recruit 46 healthy volunteers (23 volunteers per study group).
Screening visits may take place up to 30 days prior to enrolment. Informed consent will be
conducted at screening. If consent is given, the screening procedures will be undertaken,
including testing for Duffy antigen/receptor for chemokine (DARC) positivity, blood typing,
blood diseases, CBC, blood biochemistry, blood-borne infections, malaria diagnosis, malaria
exposure, malaria immunology, and serum pregnancy test. The full list of laboratory tests and
the schedules of procedures is listed in Table 4.

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Abnormal clinical findings from the medial history, physical examination, or laboratory tests at
any point in the study will be assessed according to the scales in APPENDIX C: Safety terms
and administrative information. If a test is deemed clinically significant it may be repeated to
ensure it is not a single or spurious occurrence. If an abnormal finding is deemed to be
clinically significant, the participant will be informed and appropriate medical care arranged
with the permission of the participant.
Cross-matching (Gel Coombs Card method) will be performed to check blood compatibility
between participant and the blood inoculum.
Exclusion of the participant from enrollment in the trial or withdrawal of a participant from the
trial will be at the discretion of the Investigators.
B6.2.2 Enrollment and first vaccination with PvDBPII-Matrix-MTM (D0) and days 3 (D3)
The eligibility of the participant will be reviewed at the end of the screening visit and again when
all results from the screening visit have been considered. If eligible, a day 0 visit will be
scheduled for the participant to be enrolled into the study (study vaccine group or control group).
At the day 0 visit, any new medical issues or symptoms that have arisen will be assessed.
Physical observations/examination, urinary β-HCG test in female participants and venipuncture 
for immunological assays, Immune cells & cytokines profile and safety bloods will be
undertaken according to Table 5. The inclusion and exclusion criteria for the study will be
reviewed. If the participant remains eligible and there are no contraindications to vaccination,
the PvDBPII-Matrix-MTM vaccine or 0.9% saline will be administered as described in section
6.1.
Participants will be asked to record all AEs via the diary card and bring it to the upcoming
visit.
At the day 3 visit (post-vaccination), blood will be taken for immunological assays.
B6.2.3 Reviews post-vaccination on days 7 (D7) and days 14 (D14)
On these subsequent visits, the participants will be assessed for local and systemic adverse
events, using a diary card, well-being check list, physical examination, and blood tests at the
time points indicated in the schedule of attendances (Table 5). Blood will also be taken for
immunological assays and Immune cells & cytokines profile on days 7 and 14. On the Day 7
visit participants will be asked directly about foreseeable local and systemic AEs for the 7 days
post vaccination (solicited AEs).

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B6.2.4 2nd Vaccination with PvDBPII-Matrix-MTM (D28)
This visit will include Day 28 follow up for the first vaccination and administration of the
second vaccine. Any new medical issues or symptoms that have arisen will be assessed.
Physical observations/examination, urinary β-HCG test in female participants and venipuncture 
for immunological assays, Immune cells & cytokines profile, CBC, and blood biochemistry
will be undertaken according to Table 5. The inclusion and exclusion criteria for the study will
be reviewed. If the participant remains eligible and there are no contraindications to
vaccination, the PvDBPII-Matrix-MTM vaccine or 0.9% saline will be administered
intramuscularly in the opposite arm to that used for the first vaccination and as described in
section 6.1.
B6.2.5 Reviews on 7 days post 2nd Vaccination (D35), days 42 (D42), and days 56 (D56)
On these subsequent visits, the participants will be assessed for local and systemic adverse
events, using a diary card, well-being check list, physical examination, and blood tests at the
time points indicated in the schedule of attendances (Table 5). Blood will also be taken for
immunological assays on days 35, 42, and 56 and Immune cells & cytokines profile on days 35
and 42. On the Day 35 visit participants will be asked directly about foreseeable local and
systemic AEs for the 7 days post the second vaccination (solicited AEs).
B6.2.6 3rd Vaccination with PvDBPII-Matrix-MTM (D210/ Month 7)
The 3rd vaccination will occur approximately 6 months after the second vaccination.
Any new medical issues or symptoms that have arisen will be assessed. Physical
observations/examination, urinary β-HCG test in female participants and venipuncture for 
immunological assays and safety bloods will be undertaken according to Table 5. The inclusion
and exclusion criteria for the study will be reviewed. If the participant remains eligible and
there are no contraindications to vaccination, the PvDBPII-Matrix-MTM vaccine will be
administered intramuscularly in the opposite arm to that used for the second vaccination and as
described in sections 6.1.
At this visit, laboratory tests such as blood biochemistry, CBC, immunological assays, Immune
cells & cytokines profile and urine pregnancy test (women only) will be done as described in
Table 5.
B6.2.7 Reviews on 7 days post 3rd Vaccination (D217) and days 224 (D224)
On these subsequent visits, the participants will be assessed for local and systemic adverse
events, using a diary card, well-being check list, physical examination and blood tests at the time
points indicated in the schedule of attendances (Table 5). Blood will also be taken for

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immunological assays and Immune cells & cytokines profile on days 217 and 224. On the Day
217 participants will be asked directly about foreseeable local and systemic AEs for the 7 days
post the third vaccination (solicited AEs).
Exclusion of the participant from inoculation or withdrawal of a participant from the trial will
be at the discretion of the Investigators.
B6.2.8 Day before CHMI (Admission day; C-1)
About 3-6 weeks after the 3rd vaccination, participants will attend to be admitted to the study
facility for CHMI. Any new medical issues or symptoms that have arisen will be assessed. All
participants who are eligible for the inoculation will have physical examination/observations
performed during this visit. Serum β-HCG (woman), malaria diagnosis, immunological assays, 
Immune cells & cytokines profile, complete blood count (CBC), and biochemistry will be
tested during this visit according to Table 6. Blood for malaria immunology will be taken for
baseline information only and the results will not be used as exclusion criteria. Results of the
other blood tests taken at this visit must be available and reviewed prior to challenge.
B6.2.9 Day of CHMI (C0)
All available participants who have not dropped out at this stage and are eligible will be
challenged.
CHMI will be administered according to local Standard Operating Procedure (SOP). This will
include:

Well-being checklist and examination of the injection site and any body systems felt to be
necessary by the Investigator and verification of eligibility / contraindications.

Physical observations (including respiratory rate, pulse rate, blood pressure and
temperature), with recordings to be repeated if this is longer than 60 minutes before
administration of the parasitized erythrocyte inoculum.

Intravenous access via cannulation in a forearm vein, flushed with normal saline.

For each participant, the inoculum must be injected within 4 hours of the inoculum being
thawed, followed by a further normal saline flush. “see B6.2.14 for details of the
inoculum”.

Physical observations will be repeated at 15 minutes post-administration of inoculum and
again at 1 hour, in order to assess for immediate adverse reactions, with regular visual
observation throughout, in line with The British Society for Haematology Guidelines for
the administration of blood products (23). If the participant shows signs or symptoms of a
transfusion reaction, observations will be repeated and the participant will be medically
reviewed by a physician.

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
Participants will be observed for at least 60 minutes; however, this period may be extended
if there are any clinical concerns.

If there have been no symptoms or signs indicative of a transfusion reaction, the cannula
will be removed after 1 hour.
B6.2.10 Days 1 to day of reaching treatment criteria (Dreach treatment criteria)
This section describes the clinical procedures for evaluating study participants and management
after challenge.
B6.2.10.1 Day 1 post challenge to malaria qPCR show positive (DqPCR+)
The participants will be assessed once daily from day 1 post challenge until malaria qPCR
becomes positive (DqPCR+). The assessment includes a clinical well-being check, and the
participants will be questioned whether they have experienced any malaria-related symptoms
(such as fever, chills, sweating, malaise). The once daily general vital signs and physical
examination will be done. Blood will be drawn once daily for monitoring blood parasitaemia
(malaria blood film, qPCR, and gametocyte qPCR).CBC will be performed on day C4 and
DqPCR+. The complete list of laboratory tests, immunological assays, and physical
observations/examination are shown in Table 6.
At Day 1 post challenge, if participants do not have any adverse events from the challenge
process, they may be discharged from the hospital and come to CTU for the daily study follow-
up visits. The participants will be re-admitted to the hospital at any time if clinically indicated.
To ensure that all participants will receive antimalarial treatment according to the study
protocol, participants need to stay near by the study site or convenient accommodation will be
provided to participants. In addition, participants will be asked to provide a telephone contact
of their relative or neighbour to ensure that the study team can reach participants in case of
participant loss to follow-up before they complete antimalarial treatment.
B6.2.10.2 Day after qPCR+ (DayAfter qPCR+) to day of reach treatment criteria (Dreach treatment
criteria)
Once malaria qPCR has been detected or the antimalarial treatment criteria have been met,
participants will be admitted to the hospital for close monitoring. The clinical well-being will
be checked and blood will be drawn twice daily for monitoring blood parasitaemia (malaria
blood film, qPCR, and gametocyte qPCR). For membrane feeding to assess gametocyte
transmissibility, blood will be collected once daily after qPCR positive until day discharge.
Immunological assays, Immune cells & cytokines profile, urine pregnancy test (women only),
CBC, and blood biochemistry will be assessed on Dreach treatment criteria

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Participants will be assessed after challenge according to the schedule in Table 6. Physical
observations will be performed. Venipuncture will be performed as per schedule of attendance
(Tables 6).
Participants will be questioned as to whether they have:

Experienced any of the foreseeable symptoms of malaria.

Experienced any other symptoms.

Taken any medications including over the counter medications.
Full physical examination will be performed if deemed necessary by the Investigators. The
severity of symptoms will be assessed using the grading criteria summarised in APPENDIX C:
Safety terms and administrative information.
Antimalarial treatment will be instituted when the criteria described below are met (Table 2).
If the antimalarial treatment criteria have not yet been met, the treatment shall be initiated at
day 21 after challenge (C21). Participants will be closely monitored until completion of
antimalarial therapy, as described above.
B6.2.11 Antimalarial treatment
A study physician will then immediately prescribe antimalarial treatment with chloroquine, in
accordance with Table 3. Any side effect from the antimalarials will be rapidly dealt with in a
controlled environment. They will continue to have blood films and qPCR once daily until
clinically recovered AND two consecutive malaria blood films are negative (completion of the
chloroquine treatment course). The participants will then be discharged from the hospital.
If any participant reaches day 21 post-challenge without meeting the treatment criteria, they
shall be started on a 3-day course of antimalarial treatment (chloroquine).
If any contraindications to chloroquine are identified, an alternative antimalarial
(dihydroartemisinin + piperaquine or Malarone (atovaquone + proguanil)) will be prescribed. If
a participant is unable to tolerate an oral antimalarial, the appropriate parenteral antimalarial
therapy will be prescribed.
If a participant withdraws/is withdrawn from the study after challenge but before reaching the
criteria for antimalaria treatment, then a complete, appropriate, curative course of
antimalarial therapy must be completed. The importance of this will be emphasized to
participants at screening.

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Table 2: Antimalarial treatment criteria.
Malaria-treatment Start
Criteria

Symptoms suggestive of malaria infection with the presence of
blood parasitaemia by blood film microscopy.

Asymptomatic with ≥50,000 parasites/mL by blood film
microscopy
OR

Asymptomatic and no parasitaemia until Day C21
Note: In a case where a participant develops symptoms suggestive of
malaria infection but negative for malaria (malaria blood film or
qPCR)other causes of symptoms should be considered and investigate
and treated as appropriate.
Table 3: Chloroquine treatment dose
Weight/age
Day 1
Day 2
Day 3
CQ Tab
CQ Tab
CQ Tab
50 kg or more
(above 14 years)
4
4
2
Reference: National Malaria Treatment Guideline 2019 (Thailand), CQ 150 mg base/tablet
Supportive treatments and medications
Any malaria symptoms (e.g. fever, nausea, vomiting) will be relieved accordingly. Paracetamol
(500 mg orally up to four times a day) and dimenhydrinate (50 mg orally) will be prescribed
according to clinical need. Intravenous fluid supplementation may be given following
physician decision.
In addition, all concomitant medications used in the trial will be recorded in the medical record
and CRF throughout study period.
If the participants meet the criteria for severe vivax malaria (by WHO Guidelines for malaria
13 July 2021) or fail to improve within 48 hours of starting antimalarial therapy, malaria
experts in the Faculty of Tropical Medicine will be consulted. The participants will be
managed according to National Malaria Treatment Guidelines (Thailand) as well as the
Hospital for Tropical Diseases’ standing order/ procedures / guidelines for severe malaria.

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The Investigators are able to treat any participant for malaria regardless of the blood film
microscopy or qPCR result if they are clinically concerned or a participant wishes to withdraw
from the study.
B6.2.12 Follow up assessment phase (after discharge)
On the antimalarial treatment end date, the participants will receive full counselling on the signs
and symptoms that they should observe 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 again be emphasized. After discharge from the hospital, the
follow-up assessment will be performed at FTMCTU as an out-patient on day 7 post treatment
initiation (DRx7), day C28 (C28), day C56 (C56), day C96 (C96), day C180 (C180), and day C276
(C276). On the follow-up days, diary cards will be reviewed and collected (until day C96), a history
taken using the clinical well-being checklist, a physical examination will be performed, and
AE(s) assessed. Venepuncture will also be performed to detect malaria parasites by blood film
and qPCR, and malaria gametocyte qPCR, immunological assays, malaria immunology, CBC,
and biochemistry according to Tables 6.
During this follow up phase, the participants will be contacted fortnightly (biweekly) until C96
by email / phone call / other social communications e.g. message (SMS), WhatsApp, Line, to
ensure they remain well and asymptomatic. The participant will be asked to and agree to stay
in Bangkok until 2 months after antimalarial treatment initiation, 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.

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Table 4: Screening schedule of clinical and study procedures for participants
Event
Screening
(within 30 days prior to Day 0)
Inclusion / Exclusion criteria
X
Informed Consent Questionnaire
X
Informed consent
X
Medical History
X
Physical Examination
X
Body weight and heighta
X
Urinalysis
X
Electrocardiogram
X
Vital signsb
X
Blood typing ABO, Rhesus, Crossmatching
X
Duffy antigen/receptor for chemokine (DARC)
X
CBC
X
Haemoglobinopathies and thalassaemia traits*
X
G6PD*
X
Blood biochemistryc
X
HIV, HBV, HCVd
X
Malaria diagnosis (Malaria antigen – blood film
and qPCR)
X
Malaria exposure*
X
Malaria immunology*
X
β-HCG testing (women only) 
X
(Serum pregnancy test)
Remark:
a Height will be measured only at screening visit.
b Vital signs include blood pressure, pulse rate, respiratory rate and temperature.
c Biochemistry including electrolytes (sodium, potassium, chloride, bicarbonate, calcium and
magnesium), blood urea nitrogen (BUN), creatinine (Cr), eGFR, liver function tests (LFTs), and
FBS
d HIV, HBV, and HCV will be tested for antibodies. HBs serological profile includes HBsAg, anti-
HBc, and anti-HBs.

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*This test will be done for data interpretation purpose. It will not be used as the
inclusion/exclusion criteria.

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Table 5: Schedule of Clinic Attendances during vaccination
Remark:
a Vital signs include blood pressure, pulse rate, respiratory rate and temperature.
b Biochemistry including electrolytes (sodium, potassium, chloride, bicarbonate, calcium and
magnesium), blood urea nitrogen (BUN), creatinine (Cr), eGFR, liver function tests (LFTs)
c HIV, HBV, and HCV will be tested for antibodies. HBs serological profile includes HBsAg, anti-
HBc, and anti-HBs.
d Test at Oxford
Event
Vaccination Phase
(V1)
(V2)
(V3)
Timeline
D0
D3
D7
D14
D28
D35
(7 ds after
vac)
D42
(14 ds
after vac)
D56
(28 ds
after vac)
D210
(Month 7)
D217
(7 ds after
vac)
D224
(14 ds after
vac)
Window (days)
0
0
±1
±3
±3
±1
±3
±3
±3
±1
±3
Inclusion/exclusion criteria
X
X
X
Medical history (well-being
check list)
X
X
X
X
X
X
Body weight
X
X
X
Physical
observation/examination
X
X
X
X
X
X
Urine pregnancy test
(women only)
X
X
X
Vital signsa
X
X
X
X
X
X
X
X
X
X
X
AE/SAE
X
X
X
X
X
X
X
X
X
X
X
Diary card
X
X
X
Vaccination
X
X
X
Haematology (CBC)
X
X
X
X
X
X
Biochemistryb
X
X
X
X
X
X
HIV, HBV, HCVc
X#
Malaria diagnosis (Malaria
antigen – blood film and
qPCR)
X
X#
Immunological assays
ELISAs & GIAd
X
X
X
X
X
X
X
X
X
Immunological assays:
Humoral and cellular
immune responsese
X
X
X
X
X
X
X
X
Immune cells & cytokines
profile (Phenotypic profile)
X
X
X
X
X
X
X
X
X

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e Test at Pasteur
#Check for Pre-challenge criteria

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Table 6: Schedule of Clinic Attendances since challenge (including admission C-1)
Event
Admission
Admission
Follow-up Phase
Timeline
C-1
C0*
C1
C2 – D
qPCR+
Dafter qPCR+ – D
reach treatment
criteria
DStart CQ – D
discharge
DRx7
C28
C56
C96
C180 C276
Window (days)
0
0
0
0
0
0
±2
±7
±7
±7
±14 ±14
Inclusion/exclusi
on criteria
X
Medical history
(well-being check
list)
X
X
X
(once
daily)
X
(once
daily)
X
(Twice
daily)
X
(once daily)
X
X
X
X
X
X
Body weight
X
X
X
(Dreach treatment
criteria)
X
X
X
X
X
X
Physical
observation/
examination
X
X
X
X
X
X
X
X
X
X
X
X
Urine pregnancy
test (women
only)
Xg
X
(before
treatment)
Vital signsa
X
X
X
X
X
X
X
X
X
X
X
X
AE/SAEb
X
X
X
X
X
X
X
X
X
X
X
X
Blood stage
challenge
X
Treatment for
Malariac
X
Diary card
X
Dcomplete
antimalarial
treatment
X
X
X
X
Malaria antigen
(Blood Film and
qPCR)
X
X
(daily)
X
(daily)
X
(twice daily)
X
(daily)
X
X
X
X
X
X
Malaria
Gametocyte
qPCR
X
(daily)
X
(daily)
X
(twice daily)
X
(daily)
X
X
X
X
X
X
Haematology
(CBC)
X
C4,
DqPCR+
X
(Dreach treatment
criteria)
X
X
X
X
X
X
Crossmatching
X

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Event
Admission
Admission
Follow-up Phase
Timeline
C-1
C0*
C1
C2 – D
qPCR+
Dafter qPCR+ – D
reach treatment
criteria
DStart CQ – D
discharge
DRx7
C28
C56
C96
C180 C276
Window (days)
0
0
0
0
0
0
±2
±7
±7
±7
±14 ±14
Biochemistryd
X
DqPCR+
X
(Dreach treatment
criteria)
D after complete
antimalarial
treatment
X
X
X
X
X
Immunological
assays
ELISAs & GIAe
X
X
(C+7, C+14, Dreach treatment criteria)
X
X
X
Immunological
assays: Humoral
and cellular
immune
responsesf
X
X
(C+14, C+21)
X
X
X
Immune cells &
cytokines profile
(Phenotypic
profile)
X
X
(C+21)
MFAj
X
(once daily)
X
(once daily)
COVID-19
serology test
X
Xh
CMV and EBV
serology
X
Xi
(C+14)
X
Malaria
immunology
X
C4,
DqPCR+
X
(D reach treatment
criteria)
X
X
X
X
X
X
Remark:
a Vital signs include blood pressure, pulse rate, respiratory rate and body temperature.
b AEs/SAEs will be collected until 1 year after antimalarial initiation.
c This treatment period could occur on any day between C6 and C21. Blood draws will continue as
per this Table for the relevant study day, up until the time-point of treatment. The treatment will be
done on C21 if no criteria meet.
d Biochemistry including electrolytes (sodium, potassium, chloride, bicarbonate), blood urea
nitrogen (BUN), creatinine (Cr), liver function tests (LFTs)
e Test at Oxford
f Test at Pasteur
g Serum β-HCG
h COVID-19 serology test will be done at day discharge.

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i EBV and CMV serology will be done at 2-4 weeks and at 3 months after the inoculation. EBV
and CMV serology including EBV VCA (IgM)/IgG antibody, anti EBNA and EA complex
antibody, and CMV IgM/IgG antibody
j Membrane Feeding Assay (MFA) will be conducted according to SOP Dissection of Anopheles
mosquito’s midgut to determine infection with malaria parasite and SOP Membrane feeding assay

* In case volunteer lost to follow-up after 3rd vaccination and then return, the maximum
time allowancw between 3rd vaccination and challenge duration is 6 weeks but pre-
challenge lab tests need to be repeated as appropriate at investigators discretion.

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Table 7: Blood collection during study period
Phase
Clot blood
EDTA blood/ WB Tempus
NaF
Total
blood
(mL)
Accumulate
blood
volume
(mL)
Volume
Test
Volume
Test
Volume
Test
Screening
6
Blood typing, crossmatching
2
Malaria antigen (Blood
film, qPCR)
2
FBS
31
31
Blood biochemistry
6
Duffy blood group
HIV
CBC+Thalassemia
HBs profile, anti-HCV, serum
HCG
Hemoglobinophathies
1
Malaria exposure
12
Malaria immunology
2
G6PD FST
Vaccination D0 (V1 day0)
5
Biochemistry
2
CBC
37
68
10
Immunological assays (humoral
&cellular responses)
2
Malaria antigen
10
Immunological functional assays
(ELISA&GIA)
6
Immunological assays
(humoral &cellular
responses)

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Phase
Clot blood
EDTA blood/ WB Tempus
NaF
Total
blood
(mL)
Accumulate
blood
volume
(mL)
Volume
Test
Volume
Test
Volume
Test
2
Immune cells & cytokines
profile (Phenotypic
profile)
Vaccination D3 (V1 day3)
6
Immunological assays
(humoral &cellular
responses)
6
74
Vaccination D7 (V1 day7)
5
Biochemistry
6
Immunological assays
(humoral &cellular
responses)
15
89
2
Immune cells & cytokines
profile (Phenotypic
profile)
2
CBC
Vaccination D14 (V1 day14)
4
Immunological functional assays
(ELISA&GIA)
2
Immune cells & cytokines
profile (Phenotypic
profile)
8
97
2
Immunological assays (humoral
&cellular responses)
Vaccination D28 (V2 day0)
5
Biochemistry
2
CBC
13
110
4
Immunological functional assays
(ELISA&GIA)
2

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MIST3_Research Proposal Submission Form, version 3.0, dated 14 Decemer 2022
Phase
Clot blood
EDTA blood/ WB Tempus
NaF
Total
blood
(mL)
Accumulate
blood
volume
(mL)
Volume
Test
Volume
Test
Volume
Test
Immune cells & cytokines
profile (Phenotypic
profile)
Vaccination D35 (V2 day 7)
5
Biochemistry
2
CBC
19
129
4
Immunological functional assays
(ELISA&GIA)
6
Immunological assays
(humoral &cellular
responses)
2
Immune cells & cytokines
profile (Phenotypic
profile)
Vaccination D42 (V2 day 14)
5
Immunological assays (humoral
&cellular responses)
2
Immune cells & cytokines
profile (Phenotypic
profile)
11
140
4
Immunological functional assays
(ELISA&GIA)
Vaccination D56 (V2 day 28)
4
Immunological functional assays
(ELISA&GIA)
4
144
Vaccination D210 (V3 day0)
5
Biochemistry
2
CBC
21
165
2
Immunological assays (humoral
&cellular responses)
6
Immunological assays
(humoral &cellular
responses)

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MIST3_Research Proposal Submission Form, version 3.0, dated 14 Decemer 2022
Phase
Clot blood
EDTA blood/ WB Tempus
NaF
Total
blood
(mL)
Accumulate
blood
volume
(mL)
Volume
Test
Volume
Test
Volume
Test
4
Immunological functional assays
(ELISA&GIA)
2
Immune cells & cytokines
profile (Phenotypic
profile)
Vaccination D217 (V3 day7)
5
Biochemistry
2
CBC
19
184
4
Immunological functional assays
(ELISA&GIA)
6
Immunological assays
(humoral &cellular
responses)
2
Immune cells & cytokines
profile (Phenotypic
profile)
Vaccination D224 (V3 day14)
2
HIV HBV HCV
2
Immune cells & cytokines
profile (Phenotypic
profile)
16
200
10
Immunological functional assays
(ELISA&GIA)
2
Malaria antigen (blood
film, qPCR )
Admission day (C-1)
6
Crossmatching
2
CBC
80
280
Biochemistry, serum HCG
3
Malaria antigen (blood
film, qPCR)
1
COVID-19 serology
2
Immune cells & cytokines
profile (Phenotypic
profile)

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MIST3_Research Proposal Submission Form, version 3.0, dated 14 Decemer 2022
Phase
Clot blood
EDTA blood/ WB Tempus
NaF
Total
blood
(mL)
Accumulate
blood
volume
(mL)
Volume
Test
Volume
Test
Volume
Test
4
CMV&EBV serology
30
mAB production
10
Immunological assays (humoral
&cellular responses)
12
Malaria immunology
10
Immunological functional assays
(ELISA&GIA)
Admission day C1-DqPCR+
(C1-~C8 )
5
Biochemistry (DqPCR+)
24
Malaria (blood film,
qPCR, qGAM) (once
daily)
61
341
4
Immunological functional assays
(ELISA&GIA) (C7)
4
CBC (C4, DqPCR+)
24
Malaria immunology
(C4, DqPCR+)
D afterqPCR+-D reach
treatment (~C9-~C16)
5
Biochemistry (D reach treatment)
48
Malaria (blood film,
qPCR, qGAM ) (twice
daily)
89
430
8
Immunological functional assays
(ELISA&GIA) (C14,D reach
treatment)
8
Membrane feeding assay
(once daily)
4
CMV&EBV serology (C14)
2
CBC (D reach treatment)
2
Immunological assays (humoral
&cellular responses) (C14)
12
Malaria immunology (D
reach treatment)

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MIST3_Research Proposal Submission Form, version 3.0, dated 14 Decemer 2022
Phase
Clot blood
EDTA blood/ WB Tempus
NaF
Total
blood
(mL)
Accumulate
blood
volume
(mL)
Volume
Test
Volume
Test
Volume
Test
D after start CQ- D discharge
(~C17-~C21)
5
Biochemistry (D after complete
treatment)
6
Immunological assays
(humoral &cellular
responses) (C21)
34
464
1
COVID-19 serology (D
discharge)
2
Immune cells & cytokines
profile (Phenotypic
profile) (C21)
15
Malaria (blood film,
qPCR, qGAM ) (once
daily)
5
Membrane feeding assay
(once daily)
DRx7 (~C22)
5
Biochemistry
3
Malaria (blood film,
qPCR, qGAM ) (once
daily)
22
486
12
Malaria immunology
2
CBC
C28
5
Biochemistry
3
Malaria (blood film,
qPCR, qGAM ) (once
daily)
24
510
2
Immunological assays (humoral
&cellular responses)
12
Malaria immunology

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MIST3_Research Proposal Submission Form, version 3.0, dated 14 Decemer 2022
Phase
Clot blood
EDTA blood/ WB Tempus
NaF
Total
blood
(mL)
Accumulate
blood
volume
(mL)
Volume
Test
Volume
Test
Volume
Test
2
CBC
C56
5
Biochemistry
3
Malaria (blood film,
qPCR, qGAM ) (once
daily)
28
538
4
Immunological functional assays
(ELISA&GIA)
12
Malaria immunology
2
Immunological assays (humoral
&cellular responses)
2
CBC
C96
4
CMV&EBV serology
12
Malaria immunology
32
570
5
Biochemistry
3
Malaria (blood film,
qPCR, qGAM ) (once
daily)
2
Immunological assays (humoral
&cellular responses)
2
CBC
4
Immunological functional assays
(ELISA&GIA)
C180
3
Malaria (blood film,
qPCR, qGAM ) (once
daily)
17
587
12
Malaria immunology

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Phase
Clot blood
EDTA blood/ WB Tempus
NaF
Total
blood
(mL)
Accumulate
blood
volume
(mL)
Volume
Test
Volume
Test
Volume
Test
2
CBC
C276
5
Biochemistry
3
Malaria (blood film,
qPCR, qGAM ) (once
daily)
26
613
4
Immunological functional assays
(ELISA&GIA)
12
Malaria immunology
2
CBC

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B6.2.13 Details of study clinical procedures/laboratory tests
Procedures will be performed at the time points indicated in the schedule of procedures (Tables
4 - 6). Additional procedures or laboratory tests may be performed, at the discretion of the
investigators if clinically necessary. Study procedures will be monitored by the MORU
Clinical Trials Support Group.
Clinical procedures
Body Weight and height
Body Weight and height will be measured at the screening (Table 4) and body weight will be
measured as indicated in the Table 5 and 6.
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 4 and 5).
Urinalysis
Urine will be tested for the presence of clinically significant proteinuria, glucosuria or
haematuria at the screening visit.
Electrocardiogram
An electrocardiogram will be performed at the screening visit.
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 contracted laboratories.
Samples will also be sent to collaborating laboratories within and outside Thailand for
immunomonitoring and/or harmonisation of key immunological assays. There are four
collaborating laboratories:
(1) Ehime University, Matsuyama, Japan
(2) Kenya Medical Research Institute (KEMRI), Kenya
(3) Institute Pasteur, Paris

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(4) University of Oxford, UK
The AlphaScreen assay will be performed at Ehime University 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 participant who previously had a malaria infection leading
to antibodies in their serum which are reactive to more than 10% of the malaria antigens in the
library.
B6.2.14 Challenge infection performed in the trial participants
B6.2.14.1 Description and administration of the cryopreserved P. vivax infected erythrocytes
The total volume of the diluted cryopreserved parasite that will be injected intravenously into
each participant is 5 mL.
B6.2.14.2 Source and preparation of the cryopreserved inoculum
The cryopreserved P. vivax parasitized red blood cells that will be used were produced by
investigator team in December 2020, as part of “A clinical study to assess the feasibility of a
controlled human Plasmodium vivax malaria infection model through experimental sporozoite
infection in Thai adults” (‘MIST 1 study’, certificate number 2020-038-01, approved by the
FTM-EC #TMEC19-067; OxTREC Ref 43-19. The information is as follows.
Cryopreservation and storage of blood bank
P. vivax infected blood processing was conducted within fumigated microbiological safety
cabinets. First, the red cells were separated from plasma by centrifugation of the leukodepleted
blood before mixing with GMP grade Glycerolyte 57 (at 1:2 erythrocyte to Glycerolyte 57
volume ratio). The first 20% of Glycerolyte 57 was added dropwise with gentle agitation, the
suspension was then incubated for 5 minutes at room temperature before the remaining
Glycerolyte 57 was added. The RBC-Glycerolyte mixture was aliquoted into 1.5 mL cryovials
and frozen at -80oC for one night before transferred to a dedicated liquid nitrogen tank.
Confirmation of parasitic density within the blood collected for cryopreservation was
performed via microscopy and quantitative PCR on the leukodepleted packed blood samples.
Thick film microscopy demonstrated 6 asexual parasites per 1 μL leukodepleted packed blood 
for Donor 1, and 13 asexual parasites per 1 μL leukodepleted packed blood for Donor 2. 
Quantitative PCR confirmed the presence of 155,400 genome copies/mL in Donor 1, and
988,600 genome copies/mL in Donor 2.
Sterility and screening of blood-borne and vector-borne infections of cryopreserved blood bank

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Sterility and screening for blood-borne and vector-borne infections of the cryopreserved blood
bank were performed to ensure the safety of the cryopreserved blood bank. Real time PCR for
HIV-1, HIV-2, HBV, HCV, Dengue, chikungunya, zika, Japanese encephalitis, CMV and
EBV, serology for HTLV-1, HTLV-2 and syphilis (TPHA), serology for filaria, endotoxin and
ELISA for mycoplasma were tested from plasma derived directly from blood bank. Blood
collected for cryopreservation was sent to FTM hospital for testing the bacterial contamination
using hemoculture technique. All tested results were negative for both donors.
B6.2.14.3 Preparation of the inoculum
Thawing and washing of the inoculum will be done with commercial solutions for human use
and with disposable syringes and needles according to local standard operating procedure. The
processing of the inoculum will be carried out in MVRU’s dedicated biosafety level II
laboratory at the Hospital for Tropical Diseases, Faculty of Tropical medicine. Sample
manipulations will be performed within a safety cabinet that has been fumigated, sterilized and
dedicated for this purpose. Deglycerolizing will be performed by sequentially adding 0.1
volume of 12%, 10 volume of 1.6% and 10 volume of 0.9% saline solution. The inoculum will
be reconstituted in 0.9% saline, to a total volume of 5 mL. Further dilution of the incoculum
will be undertaken based on the results of the MIST2, which at the time of writing the first
version of this protocol are still pending. Five milliliters of the diluted inoculum will be drawn
in 5 mL syringe and closed with sterile cap. The inoculum will be kept in the warm transport
box until inoculation in the participants. The inoculum must be injected intravenously into the
participant within 4 hours of the inoculum being thawed.
B6.2.14.4 Administering the study agent to the participants:
The inoculation will take place at the Hospital for Tropical Diseases. The inoculum will be
administered by injection into an indwelling intravenous cannula.
The challenge steps:
The participant(s) will be hospitalized a night before the inoculation day (C0) to ensure their
safety /eligibility and to avoid the traffic burden.
Prior to challenge, all participants are briefed. The arms will be cleaned by using 70% alcohol
according to the Inoculum Administration Work Instruction (WI).
The inoculum will be transported directly from the MVRU clean room lab at 9th floor in a
warm box to the admission facility within the Hospital for Tropical Diseases where the
injection of the inoculum being take place.

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All groups will receive a total of 5 ml of inoculum diluted with 0.9% normal saline. This
dilution selected will be based on the optimal dose finding result of the MIST2 study (currently
still pending).
The reconstituted inoculum will be injected via an indwelling cannula, followed by a saline
flush.

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B6.3 Schematic diagram of study design, procedures and stages, step-by-step
Screening and eligibility assessment (Day-30 to Day 0)

History taking, vital signs and physical
examination, body weight and height, ECG

Blood group ABO, RH Rhesus, Duffy blood group

CBC, blood diseases, FBS, blood biochemistry

HBV serology, HIV serology, HCV antibody, and
HBs profile

Malaria diagnosis (blood film, qPCR), Malaria
exposure and Malaria immunology

Serum pregnancy test (women only)

Crossmatching
Vaccination Phase

Well-being check list, body weight, vital signs,
physical observation/examination, local &
systemic events (AE/SAE)

CBC, blood biochemistry, Urine pregnancy test
(women only) and venipuncture for immunology
assays and Immune cells & cytokines profile

Diary card

HIV, HBV, HCV serology and HBs profile

Malaria diagnosis (blood film, qPCR)
Before vaccination, the inclusion and exclusion criteria
will be reviewed (participants are eligible and no
contraindications to vaccination)
Day before CHMI (C-1)

Well-being check list, physical observation/examination, body weight,
local & systemic events (AE/SAE), vital signs, CBC, crossmatching,
blood biochemistry, serum pregnancy test (women only)

Malaria blood film, malaria qPCR, malaria immunology,
immunological assays, and Immune cells & cytokines profile

COVID-19 serology test, CMV and EBV serology
Discharge if volunteers do not have any adverse event from the challenge
process
Recruiting research participants
Day of CHMI (C0)

CHMI blood stage challenge (at Day 0)

Well-being check list, vital signs, body weight and physical
observation/examination, local & systemic events (AE/SAE)
Obtaining approval from the Ethics Committees
Dafter qPCR+ - Day discharge

Well-being check list, physical observation/examination, body
weight, local & systemic events (AE/SAE), vital signs, diary
card

CBC, blood biochemistry

Urine pregnancy test (before treatment for malaria)

Malaria blood film, malaria qPCR and gametocyte qPCR,
immunological assays, Immune cells & cytokines profile,
membrane feeding assay, and malaria immunology

COVID-19 serology test (at day discharge)

CMV and EBV serology (at 2-4 weeks after the inoculation)
Day 1 post challenge (C1)

Well-being check list, physical observation/examination, local &
systemic events (AE/SAE), vital signs

Malaria blood film, malaria qPCR and gametocyte qPCR
C2 - Day qPCR positive (DqPCR+)

Well-being check list, physical observation/examination, local
& systemic events (AE/SAE), vital signs

CBC, blood biochemistry, Malaria immunology

Malaria blood film, malaria qPCR and gametocyte qPCR, and
immunological assays
If participants meet the criteria for treatment, the
standard treatment for malaria will be initiated.
Discharge when clinically recovered, two negative consecutive
malaria blood films and completion of chloroquine treatment
course
Informed consent be obtained

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B6.4 Specimen and data management
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.
The investigators will maintain and retain appropriate medical and research records and
essential documents for this trial in compliance with ICH Good Clinical Practice (ICH GCP)
E6 (R2) and regulatory and institutional requirements for safety and protection of
confidentiality of participants. The Co-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
participants’ 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 documentation, data and all other information generated will be held in strict confidence.
Only authorized, trained study staff as noted on the delegation log 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.
B6.5 Data analysis
A brief overview of the analytical plan is given below. A detailed Statistical Analysis Plan
(SAP) will be developed as a separate document and finalized before database lock.
Follow up phase

Well-being check list, and physical observation/examination, vital
signs body weight, local & systemic events (AE/SAE), diary card

CBC and blood biochemistry

Malaria blood film, malaria qPCR and gametocyte PCR,
immunological assays, and malaria immunology
During follow up phase volunteer will be contacted
fortnight until C96 by email/ phone call/ other social
communications e.g. message (SMS), WhatsApp,
Line to ensure they remain well and asymptomatic.

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Proportions
Frequency data will be compared using chi squared or Fisher’s exact test, as appropriate. Crude
proportions will be calculated with the exact 95% confidence intervals (CI), where relevant.
Continuous data
For the primary efficacy outcome, the geometic means of the three replicate PCR results
obtained for each individual at each timepoint will be used for model-fitting. Negative
individual replicates will be handled as specified in the MIST3 SAP. qPCR data points which,
based upon the geometric mean of the three replicates, are negative or below the limit of
quantification will be handled as specified in the MIST qPCR SAP. PMR will be calculated
using a linear model fitted to log10-transformed qPCR data.
The distribution of PMRs will be assessed for deviation from normality visually and using the
d’Agostino-Pearson test. Standard ladder-of-power transformations may be applied to achieve
normality. Equality of variance will be tested with an F-test. In the event of deviation from
normality, PMRs will be compared by two-tailed Mann-Whitney test; otherwise a two-tailed
two-sample t-test will be used, with Welch’s correction if variances are non-equal as assessed
by the F-test. Test of significance will be performed at 5% significance level. Data will be
analysed in Stata version 16 and above or using a comparable standard statistical analysis
software. This will be detailed in the Statistical Analysis Plan.
B7. DATA AND SPECIMEN STORAGE AND/OR SHARING
B7.1 Management of specimen/data archiving and management of left-over samples
Specimen storage
With the participants’ informed consent, any leftover cells and serum will be stored for 10
years in the FTM/MORU Sample Management System for future immunological and genetic
analysis of malaria-specific responses.
Data handling and record keeping
Medical records and essential documents will be kept at the study site for approximately 5
years after study completion. Files containing identifiable information will be stored separate
from other study data, in secure locations. Data recorded on paper records such as clinical
worksheets and laboratory results printouts will be stored in locked cabinets. Electronic data
will be stored on password controlled computers and in the study database that is hosted on a
secure, access-controlled server in MORU. Physical access to the server is restricted to
authorised and authenticated individuals only. A key card with special permissions is required
to gain access to the server room . Only the Sponsor’s representative, Investigators, the clinical

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monitor, the ethical committee(s) and the regulatory authorities will have access to these
records.
Data recorded in the CRF will be entered into the study database for storage and analysis.
Personal identifying information such as names and telephone numbers will not be stored in
the study database or used for analysis. Data will be pseudonymized, each participant will be
identified by a unique study ID. Pseudonymised data will be stored indefinitely.
B7.2 Specimen/data sharing plan
With participants’ consent, de-identified data and results from blood analyses stored in our
database may be shared with other researchers to use in the future. Access to study data will be
provided following the MORU data sharing policy.
With participants’ consent, specimen/leftover specimen from this research project may be
stored for 10 years for future use in the scope related or not related to this project.
If any data and/or specimen/leftover specimen from this current study to be used in any future
research project, the investigator will submit the project to the Ethics Committee affiliated to
the investigator for due consideration before use.
PART C: ETHICAL CONSIDERATION (DESCRIBE ONLY THE RESPONSIBILITIES
OF THE PI)
C1. SIGNIFICANCE OF THE STUDY
This study is carried out to determine the safety, immunogenicity and efficacy of the
PvDBPII/Matrix-M vaccine for P. vivax malaria.
C2. BALANCE OF RISK AND BENEFIT
C2.1 Risk of the study
This section describes the foreseen risks to the participant including risk related to:
Phlebotomy
There may be minor bruising, local tenderness or pre-syncopal symptoms associated with
venipuncture, which will not be documented as AEs if they occur.
PvDBPII/Matrix-MTM vaccination
Foreseeable risks from vaccination, which include local and systemic reactions, are specific to
each IMP and full details available in the respective IBs.

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As with any vaccine, Guillain-Barré syndrome or immune-mediated reactions that can lead to
organ damage including serious allergic reactions may occur but this should be extremely rare.
Serious allergic reactions including anaphylaxis could also occur and for this reason
participants will be vaccinated in a clinical area where Advanced Life Support trained
physicians, equipment and drugs are immediately available for the management of any serious
adverse reactions.
Based on the safety data available from the 27 volunteers who received PvDBPII vaccine
formulated in GLA-SE in the Phase Ia study (24), as well as data available from more than
37,734 individuals who have previously received vaccines formulated in Matrix-M1, reversible
reactogenicity will occur at a higher rate in subjects receiving the Matrix-M-adjuvanted
vaccine than among those receiving vaccine with the same antigen without the adjuvant or
placebo, although the reactogenicity is transient and generally mild or moderate in severity.
The following adverse event profile is expected following vaccination with PvDBPII-Matrix-
M1:
Local adverse events (AEs) such as injection site pain would be foreseeable to occur
frequently. Less frequent local AEs are likely to include erythema, swelling and bruising.
Local AEs are likely to be mild in nature and should resolve rapidly, although there is the
possibility of moderate or severe arm pain in some cases. Among the solicited systemic
complaints following Matrix M1-adjuvanted vaccines, headache, muscle pain, fatigue, chills,
joint pain, wheezing, and eyelid swelling all occur at higher rates among adjuvanted vaccinees,
when compared to placebo vaccinees at an excess of ≥ 1% of subjects.  
Inoculation
This controlled blood-stage challenge will involve a transfusion of a blood inoculum. As with
any transfusion, there are risks of blood-borne infections and allergic reaction to blood
products. The risk of blood-borne infection was reduced during the collection of blood
inoculum in MIST 1 by extensive screening for blood-borne infection in all three of: the source
patients; the CHMI infected blood donation volunteers from MIS1; and the stored inoculum.
Serious allergic reactions including anaphylaxis have not been seen in challenge studies to
date, but for safety reason participants will be inoculated in an area where physicians and a
defibrillator are immediately available.
As the inoculum is prepared from packed red cells, the participants have a risk of developing a
transfusion reaction. This risk has been minimized by preparing the inoculum from donors with
blood Group O (universal donors), and the blood compatibility between the inoculum and
volunteers will be checked. In addition, the volume of blood to be used (0.5 mL packed red
cells) is much smaller than that given in transfusion of one unit packed red cells (470 mL).

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Nevertheless, the participants will be monitored for this possibility during the first hour after
the administration of the inoculum.
Plasmodium vivax infection
Base on previous studies, volunteers are likely to develop symptomatic malaria infection
following challenge (25). The participants will be followed up closely post-challenge and only
enrolled in the study if they are deemed reliable and capable of complying with the study
schedule. We will admit the participants to the Hospital for Tropical Diseases, Faculty of
Tropical Medicine, Mahidol University for close clinical monitoring, treatment (antimalarials)
and symptomatic relief. 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.
C2.2 Preventive and alleviative measures for risk
Phlebotomy
Participants’ blood samples will be collected by well - trained medical personnel with aseptic
technique to prevent infection or complications.
PvDBPII-Matrix-MTM vaccination
Participants will be vaccinated in a clinical area where Advanced Life Support trained
physicians, equipment and drugs are immediately available for the management of any
immediate postvaccination serious adverse reactions. Participants will be observed closely for at
least 30 minutes following administration of the vaccine.
Plasmodium vivax infection
Participants 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 participants will be hospitalized in the Hospital for Tropical Medicine,
Faculty of Tropical Medicine, Mahidol University for close clinical monitoring, antimalarial
treatment and symptomatic relief (e.g. fluids, analgesia and/or anti-emetics) until parasitaemia
becomes negative. The treatment algorithm as well as the hospital guideline will be fully
reviewed and strictly followed. The hospital will be officially informed prior to study initiation
to ensure all the required medical facilities are in place. Clinical malaria expert at the Faculty
of Tropical Medicine will be officially informed prior to study initiation. The medical monitors
will be reachable throughout the whole study process.

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In addition, this blood-stage inoculation will cause P. vivax infection without liver-stage
parasites, thus removing the risk of relapse.
C2.3 Benefits of the study
There are no direct benefits to the participants taking part in this study. The expected benefit is
to provide information about the safety and tolerability of the PvDBPII/Matrix-MTM vaccine
compared to controls (placebo vaccine) in a P. vivax blood-stage controlled human malaria
infection model and immunology data. All of these will benefit patients with malaria infections
and population who are at risk of malaria infection in the future.
C3 SAFETY
Participant safety is of paramount importance, and we will follow these guidelines. The following
measures are in place to safeguard participant safety:

Participants will only be enrolled in the study if investigator judges this is appropriate.

Participants’ 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 participant will be documented, including
home address and mobile telephone numbers. Mobile telephone numbers will be verified
prior to challenge to ensure the participants are easily contactable. Home and work landline
telephone numbers where available and next-of-kin address and telephone numbers will
also be documented. Participants 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.

The participants will be hospitalized when malaria parasite is identified (by qPCR) and will
stay in the hospital until clinically recovered AND the completion of the antimalarial
course (chloroquine) AND until two consecutive negative blood films.

Participants will be able to contact a medically qualified member of the study team 24
hours a day throughout the study period.
Expectations of participant events following blood stage 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.

According to references No. (25-28) and personal communication with other investigators,
all volunteers in P. vivax blood stage challenge studies developed infection within 3 weeks

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following the challenge. Most of AEs were mild to moderate grade and all were transient.
No serious adverse events were reported.
C3.2 Safety Monitoring
C3.2.1 Medical Monitor
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 appointed medical monitor for the MIST 3 study is Dr. Lorenz Von Seidlein M.D.
C3.2.2 Data and Safety Monitoring Board
The Data Safety Monitoring Board (DSMB) will review the study prior to initiation; review the
interim safety data reports; and review all serious adverse events (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 Co-
Principal Investigator(s) to the DSMB at the same time as they are submitted to the ethics
committees. The Co-Principal Investigator(s) will notify the Board and obtain a
recommendation concerning continuation, modification, or termination of the study. The Co-
Principal Investigator(s) 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.
The DSMB will be independent of the Clinical Trials and Research Governance, Oxford
University (Sponsor) and MIST programme team. The DSMB is charged with monitoring and
evaluating the clinical data generated by this study, with a focus on safety, in and independent
and objective manner. The main roles and responsibilities of the DSMB, DSMB procedures,
and the DSMB meeting schedule are defined in the Data and Safety Monitoring Board Charter
for the Malaria Infection Study in Thailand (MIST) Programme.
C4. CONSIDERATION FOR VULNERABLE RESEARCH PARTICIPANTS
Check whether your study involves any of the following vulnerable research participants.
Prisoners

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Pregnant women
Mentally ill persons
Cancer or terminally ill patients
Neonates/infants/children (aged <18)
HIV/AIDS patients
Institutionalized persons e.g. military, students, etc.
Others (please specify)………………………………….….
Not applicable
C5. INFORMED CONSENT ISSUES
C5.1 Informed consent process
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 participant 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 in 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 participants’ 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.

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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 from of Hospital for Tropical Diseases. Pre and post counselling for
HIV screening and reporting will 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.

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C5.2 Informed consent documentation
Age
Informed Consent
Participant Information Sheet
20 years and over
(Adult)
ICF for Adult
Version 3.0 , dated 14 Dec 2022
PIS for Adult
Version 3.0, dated 14 Dec 2022
ICF for Data Sharing/ Data and
Leftover Specimen Storage from
Current Study for Future Use
Version 3.0 , dated 14 Dec 2022
ICF for Leftover Specimen
Storage from Current Study for
future genetic analysis
Version 3.0, dated 14 Dec 2022
20 years and over
(Adult)
HIV Pre-test counseling form, dated 1 October 2009
C5.3 Compensation for research participants
Yes, please provide details:
Participants will be compensated for their time and for the inconvenience caused by procedures as
below (Table 8). 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, compensation specifically for risk was also advised (29).
Table 8: Estimated compensation amounts.
Activity
Compensation
(THB)
Number of visits
Total
(THB)
1. Screening visit (D-30)
2,000
1
2,000
2. 1stvaccination & D3&7&14
1,500
4
6,000
3. 2ndvaccination & D35&42&56
1,500
4
6,000
4. 3rdvaccination & D217&224
1,500
3
4,500

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Activity
Compensation
(THB)
Number of visits
Total
(THB)
5. Admission per night
(C-1&C0)
2,000
2 nights
4,000
6. Follow up (after challenge)
1,500
Around x 5 visits
7,500
7. Admission per night
2,000
Around x 10 nights
20,000
8. DRx7
1,500
1
1,500
9. C28
1,500
1
1,500
10. C56
1,500
1
1,500
11. C96
1,500
1
1,500
12. C180
1,500
1
1,500
13. C276
1,500
1
1,500
Time in Trial
(approx.)
Maximum no. of
visits
Maximum volume of
blood taken1 (mL)
Total compensation amount (THB)
1 year
13
613 mL
59,000
Remark:
1Maximum volume of blood taken is 613 mL in case participant received antimalarial treatment on
day 16.
In case a participant has to come for extra visit(s), they will be compensated for their time and for
the inconvenience with 1,000 THB per day.
No, please provide reasons: ………………………………………………..………
C5.4 Responsible and contact persons
Person(s) responsible for payment for treatment of complications and adverse effects
Person(s) including doctor(s) and/or contact address(es) and telephone number(s) for
emergency use

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1. Dr. Kittiyod Poovorawan, Department of Clinical Tropical Medicine, Faculty of Tropical
Medicine, Mahidol University, Tel. (02) 354-9100 ext. 1435, Mobile (083) 149-6864
2. Dr. Podjanee Jittamala, Department of Tropical Hygiene, Faculty of Tropical Medicine,
Mahidol University, Tel. (02) 306-9157, Mobile (081) 956-3371.
3. Dr. Borimas Hanboonkunupakarn, Department of Clinical Tropical Medicine, Faculty of
Tropical Medicine, Mahidol University, Tel. (02) 354-9100 ext. 3160, Mobile (086) 970-
5705

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PART D: APPENDIX A
REFERENCES
1.
Baird JK. Evidence and implications of mortality associated with acute Plasmodium vivax malaria. Clin
Microbiol Rev. 2013;26(1):36-57.
2.
WHO. World malaria report 2017.2017.
3.
Rahimi BA, Thakkinstian A, White NJ, Sirivichayakul C, Dondorp AM, Chokejindachai W. Severe
vivax malaria: a systematic review and meta-analysis of clinical studies since 1900. Malar J.
2014;13:481.
4.
Price RN, Tjitra E, Guerra CA, Yeung S, White NJ, Anstey NM. Vivax malaria: neglected and not
benign. Am J Trop Med Hyg. 2007;77(6 Suppl):79-87.
5.
Watson J, Taylor WRJ, Bancone G, Chu CS, Jittamala P, White NJ. Implications of current therapeutic
restrictions for primaquine and tafenoquine in the radical cure of vivax malaria. PLoS Negl Trop Dis.
2018;12(4):e0006440.
6.
Pybus BS, Marcsisin SR, Jin X, Deye G, Sousa JC, Li Q, et al. The metabolism of primaquine to its
active metabolite is dependent on CYP 2D6. Malar J. 2013;12:212.
7.
Bennett JW, Pybus BS, Yadava A, Tosh D, Sousa JC, McCarthy WF, et al. Primaquine failure and
cytochrome P-450 2D6 in Plasmodium vivax malaria. N Engl J Med. 2013;369(14):1381-2.
8.
Grabowsky M. The billion-dollar malaria moment. Nature. 2008;451(7182):1051-2.
9.
Moorthy VS, Newman RD, Okwo-Bele JM. Malaria vaccine technology roadmap. Lancet.
2013;382(9906):1700-1.
10. Mueller I, Shakri AR, Chitnis CE. Development of vaccines for Plasmodium vivax malaria. Vaccine.
2015;33(52):7489-95.
11. Payne RO, Griffin PM, McCarthy JS, Draper SJ. Plasmodium vivax Controlled Human Malaria
Infection - Progress and Prospects. Trends Parasitol. 2017;33(2):141-50.
12. Hou MM, Barrett JR, Themistocleous Y, Rawlinson TA, Diouf A, Martinez FJ, et al. Impact of a blood-
stage vaccine on Plasmodium vivax malaria. medRxiv. 2022.
13. Minassian AM, Themistocleous Y, Silk SE, Barrett JR, Kemp A, Quinkert D, et al. Controlled human
malaria infection with a clone of Plasmodium vivax with high-quality genome assembly. JCI Insight.
2021;6(23).
14. Miller LH, Mason SJ, Clyde DF, McGinniss MH. The resistance factor to Plasmodium vivax in blacks.
The Duffy-blood-group genotype, FyFy. N Engl J Med. 1976;295(6):302-4.
15. Horuk R, Colby TJ, Darbonne WC, Schall TJ, Neote K. The human erythrocyte inflammatory peptide
(chemokine) receptor. Biochemical characterization, solubilization, and development of a binding assay
for the soluble receptor. Biochemistry. 1993;32(22):5733-8.
16. Chitnis CE, Miller LH. Identification of the erythrocyte binding domains of Plasmodium vivax and
Plasmodium knowlesi proteins involved in erythrocyte invasion. J Exp Med. 1994;180(2):497-506.
17. Chitnis CE, Chaudhuri A, Horuk R, Pogo AO, Miller LH. The domain on the Duffy blood group
antigen for binding Plasmodium vivax and P. knowlesi malarial parasites to erythrocytes. J Exp Med.
1996;184(4):1531-6.

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18. King CL, Michon P, Shakri AR, Marcotty A, Stanisic D, Zimmerman PA, et al. Naturally acquired
Duffy-binding protein-specific binding inhibitory antibodies confer protection from blood-stage
Plasmodium vivax infection. Proc Natl Acad Sci U S A. 2008;105(24):8363-8.
19. Keith JA, Agostini Bigger L, Arthur PA, Maes E, Daems R. Delivering the promise of the Decade of
Vaccines: opportunities and challenges in the development of high quality new vaccines. Vaccine.
2013;31 Suppl 2:B184-93.
20. Pronker ES, Weenen TC, Commandeur H, Claassen EH, Osterhaus AD. Risk in vaccine research and
development quantified. PLoS One. 2013;8(3):e57755.
21. Knezevic I, Moorthy V, Sheets R. WHO consultation on clinical evaluation of vaccines, 17-18 July
2014, WHO Headquarters, Geneva, Switzerland. Vaccine. 2015;33(17):1999-2003.
22. Stanisic DI, McCarthy JS, Good MF. Controlled Human Malaria Infection: Applications, Advances,
and Challenges. Infect Immun. 2018;86(1).
23. Robinson S, Harris A, Atkinson S, Atterbury C, Bolton-Maggs P, Elliott C, et al. The administration of
blood components: a British Society for Haematology Guideline. Transfus Med. 2018;28(1):3-21.
24. Singh K, Mukherjee P, Shakri AR, Singh A, Pandey G, Bakshi M, et al. Malaria vaccine candidate
based on Duffy-binding protein elicits strain transcending functional antibodies in a Phase I trial. NPJ
Vaccines. 2018;3:48.
25. McCarthy JS, Griffin PM, Sekuloski S, Bright AT, Rockett R, Looke D, et al. Experimentally induced
blood-stage Plasmodium vivax infection in healthy volunteers. J Infect Dis. 2013;208(10):1688-94.
26. Griffin P, Pasay C, Elliott S, Sekuloski S, Sikulu M, Hugo L, et al. Safety and Reproducibility of a
Clinical Trial System Using Induced Blood Stage Plasmodium vivax Infection and Its Potential as a
Model to Evaluate Malaria Transmission. PLoS Negl Trop Dis. 2016;10(12):e0005139.
27. Collins KA, Wang CY, Adams M, Mitchell H, Robinson GJ, Rampton M, et al. A Plasmodium vivax
experimental human infection model for evaluating efficacy of interventions. J Clin Invest.
2020;130(6):2920-7.
28. Odedra A, Mudie K, Kennedy G, Watts RE, Rossignol E, Mitchell H, et al. Safety and feasibility of
apheresis to harvest and concentrate parasites from subjects with induced blood stage Plasmodium
vivax infection. Malar J. 2021;20(1):43.
29. Bambery B, Selgelid M, Weijer C, Savulescu J, Pollard AJ. Ethical Criteria for Human Challenge
Studies in Infectious Diseases. Public Health Ethics. 2016;9(1):92-103.

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APPENDIX B: SUBMISSION PACKAGE INCLUDES
No.
Submitted Documents
1.
Research Proposal Submission Form for a study involving human subject enrollment
WITH specimen collection (FTM ECF-033-RR)
2.
Informed Consent Form and Participant Information Sheet
3.
Informed Consent Form for Data Sharing/ Data and Leftover Specimen Storage from
Current Study for Future Use
4.
Informed Consent Form for Leftover Specimen Storage from Current Study for future
genetic analysis
5.
HIV Pre-test Counseling Form
6.
Questionnaire to evaluate participant’s understanding of the study
7.
Recruitment materials
8.
Diary card
9.
Screening Form
10.
Case Record Form

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APPENDIX C: SAFETY TERMS AND ADMINISTRATIVE INFORMATION
1. 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 adverse event
(AE), or serious adverse event (SAE) as provided in this protocol. Participants will be instructed to
contact the investigator immediately should the participant manifest any signs or symptoms they
perceive.
1.1 Definitions
1.1.1 Adverse Event (AE):
An AE is any untoward medical occurrences in a participant, 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 C-1 will be considered as
baseline/, pre-existing conditions. This information will be recorded in the medical records.
1.1.2 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 participant 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, 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

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participant and/or require medical or surgical intervention to prevent one of the outcomes
listed above.

Congenital anomaly or birth defect.
All pregnancies occurring in participants within 3 months of challenge will be considered a
SAE.
1.2 Clinical laboratory parameters and other abnormal assessments qualifying as adverse events
or serious adverse events:
In absence of a 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 an 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 participant will be informed and
appropriate medical care arranged as appropriate with the permission of the participant.
Decisions to exclude the participant from enrolling in the trial or to withdraw a participant
from the trial will be at the discretion of the Investigator.
The study will cover all expense of the treatment for SAE.
1.2.1 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 C1). 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.

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Table C1: 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.
1
Unlikely to be related
Unlikely temporal relationship to study intervention and
Alternate aetiology 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.
1.2.2 Assessment of AE 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

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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. All AEs that result in a participant’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 participant consents to this). In cases where the participant has to do AE self-
detection and self- assessment, a simple grading system for the self-assessment on the diary
card will be used (Table C2).
Table C2: Severity grading criteria for the self-assessment for AEs.
GRADE 0
None
GRADE 1
Noticeable, but no impact on activities
GRADE 2
Noticeable and alters, but does not prevent daily activities
GRADE 3
Prevents normal daily activities, and/or requires visits with a medical
professional
GRADE 4
Precipitates ER visit or hospitalization (these are, of course, also SAEs; Serious
Adverse Effects).
1.2.3 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.

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While the participant is followed as an outpatient, a diary card will be provided, and the
participant 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.
For all AEs , appropriate follow-up visits or medical care will be arranged, with the agreement
of the volunteer, until the AE has resolved, stabilized, or a non-trial related causality has been
assigned.
1.3 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 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.
1.4 Events or outcomes not qualifying as adverse events or serious adverse events
Pregnancy
Participants are informed that for the safety of participant and their child, pregnancy and
breastfeeding are prohibited until 3 months after the challenge. Female study participants are
asked to use appropriate contraceptive methods to prevent pregnancy while they participate in
the studyfrom the first vaccination until 3 months after the injection of malaria infected
erythrocytes.
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)

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
True abstinence, when this is in line with the preferred and usual lifestyle of the participant.
Periodic abstinence (e.g., calendar, ovulation, symptothermal, post-ovulation methods) and
withdrawal are not acceptable methods of contraception.
Female participants will be tested for pregnancy at screening, prior to receive each vaccine
dose, prior to challenge, prior to receive antimalarial drug, and during follow up as indicated in
the schedule of procedures (Tables 4 and 5).
Should a participant become pregnant during vaccination she will be winthdrawn from the
study, if she becomes pregnant after challenge, she will be treated with antimalarials
immediately and will be withdrawn from the study. We will not routinely perform
venepuncture on such participants, other than blood films to check that any parasitaemia has
been cleared by the antimalarial treatment. With the participant’s permission she shall be
followed up until pregnancy outcome. The management of any participant 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 participant 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 participant will still be followed up until 3 months after
challenge and pregnancy outcome will be followed up. For male participant whose partner
become pregnant during the study, the partner will be followed up pregnancy outcome.
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).

Any congenital anomaly or birth defect identified in the offspring of a study participant
(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, all pregnancies 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

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investigator is not obligated to actively seek this information from former study participants,
he/she may learn of a pregnancy through spontaneous reporting.
Male study participants are asked to use condom during sexual intercourse or abstinent to
prevent pregnancy in their partners while they participate in the study from the first vaccination
until 3 months after the injection of malaria infected erythrocytes.
All pregnancies occurring during the study will be reported to the DSMB.
1.5 Safety monitoring
Monitoring
Monitoring will be performed using an 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 monitoring and auditing by the Sponsor and
inspection by local and regulatory authorities.
2. RANDOMIZATION
This will be a randomized double-blinded controlled design. Volunteers who fulfil the inclusion
criteria and have none of the exclusion criteria will be randomised in a 1:1 ratio to either the study
vaccine group or the sham vaccine control group in accordance with the randomisation schedule.
Randomisation will be in blocks of 8. Allocation will be done by drawing the next sequential
numbered opaque envelope, which contains the study number and treatment allocation. The
randomization list and numbered envelopes will be prepared by members of the MORU CTSG
statistical staff.
Only the CTSG statistical staff and the study pharmacist will know the study group allocation of
each participant. The study pharmacist will prepare the syringes containing either the vaccine or
0.9% saline. These individuals will not be involved in providing participant care, conducting study
investigations, or performing laboratory tests necessary for determining the study endpoint. They
will not be involved in any other study assessment or evaluation.
3. QUALITY CONTROL AND QUALITY ASSURANCE PROCEDURES
Data will be evaluated for compliance with the protocol and accuracy in relation to source
documents. Following written standard operating procedures and prespecified monitoring plan, the
monitors will verify that the clinical study is conducted and data are generated, documented and

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reported in compliance with the current approved protocol, standard operating procedures, ICH
GCP and relevant regulations.
The Sponsor and authorised individuals may carry out audit to ensure compliance with the
protocol, standard operating procedures, ICH GCP and relevant regulations. The study audits will
be managed by an independent function according to standard operating procedures a prespecified
audit plan.
GCP inspections may also be undertaken by the regulatory authority or ethical committee(s) to
ensure compliance with protocol and national regulations. The sponsor will assist in any
inspections.
4. 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, DSMB, and the 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 E6 (R2).
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 Co-
Principal Investigator (Co-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) in accordance with
procedures.
The Co-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.
5. SPONSOR: UNIVERSITY OF OXFORD
Contact information: University Offices, Wellington Square, Oxford, OX1 2JD, United Kingdom
The study sponsor has a role in the design of the study; collection of data; management, analysis,
and interpretation of data; writing of the report; and the decision to submit the report for
publication.

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6. FUNDER
The funder, the Welcome Trust, had no role in the design of the study. The funder will not have
any role in its execution, analyses, and interpretation of data or decision to submit results.
7. TRIAL COMMITTEES
Programme Steering Committee (PSC): The role of PSC is to provide overall supervision for the
project on behalf of the Project Sponsor and Project Funder and to ensure that the project is
conducted to the rigorous standards set out in the Research Governance Framework for Health and
Social Care, and in the Guidelines for Good Clinical Practice. The composition and roles and
responsibilities of the PSC, and the PSC meeting schedule, are defined in the PSC Terms of
Reference.
8. INSURANCE
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 participant
suffering harm as a result of their involvement in the research.
9. 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.
The trial results for each participant will be communicated individually to that individual to inform
his/her health history.
10. IMMUNOLOGICAL ASSAYS (BLOOD VOLUME TABLE)
10.1 Immunological assays (humoral & cellular responses)
Time point
Sample
Amount
Test
Vaccination
D0
Serum
10ml
Binding inhibitory titers, Antibody titers, IgG purification
for growth inhibition assays (control for all assays –
therefore large vol required)
Plasma
2ml
Cytokines/chemokine by Luminex (control for all assays)
WB Tempus
2ml
Gene expression by NanoString (control)
WB Cyto
2ml
Immune cell populations by flow cytometry (control)
Vaccination
D3
Plasma
2ml
Cytokines/chemokine by Luminex
WB Tempus
2ml
Gene expression by NanoString

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Time point
Sample
Amount
Test
WB Cyto
2ml
Immune cell populations by flow cytometry
Vaccination
D7
Plasma
2ml
Cytokines/chemokine by Luminex
WB Tempus
2ml
Gene expression by NanoString
WB Cyto
2ml
Immune cell populations by flow cytometry
Vaccination
D14
Serum
2ml
Antibody titers
Vaccination
D35
Plasma
2ml
Cytokines/chemokine by Luminex
WB Tempus
2ml
Gene expression by NanoString
WB Cyto
2ml
Immune cell populations by flow cytometry
Vaccination
D42
Serum
5ml
Antibody titers, Antibody affinity
Vaccination
D210
Serum
2ml
Antibody titers, Antibody affinity
Plasma
2ml
Cytokines/chemokine by Luminex
WB Tempus
2ml
Gene expression by NanoString
WB Cyto
2ml
Immune cell populations by flow cytometry
Vaccination
D217
Plasma
2ml
Cytokines/chemokine by Luminex
WB Tempus
2ml
Gene expression by NanoString
WB Cyto
2ml
Immune cell populations by flow cytometry
C-1
Serum
10ml
Binding inhibitory titers, Antibody titers, isotyping,
avidity, antibody affinity, C1q and Fc receptor binding
(including
FcRI,
FcRIIA,
FcRIIB,
FcRIIIA,
FcRIIIB, Fcand ), cellular phagocytosis (antibody
dependent cellular phagocytosis, ADCP), neutrophil burst
(antibody
dependent
neutrophil
burst,
ADNB),
complement deposition (ADCD) and NK cell activation
(ADNKA)
WB for PBMCs
for mAbs
30ml
Monoclonal antibody development
C+14
Serum
2ml
Antibody titers
C+21
Plasma
2ml
Cytokines/chemokine by Luminex
WB Tempus
2ml
Gene expression by NanoString
WB Cyto
2ml
Immune cell populations by flow cytometry
C+28
Serum
2ml
Antibody titers
C+56
Serum
2ml
Binding inhibitory titers, Antibody titers
C+96
Serum
2ml
Binding inhibitory titers, Antibody titers
10.2 Immunological functional assays (ELISA&GIA)
Time point
Sample
Amount
Test
Vaccination
D0
Clot
blood
4ml
Humoral and B cell responses to the P. vivax Duffy-binding
protein region II (PvDBPII) by ELISA

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Time point
Sample
Amount
Test
Clot
blood
6ml
Humoral and B cell responses to the P. vivax Duffy-binding
protein region II (PvDBPII) by ELISA
Vaccination
D14
Clot
blood
4ml
Humoral and B cell responses to the P. vivax Duffy-binding
protein region II (PvDBPII) by ELISA
Vaccination
D28
Clot
blood
4ml
Humoral and B cell responses to the P. vivax Duffy-binding
protein region II (PvDBPII) by ELISA
Vaccination
D35
Clot
blood
4ml
Humoral and B cell responses to the P. vivax Duffy-binding
protein region II (PvDBPII) by ELISA
Vaccination
D42
Clot
blood
4ml
Humoral and B cell responses to the P. vivax Duffy-binding
protein region II (PvDBPII) by ELISA
Vaccination
D56
Clot
blood
4ml
Humoral and B cell responses to the P. vivax Duffy-binding
protein region II (PvDBPII) by ELISA
Vaccination
D210
Clot
blood
4ml
Humoral and B cell responses to the P. vivax Duffy-binding
protein region II (PvDBPII) by ELISA
Vaccination
D217
Clot
blood
4ml
Humoral and B cell responses to the P. vivax Duffy-binding
protein region II (PvDBPII) by ELISA
Vaccination
D224
Clot
blood
4ml
Humoral and B cell responses to the P. vivax Duffy-binding
protein region II (PvDBPII) by ELISA
Clot
blood
6ml
Humoral and B cell responses to the P. vivax Duffy-binding
protein region II (PvDBPII) by ELISA
C-1
Clot
blood
4ml
Humoral and B cell responses to the P. vivax Duffy-binding
protein region II (PvDBPII) by ELISA
Clot
blood
6ml
Humoral and B cell responses to the P. vivax Duffy-binding
protein region II (PvDBPII) by ELISA
C7
Clot
blood
4ml
Humoral and B cell responses to the P. vivax Duffy-binding
protein region II (PvDBPII) by ELISA
C14
Clot
blood
4ml
Humoral and B cell responses to the P. vivax Duffy-binding
protein region II (PvDBPII) by ELISA
D reach
treatment
Clot
blood
4ml
Humoral and B cell responses to the P. vivax Duffy-binding
protein region II (PvDBPII) by ELISA
C56
Clot
blood
4ml
Humoral and B cell responses to the P. vivax Duffy-binding
protein region II (PvDBPII) by ELISA
C96
Clot
blood
4ml
Humoral and B cell responses to the P. vivax Duffy-binding
protein region II (PvDBPII) by ELISA
C276
Clot
blood
4ml
Humoral and B cell responses to the P. vivax Duffy-binding
protein region II (PvDBPII) by ELISA

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10.3 Immune cells & cytokines profile (phenotypic profile)
Time point
Sample
Amount
Test
D0
EDTA blood
2ml
Host response to the blood stage infection by phenotypic
profile of the immune cells and cytokines profile
D7
EDTA blood
2ml
Host response to the blood stage infection by phenotypic
profile of the immune cells and cytokines profile
D14
EDTA blood
2ml
Host response to the blood stage infection by phenotypic
profile of the immune cells and cytokines profile
D28
EDTA blood
2ml
Host response to the blood stage infection by phenotypic
profile of the immune cells and cytokines profile
D35
EDTA blood
2ml
Host response to the blood stage infection by phenotypic
profile of the immune cells and cytokines profile
D42
EDTA blood
2ml
Host response to the blood stage infection by phenotypic
profile of the immune cells and cytokines profile
D210
EDTA blood
2ml
Host response to the blood stage infection by phenotypic
profile of the immune cells and cytokines profile
D217
EDTA blood
2ml
Host response to the blood stage infection by phenotypic
profile of the immune cells and cytokines profile
D224
EDTA blood
2ml
Host response to the blood stage infection by phenotypic
profile of the immune cells and cytokines profile
C-1
EDTA blood
2ml
Host response to the blood stage infection by phenotypic
profile of the immune cells and cytokines profile
C21
EDTA blood
2ml
Host response to the blood stage infection by phenotypic
profile of the immune cells and cytokines profile

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APPENDIX D: LIST OF ABBREVIATIONS
AE
adverse event
β-HCG 
beta–Human Chorionic Gonadotropin
BP
blood pressure
BT
body temperature
BUN
blood urea nitrogen
CBC
complete blood count
CBF
Clinical Bio-Manufacturing Facility
CCVTM
Centre for Clinical Vaccinology and Tropical Medicine
CHIK
Chikungunya
CHMI
controlled human malaria infection
CI
Chief Investigator
CMV
Cytomegalovirus
Cr
Creatinine
CRF
case record form
CSP
Circumsporozoite
CTCAE
Common Terminology Criteria for Adverse Events
CTSG
Clinical Trials Supporting Group
CYP2D6
enzyme predicting Primaquine metabolism (Cytochrome p450 2D6)
DARC
Duffy antigen receptor for chemokines
DBP
Duffy binding protein
DEN
Dengue

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DSMB
Data Safety Monitoring Board
EBV
Epstein-Barr virus
ELISA
Enzyme-linked Immunosorbent Assay
ECG
Electrocardiogram
ELISPOT
Enzyme linked immunospot assay
EPI
Expanded Program of Immunization
FBS
fasting blood sugar
FDA
United States Food and Drug Administration
FTM
Faculty of Tropical Medicine
FTMCTU
Clinical Therapeutics Unit (healthy volunteer ward), Faculty of
Tropical Medicine
FTMEC
Ethics Committee, Faculty of Tropical Medicine
G6PD
glucose-6-phosphate dehydrogenase
GCP
Good Clinical Practice
GMP
Good Manufacturing Practice
HBsAg
Hepatitis B surface antigen
HBV
Hepatitis B virus
HCV
Hepatitis C virus
HIV
Human Immunodeficiency virus
HLA
Human Leukocyte antigen
HTLV
Human T cell Lymphotrophic virus
ICF
Informed Consent Form

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ICH
International Conference on Harmonisation
ICMJE
International Committee of Medical Journal Editors
IFN-α 
Interferon-gramma
IM
Intramuscular
IMP
Investigational Medicinal Product
JE
Japanese Encephalitis
KEMRI
Kenya Medical Research Institute
LFT
liver function test
LPS
Lipopolysaccharide
MFA
Membrane Feeding Assay
MORU
Mahidol Oxford Tropical Medicine Research Unit
MVRU
Mahidol Vivax Research Unit
MSP
Merozoite surface proteins
µg
Microgram
OxTREC
Oxford Tropical Research Ethics Committee
PCR
polymerase chain reaction
P. falciparum
Plasmodium falciparum
PI
Principal Investigator
PIS
Participant Information Sheet
PMR
parasite multiplication rate
PQ
Primaquine
PR
pulse rate

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P. vivax
Plasmodium vivax
PvAMA1
P. vivax apical merozoite antigen 1
PvCSP
P. vivax circumsporozoite
PvDBPII
receptor-binding region II of P. vivax Duffy binding protein
PvRBP
P. vivax reticulocyte binding protein
PvTRAP
P. vivax thrombospondin related adhesive protein
QIMR
Queensland Institute of Medical Research
qPCR
quantitative polymerase chain reaction
RBC
red blood cell
REC
Research Ethics Committee
RR
respiratory rate
RUNMC
Radboud University Nijmegen Medical Centre
SAE
serious adverse event
SAP
Statistical Analysis Plan
SAR
Serious Adverse Reaction
SOP
Standard Operating Procedure
TMDR
Tropical Medicine Diagnostic Reference Laboratory
USMMVP
U.S. Military Malaria Vaccine Program
VLP
Virus-like particle
WHO
World Health Organization
WI
Work Instruction
ZIK
Zika virus

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APPENDIX E: AMENDMENT HISTORY
Amendment
No.
Proposal
Version No.
Date issued
Author(s) of
changes
Details of Changes made
NA
NA
NA
NA
NA