VAC069: A Clinical Study to Assess the Safety and Feasibility of Controlled Blood-stage Plasmodium Vivax Human Malaria Infection Through Experimental Inoculation of Cryopreserved Infected Erythrocytes in Healthy Malaria-naïve UK Adults
Phase: EARLY_PHASE1
Conditions: Malaria, Vivax
Sponsor: University of Oxford

VAC069: A study of blood-stage controlled human Plasmodium vivax malaria infection. IRAS 
project ID: 252499 
VAC069 Clinical Trial Protocol, V8.0, 23rd June 2022, University of Oxford.  
Page 1 of 122  
 
 
 
 
 
 
 
VAC069: A clinical study to assess the safety and feasibility of controlled blood-stage 
Plasmodium vivax human malaria infection through experimental inoculation of cryopreserved 
infected erythrocytes in healthy malaria-naïve UK adults 
 
 
Study Reference: VAC069 
Protocol Version: 8.0 
Date: 23rd June 2022 
 
 
Chief Investigator: Angela M. Minassian 
Sponsor: University of Oxford 
 
 
 
 
 
 

VAC069: A study of blood-stage controlled human Plasmodium vivax malaria infection. IRAS 
project ID: 252499 
VAC069 Clinical Trial Protocol, V8.0, 23rd June 2022, University of Oxford.  
Page 2 of 122  
 
 
MODIFICATION HISTORY 
Version Date 
Author(s) 
Modifications 
1.0 
19 September  
2018 
Yrene 
Themistocleous 
Simon Draper 
Angela Minassian 
Created 
1.1 
10th December 
2018 
Yrene 
Themistocleous 
Minor clarification regarding procedure for 
preparation of inoculum , referencing the 
standard operating procedure 
2.0 
12th February 
2019 
Yrene 
Themistocleous 
Thomas Rawlinson 
Angela Minassian 
Substantial amendment. 
Number of volunteers increased from 6 to 25 
Number of challenges increased from 3 to 5 
Amendments to the schedule of clinic visits and 
blood tests 
Inclusion of mosquito transmission studies 
3.0 
10th September 
2019 
Susanne Hodgson 
Yrene 
Themistocleous 
Substantial amendment.  
Addition of testing for blood borne viruses at 
C+90 visits 
4.0 
23rd October 
2019 
Susanne Hodgson 
Angela Minassian 
Substantial amendment 
Removal of microscopy from diagnostic criteria 
for VAC069C-E and from study endpoints 
Updates to schedule of visits and bleeds, 
simplification of bleed tables and reduction in 
total blood draw volume for VAC069C-E 
Change in exclusion criteria regarding prior use 
of immunoglobulin & GP response 
Removal of AEs of special interest section 
Modification to collection of adverse event data 
Modification to timing of observations on day of 
challenge procedures 
Addition of potential for pre-screening via 
telephone 
Potential for use of alternative antiemetic to 
cyclizine.  

VAC069: A study of blood-stage controlled human Plasmodium vivax malaria infection. IRAS 
project ID: 252499 
VAC069 Clinical Trial Protocol, V8.0, 23rd June 2022, University of Oxford.  
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Ranges added for Group sizes  
Local safety monitor takes on role previously 
taken by local safety committee  
Consent form only required to be completed 
once at screening (and not routinely again prior 
to each challenge) 
Update of bleed tables to show that 4ml rather 
than 5mls was collected for HLA and 
haemoglobinopathy testing for VAC069A&B.  
5.0 
19th August 
2020 
Yrene 
Themistocleous 
Mimi Hou 
Angela Minassian 
Corrections to blood draw volumes for 
VAC069C-F  
Addition of VAC069F and additional secondary 
and tertiary challenges in phases VAC069E and 
VAC069F 
Adjustment of study group sizes for VAC069C-F 
Extension of time window between primary and 
subsequent challenges 
Extension of time window for follow-up study 
visits 
Changes to trial procedures to account for 
possibility of COVID-19 infection during CHMI. 
Addition of COVID-19 PCR swab test prior to 
challenge and on day of malaria diagnosis. 
Guidance on testing for COVID-19 if fever post 
challenge. 
Added option of using Malarone as first line 
anti-malaria treatment 
Correction of typographical errors. Addition of 
relevant references. Clarification of study 
procedures in text: G6PD, DARC & 
haemoglobinopathy screen are done by NHS 
labs only; added description of T+3 and T+9 
visits within text; added reasoning for selecting 
1:10 dose of blood inoculum for all subsequent 
malaria challenges after VAC069A. 
6.0 
4th March 2021 
Mimi Hou 
Jee-Sun Cho 
Addition of Wellcome Trust as Funder of 
VAC069C. 
Addition of exclusion criteria on concomitant 
COVID-19 vaccination around time of CHMI 

VAC069: A study of blood-stage controlled human Plasmodium vivax malaria infection. IRAS 
project ID: 252499 
VAC069 Clinical Trial Protocol, V8.0, 23rd June 2022, University of Oxford.  
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Updated section on Conduct of CHMI in the 
context of COVID-19 pandemic. 
Correction of total blood volumes in VAC069C-F 
schedule of attendance tables. 
Addition of DARC to schedule of attendance 
table. 
Change of C-1 visit to C-2. 
Increased number of back-up volunteers that 
can be recruited into each group. 
Correction in section 6.4 that there is no specific 
timeframe that observations are taken pre-
challenge. 
7.0 
28th July 2021 
Mimi Hou 
For phase VAC069D onwards:  
Post-challenge follow-up visits changed to once 
a day until parasite count reaches >1000 
genome copies/ml, then to continue twice a day 
visits until diagnosis. Latest day of treatment 
reduced to C+21. 
Removal of T+2 post malaria treatment visit. 
Reduction in number of observed doses of 
antimalarial medication to two observed doses. 
Updated compensation table 
Addition of reticulocyte count to FBC taken at C-
2 visit. 
Removal of need to collect unused medications 
after completion of challenge. Dose of 
lumefantrine corrected in section 6.4.4 
Clarification that only weight is re-measured at 
C-2 visit. 
Changed Senior Laboratory Investigator 
address. Addition of New Biochemistry Building 
as location for processing of research bloods. 
8.0 
23rd June 2022 
Mimi Hou 
Angela Minassian 
Phase VAC069E changed to CHMI with P. 
falciparum instead of P. vivax. Addition to 
section on study rationale, details on P. 
falciparum inoculum to be used, secondary 
objective, immunological blood tests and study 
visits for VAC069E.  

VAC069: A study of blood-stage controlled human Plasmodium vivax malaria infection. IRAS 
project ID: 252499 
VAC069 Clinical Trial Protocol, V8.0, 23rd June 2022, University of Oxford.  
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Clarification of immunological tests on blood 
samples and assays to be used to include both 
vivax and falciparum CHMIs. Cell phenotyping 
by mass cytometry added to immunological 
tests.  
Addition of red cell alloantibody testing pre and 
post CHMI on either stored serum or from 
whole blood. 
Removal of phase VAC069F 
Increased time window for re-screening visit for 
VAC069E. 
Change to exclusion criteria on day of CHMI – 
Covid infection now an exclusion criteria if 
within 7 days.  
Covid testing pre-CHMI can now be performed 
by lateral flow test or PCR test. Requirement for 
volunteers to undergo Covid test on day of 
malaria diagnosis removed. 
Samples will now be stored long term under 
University of Oxford’s HTA license at 
Department of Biochemistry instead of Oxford 
Vaccine Centre Biobank. 
Organisation name changes: sponsor name 
change from Clinical Trials and Research 
Governance to Research Governance Ethics and 
Assurance; Public Health England changed to UK 
Health Security Agency 
 
 
 

VAC069: A study of blood-stage controlled human Plasmodium vivax malaria infection. IRAS 
project ID: 252499 
VAC069 Clinical Trial Protocol, V8.0, 23rd June 2022, University of Oxford.  
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A clinical study to assess the safety and feasibility of controlled blood-stage Plasmodium vivax 
human malaria infection through experimental inoculation of cryopreserved infected 
erythrocytes in healthy malaria-naïve UK adults 
Study Code:  VAC069 
 
Chief Investigator 
Dr Angela M Minassian 
 
 
 
 
 
 
Principal Investigator 
 
 
 
 
 
 
Trial sites 
 
 
 
 
 
 
 
 
Centre for Clinical Vaccinology and Tropical Medicine, 
Jenner Institute, University of Oxford,  
Churchill Hospital, Old Road, Headington, 
Oxford, OX3 7LE 
Email: angela.minassian@ndm.ox.ac.uk 
 
Prof Simon J Draper  
New Biochemistry Building, 
Department of Biochemistry, 
South Parks Road 
Oxford, OX1 3QU 
Email: simon.draper@bioch.ox.ac.uk 
 
Jenner Institute, University of Oxford 
Comprising:  
Centre for Clinical Vaccinology & Tropical Medicine 
Churchill Hospital, Old Road, Headington,  
Oxford, OX3 7LE 
And 
Old Road Campus Research Building, 
Roosevelt Drive, Headington,  
Oxford, OX3 7DQ 
PI at Site: Dr Angela M Minassian 
 
Sponsoring Institution 
University of Oxford 

VAC069: A study of blood-stage controlled human Plasmodium vivax malaria infection. IRAS 
project ID: 252499 
VAC069 Clinical Trial Protocol, V8.0, 23rd June 2022, University of Oxford.  
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Research Governance Ethics and Assurance,  
Research Services 
University of Oxford 
Joint Research Office 
Boundary Brook House 
Churchill Drive 
Headington 
OX3 7GB 
Tel: 01865 616480  
Email: ctrg@admin.ox.ac.uk 
 
External Monitor  
 
 
 
 
 
Research Governance, Ethics and Assurance (RGEA) 
Research Services 
University of Oxford 
Joint Research Office 
Boundary Brook House 
Churchill Drive 
Headington 
OX3 7GB 
Tel: 01865 616480 
 
 
 
Local Safety Monitor  
 
Prof Brian Angus 
Centre for Clinical Vaccinology and Tropical Medicine, 
Churchill Hospital, Old Road, Headington, 
Oxford, OX3 7LE 
Tel: 01865 553 20289 
Email: brian.angus@ndm.ox.ac.uk 
 
 
 
 

VAC069: A study of blood-stage controlled human Plasmodium vivax malaria infection. IRAS 
project ID: 252499 
VAC069 Clinical Trial Protocol, V8.0, 23rd June 2022, University of Oxford.  
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Confidentiality Statement 
This document contains confidential information that must not be disclosed to anyone other 
than the Sponsor, the Investigator Team, and members of the Independent Ethics Committee.  
This information cannot be used for any purpose other than the evaluation or conduct of the 
clinical investigation without the prior written consent of Dr Angela M. Minassian. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

VAC069: A study of blood-stage controlled human Plasmodium vivax malaria infection. IRAS 
project ID: 252499 
VAC069 Clinical Trial Protocol, V8.0, 23rd June 2022, University of Oxford.  
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Investigator Agreement 
“I have read this protocol and agree to abide by all provisions set forth therein. 
I agree to comply with the principles of the International Conference on Harmonisation Tripartite 
Guideline on Good Clinical Practice.”   
Dr Angela M. Minassian 
 
 
 
23/06/2022 
Chief Investigator 
 
 
Investigator Signature 
 
Date  
 
Conflict of Interest 
1. “According to the Declaration of Helsinki, 2008, I have read this protocol, and declare 
no/the following (delete as appropriate) conflict of interest” 
Dr Angela M. Minassian 
 
 
 
23/06/2022 
Chief Investigator 
 
 
Investigator Signature 
 
Date  
 
Details:_________________________________________________________________________ 
 
2.  “According to the Declaration of Helsinki, 2008, I have read this protocol, and declare 
no/the following (delete as appropriate) conflict of interest” 
Prof Simon J. Draper 
 
 
 
23/06/2022 
Principal Investigator 
 
 
Investigator Signature 
 
 
Date  
 
Details:________________________________________________________________________ 
 
 
 
 

VAC069: A study of blood-stage controlled human Plasmodium vivax malaria infection. IRAS 
project ID: 252499 
VAC069 Clinical Trial Protocol, V8.0, 23rd June 2022, University of Oxford.  
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TABLE OF CONTENTS 
Modification History .......................................................................................................................... 2 
1 Synopsis ........................................................................................................................................ 15 
2 Abbreviations ............................................................................................................................... 17 
Background & Rationale .................................................................................................................. 19 
3.1 
Impact of P. vivax malaria and the need for a vaccine ................................................... 19 
3.2 
Lifecycle of the Plasmodium vivax malaria parasite ....................................................... 20 
3.3. 
 Challenges and directions in P. vivax vaccine development .......................................... 21 
3.4 
Rationale for the development of a blood-stage challenge model ................................ 22 
3.5 
Study rationale ................................................................................................................ 25 
3.6 
Rationale for re-challenge with P. falciparum ................................................................ 25 
4 Objectives ..................................................................................................................................... 27 
5 Controlled human malaria infection studies ................................................................................ 28 
5.1 
Microbial challenge studies of human volunteers .......................................................... 28 
5.2  
Controlled human malaria infection with Plasmodium falciparum ................................ 28 
5.3  
Controlled human malaria infection with Plasmodium vivax ......................................... 28 
5.4 
P. vivax sporozoite challenge in the modern era ............................................................ 29 
5.5 
Modern blood-stage controlled human P. vivax infection ............................................. 33 
5.6 
 Re-challenge in controlled human malaria infection ..................................................... 34 
5.6.1 
Safety of re-challenge in controlled human malaria infection ................................... 34 
5.7 
Source and preparation of the P. vivax cryopreserved inoculum ................................... 35 
5.7.1 
Collection of P. vivax infected erythrocytes ................................................................ 35 
5.7.2 
Cryopreservation and storage of blood bank ............................................................. 36 
5.7.3 
Infectivity and characterisation of parasite stock ....................................................... 36 
5.7.4 
Testing of source patient and blood donors for blood-borne and mosquito-borne 
infections .................................................................................................................................. 37 
5.7.5 
Sterility and screening for blood-borne infections of cryopreserved blood bank ...... 38 
5.8 
Source and preparation of the P. falciparum cryopreserved inoculum ......................... 38 
5.8.1 
Collection and cryopreservation of inoculum ............................................................. 38 
5.8.2 Previous Studies .............................................................................................................. 39 
5.9 
Conduct of CHMI trials .................................................................................................... 40 
5.9.1 
Clinical presentation post-CHMI ................................................................................. 41 
5.9.2 
Ethical considerations of CHMI trials .......................................................................... 42 

VAC069: A study of blood-stage controlled human Plasmodium vivax malaria infection. IRAS 
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5.9.3 
Oxford’s experience conducting CHMI trials............................................................... 42 
5.9.4 
Conducting CHMI in the context of the COVID-19 pandemic ..................................... 43 
6 Description and justification of study design ............................................................................... 45 
6.1 
Overview .......................................................................................................................... 45 
6.2 
Study groups .................................................................................................................... 45 
6.2.1 VAC069A Initial challenge and dose-finding: Groups 1 (n=2), 2 (n=2) and 3 (n=2). ....... 46 
6.2.2 VAC069B Secondary re-challenge with new primary controls: Groups 4 (n=6) and 6 
(n=2-3). ..................................................................................................................................... 47 
6.2.3 VAC069C Tertiary (n=6) and secondary (n=2) re-challenges with new primary challenge 
controls (n=4-8): Groups 5, 7 and 9. ........................................................................................ 47 
6.2.4 VAC069D Tertiary (n=2) and secondary (n=4-8) re-challenges with new primary 
challenge controls (n=4-8): Groups 8, 10 and 12. .................................................................... 48 
6.2.5 VAC069E Re-challenge with P. falciparum: Group 13. ................................................... 48 
6.3 
Duration of volunteer participation ................................................................................ 50 
6.3.1 
Definition of the start and end of the trial ...................................................................... 50 
6.4 
Potential risks for volunteers .......................................................................................... 50 
6.4.1 
Phlebotomy ................................................................................................................. 50 
6.4.2 
Risk of Infection with Blood Borne Organisms ............................................................ 51 
6.4.3 
Plasmodium vivax and falciparum infection ............................................................... 53 
6.4.4 
Medications dispensed to volunteers in course of trial .............................................. 53 
6.4.5 
Risk of reaction to the blood sample .......................................................................... 54 
6.4.6 Mosquito bites (for volunteers partaking in direct feeding assays only) ....................... 54 
6.5 
Potential benefits for volunteers .................................................................................... 55 
7 Recruitment and withdrawal for trial volunteers ........................................................................ 56 
7.1 
Informed consent ............................................................................................................ 56 
7.1.1 
Informed consent ........................................................................................................ 56 
7.2  
Inclusion and exclusion criteria ....................................................................................... 57 
7.2.1 
Inclusion criteria .......................................................................................................... 57 
7.2.2 
Exclusion criteria ......................................................................................................... 58 
7.2.3 
Prevention of ‘Over Volunteering’ .............................................................................. 59 
7.2.4 
Exclusion criteria on day of CHMI ............................................................................... 59 
7.3 
Withdrawal of volunteers ............................................................................................... 60 
7.4 
Pregnancy ........................................................................................................................ 60 
8 Controlled Blood-Stage Malaria Infection Inoculum .................................................................... 62 

VAC069: A study of blood-stage controlled human Plasmodium vivax malaria infection. IRAS 
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8.1 
Preparation of the inoculum ........................................................................................... 62 
8.2 
Administration of the inoculum ...................................................................................... 62 
9 Treatment of trial volunteers ....................................................................................................... 63 
9.1 
Trial sites .......................................................................................................................... 63 
9.2 
Study procedures ............................................................................................................ 63 
9.2.1 
Observations................................................................................................................ 63 
9.2.2 
Blood tests ................................................................................................................... 63 
9.2.3 
 Urinalysis .................................................................................................................... 64 
9.2.4 
 Electrocardiogram ...................................................................................................... 65 
9.2.5 
SARS-COV-2 testing ..................................................................................................... 65 
9.3 
Clinical reviews ................................................................................................................ 65 
9.3.1 
Screening visits ............................................................................................................ 65 
9.3.2 
Re-screening visits for participants not yet enrolled in the study .............................. 66 
9.3.3 
Re-screening visits prior to 2nd and 3rd Challenges ..................................................... 66 
9.3.3 
Two days before CHMI (C-2) ....................................................................................... 66 
9.3.4 
Day of CHMI (C0) ......................................................................................................... 66 
9.3.5 
Days 1-28 post-CHMI (C+1 – C+28) ............................................................................. 67 
9.3.6 Direct feeding of mosquitos ............................................................................................ 69 
9.4 
Algorithm for initiation of treatment .............................................................................. 69 
9.4.1 Algorithm for initiation of treatment in Groups VAC069A&B ........................................ 69 
9.4.2 Algorithm for initiation of treatment in Groups VAC069C-E .......................................... 72 
9.4.3 Participants with fever post malaria challenge and COVID-19 testing ........................... 75 
9.5 
Malaria management ...................................................................................................... 76 
9.5.1 
Malaria management – Riamet ................................................................................... 76 
9.5.1 
Malaria management – Malarone .............................................................................. 76 
9.5.2 
Malaria management – supportive medications ........................................................ 76 
9.5.3 
Follow-up after commencing malaria treatment ........................................................ 77 
9.5.3 
Criteria for inpatient transfer to the NHS ................................................................... 77 
9.6 
Safety measures for conduct of CHMI ............................................................................ 77 
9.6.1 
Measures to be taken if a volunteer goes missing post-CHMI ................................... 78 
9.7 
Follow-up post-treatment ............................................................................................... 79 
9.7.1 
Six days after initiation of treatment (T+6) ................................................................. 79 
9.7.2 
Nine days after initiation of treatment (for VAC069C-D) (T+9 (+2)) ........................... 79 

VAC069: A study of blood-stage controlled human Plasmodium vivax malaria infection. IRAS 
project ID: 252499 
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9.7.3 
Ten days after initiation of treatment (for VAC069E) (T+10 (±2)) .............................. 79 
9.7.5 Days 56 and 96 post CHMI (for VAC069B-E) (C+56 (±14) and C+96 (±14)) .................... 79 
9.8 
Secondary and tertiary blood-stage CHMI ...................................................................... 79 
10 Assessment of scientific objectives .......................................................................................... 104 
10.1 
Primary endpoints ......................................................................................................... 104 
10.2 
Secondary endpoints ..................................................................................................... 104 
11 Assessment of safety ................................................................................................................ 105 
11.1 
Definitions ..................................................................................................................... 105 
11.2 
Causality assessment ..................................................................................................... 106 
11.3 
Reporting procedures for all AEs (see SOP VC027) ....................................................... 107 
11.3.1 
Severity grading for laboratory AEs ...................................................................... 108 
11.3.2 
Reporting procedures for serious AEs ................................................................... 108 
11.3.4 
Reporting procedures for SUSARs ......................................................................... 108 
11.3.5 
Procedures to be followed in the event of abnormal findings ............................. 108 
11.4 
Foreseeable medical occurrences ................................................................................. 108 
11.5 
Safety profile review of CHMI and antimalarial treatment .......................................... 109 
11.6 
Local safety monitor ...................................................................................................... 109 
12 Statistics .................................................................................................................................... 110 
12.1  
Sample size .................................................................................................................... 110 
13 Quality control and quality assurance procedures .................................................................. 111 
13.1  
Investigator procedures ................................................................................................ 111 
13.2 
Monitoring ..................................................................................................................... 111 
13.3 
Modification to protocol ............................................................................................... 111 
13.4 
Protocol deviation ......................................................................................................... 111 
13.5 
Audit & inspection ......................................................................................................... 111 
13.6 
Serious breaches ........................................................................................................... 111 
13.7 
Trial progress ................................................................................................................. 112 
13.8 
Publication policy .......................................................................................................... 112 
13.9 
Intellectual Property ...................................................................................................... 112 
14 Ethics ........................................................................................................................................ 113 
14.1 
Declaration of Helsinki .................................................................................................. 113 
14.2 
ICH guidelines for good clinical practice ....................................................................... 113 
14.3 
Approvals ....................................................................................................................... 113 

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14.5 
Volunteer confidentiality .............................................................................................. 113 
15 Data handling and record keeping ........................................................................................... 114 
15.1 
Data handling ................................................................................................................ 114 
15.2 
Record keeping .............................................................................................................. 114 
15.3 
Source data and electronic case report forms (eCRFs) ................................................. 114 
15.4 
Data protection ............................................................................................................. 114 
16 Financing and insurance ........................................................................................................... 115 
16.1 
Financing ....................................................................................................................... 115 
16.2 
Insurance ....................................................................................................................... 115 
16.3 
Compensation ............................................................................................................... 115 
17 Appendices ............................................................................................................................... 116 
18 References ................................................................................................................................ 117 

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1 SYNOPSIS 
Title 
A clinical study to assess the safety and feasibility of controlled blood-stage 
Plasmodium vivax human malaria infection through experimental 
inoculation of cryopreserved infected erythrocytes in healthy malaria-naïve 
UK adults 
Trial Centre 
Centre for Clinical Vaccinology & Tropical Medicine, Jenner Institute, 
University of Oxford, Churchill Hospital, Old Road, Headington, Oxford, OX3 
7LE 
 
Trial Identifier 
VAC069     
Clinical Phase  
N/A 
Design 
Controlled human malaria infection (CHMI) study 
Population 
Healthy adults aged 18 – 50 years 
Sample Size 
Total: 20 - 24 healthy malaria-naïve volunteers  
Group 1: 2 volunteers, undiluted inoculum 
Group 2: 2 volunteers, 1:5 diluted inoculum 
Group 3: 2 volunteers, 1:20 diluted inoculum 
Group 4: secondary challenge of all 6 volunteers from Groups 1-3 above 
Group 5: tertiary challenge of all 6 volunteers from Group 4 
Group 6: 2-3 volunteers primary challenge 
Group 7: secondary challenge of all 2 volunteers from Group 6 
Group 8: tertiary challenge of all 2 volunteers from Group 7 
Group 9: primary challenge of 4-8 volunteers 
Group 10: secondary challenge of all 4-8 volunteers from Group 9 
Group 12: primary challenge of 4-8 volunteers 
Group 13: repeat challenge with P. falciparum of volunteers from Groups 
10 and 12 
 
Follow-up 
duration 
Groups 1-8: Approximately 2.5 years from the time of primary challenge 
Groups 9-12: Approximately 18 months 
Group 13: Approximately 3 months  

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These estimates may vary in accordance with the specified time windows 
for each attendance. 
Planned Trial 
Period 
The start date of the trial will be in January 2019. The end date of the trial 
will be within approximately 4 years after primary CHMI of groups 1-3.  
Primary Objectives To assess the safety and feasibility of blood-stage controlled human P. 
vivax malaria infection in healthy human volunteers, through experimental 
injection of cryopreserved P. vivax infected erythrocytes at different doses 
To choose the optimal inoculation dose to take forward to future P. vivax 
CHMI studies. 
Secondary 
Objectives 
 
 
To assess safety and feasibility of a secondary and tertiary homologous 
controlled blood-stage human P. vivax malaria infection 
To assess the immune response to primary, secondary and tertiary 
homologous and heterologous Plasmodium infection, through 
experimental injection of P. vivax and P. falciparum-infected erythrocytes  
To assess gametocytaemia following primary, secondary and tertiary P. 
vivax infection through experimental injection of P. vivax infected 
erythrocytes. 
To assess transmission of gametocytes from infected volunteers to 
Anopheles mosquitoes. 
Investigational 
products 
None 
 
 
 
 

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2 ABBREVIATIONS 
ACT 
 
  
Artemisinin-based combination therapies 
AE 
 
 
Adverse event 
AR 
 
 
Adverse reaction 
CBF  
 
 
Clinical Bio-Manufacturing Facility 
CCVTM 
 
Centre for Clinical Vaccinology and Tropical Medicine 
CHMI  
 
Controlled human malaria infection 
CI 
 
 
Chief Investigator 
CMV 
 
 
Cytomegalovirus 
CRF 
 
 
Case Report Form or Clinical Research Facility 
CYP2D6 
 
Enzyme predicting Primaquine metabolism – Cytochrome P450 2D6 
DARC  
 
Duffy Antigen Receptor for Chemokines 
DFA 
 
 
Direct Feeding Assay 
DSUR  
 
Development Safety Update Report 
EBV 
 
 
Epstein-Barr Virus 
ELISA  
 
Enzyme linked immunosorbent assay 
ELISPOT 
 
Enzyme linked immunospot assay 
EPI 
 
 
Expanded Programme of Immunisation 
FDA 
 
 
United States Food and Drug Administration 
GCP 
 
 
Good Clinical Practice 
GMP  
 
Good Manufacturing Practice 
HBsAg  
 
Hepatitis B surface antigen 
HCV 
 
 
Hepatitis C virus 
HIV 
 
 
Human Immunodeficiency virus 
HTLV  
 
Human T cell Lymphotrophic virus 
HLA 
 
 
Human Leukocyte Antigen 
ICH 
 
 
International Conference on Harmonisation 
IFN-

Interferon-gamma 
IM 
 
 
Intramuscular 
IMP 
 
 
Investigational Medicinal Product 
LPS 
 
 
Lipopolysaccharide 
LSM 
 
 
Local Safety Monitor 
MFA 
 
 
Membrane Feeding Assay 
MHRA  
 
Medicines and Healthcare products Regulatory Agency 
MRC 
 
 
Medical Research Council 

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g 
 
 
Microgram 
NIHR  
 
National Institute for Health Research  
pIMD  
 
potential Immune-Mediated Diseases 
PCR 
 
 
Polymerase Chain Reaction 
PIS 
 
 
Participant Information Sheet 
QP 
 
 
Qualified Person 
RUNMC 
 
Radboud University Nijmegen Medical Center 
REC 
 
 
Research Ethics Commitee 
RGEA  
 
Research Governance, Ethics and Assurance Team 
SAE 
 
 
Serious Adverse Event 
SAR 
 
 
Serious Adverse Reaction 
SOP 
 
 
Standard Operating Procedure 
SUSAR  
 
Suspected Unexpected Serious Adverse Reaction 
TMF 
 
 
Trial Master File 
UKHSA  
 
UK Health Security Agency 
UOXF  
 
University of Oxford 
USMMVP 
 
United States Military Malaria Vaccine Program 
WHO  
 
World Health Organisation 
WTCRF 
 
Wellcome Trust Clinical Research Facility 
 

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BACKGROUND & RATIONALE 
3.1 
Impact of P. vivax malaria and the need for a vaccine 
Plasmodium vivax (P. vivax) is one of the five Plasmodium species to cause human malaria and 
accounts for the most cases of non-P. falciparum malaria worldwide. Geographically, it is the 
most widespread human malarial infection and approximately 2.5 billion people are at risk of 
contracting the infection in endemic areas [1]. It is estimated over one third of malaria cases 
outside of the African continent are due to P. vivax and that it accounts for 64% of malaria cases 
in the Americas, 30% in South-East Asia and 40% in the Eastern Mediterranean WHO regions.  
The burden is highest in Afghanistan, Ethiopia, India, Indonesia and Pakistan, which together 
account for 85% of P. vivax cases [2]. 
P. vivax has long been considered, and termed, ‘benign’ malaria, but more recently, large case 
series demonstrate that P. vivax infection is associated with significant morbidity and mortality. 
Complications reported include severe anaemia, respiratory and hepatic dysfunction, severe 
thrombocytopenia and coagulopathies, such as disseminated intravascular coagulation (DIC) [3]. 
P. vivax infection has also been associated with important sequelae for maternal and foetal 
health, correlating with increased rates of low birthweight, preterm delivery, and reduced foetal 
growth, as well as maternal and foetal deaths [4].  
The socio-economic impact of this infection is not known, but in terms of overall global cost to 
the individual due to lost productivity this is estimated at US$ 1.4 to 4.0 billion per year [5]. 
Those affected by P. vivax malaria are typically poor with inadequate access to affordable 
healthcare and with little financial reserve, perpetuating the cycle of poverty [6].  
Clinical cases are not only due to primary infection but relapses, which occur weeks to years after 
primary infection. Relapses are due to the ability of the P. vivax parasite to remain dormant in 
the liver in the hypnozoite stage, a lifecycle stage not seen with P. falciparum malaria [5]. 
Standard schizonticidal regimes are not effective against the hypnozoite stage. Radical cure of 
the hypnozoite stage requires therapy with the 8-aminoquinolone primaquine, currently the only 
licensed hypnozoitcidal anti-malarial, although tafenoquine, a new drug of the same class, given 
as a single dose, has recently been FDA-approved in the United States in patients aged 16 years 
and over. However, both 8-aminoquinolones carry a significant risk of severe haemolytic 
anaemia in individuals who are deficient in the glucose-6-phosphate dehydrogenase enzyme 
(G6PD), an inherited X-linked red blood cell enzyme disorder common in tropical and sub-tropical 
areas [7]. More recently, the importance of the cytochrome P450 enzyme, CYP2D6 in metabolism 
of primaquine to the active metabolite has been recognised [8, 9]. A common polymorphism in 
the CYP2D6 gene for the cytochrome P450 enzyme results in poor conversion of primaquine to 
the active form, resulting in higher treatment failure rates. It is estimated that these two factors 
combined may make nearly 40% of the population at risk of P. vivax infection ineligible for 
primaquine therapy [7].   
Control of P. vivax is also challenging, with re-emergence in areas where it has previously been 
eradicated. This is due to multiple factors including relapses, difficulty detecting asymptomatic 
infection, resistance to antimalarials and a lack of understanding of parasite biology [10]. Recent 
calls for control and ‘eradication’ of malaria worldwide [11] 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 [12]. 
Consequently, the revised Malaria Vaccine Technology Roadmap to 2030 [12] now recognises the 
importance of P. vivax and calls for a vaccine to achieve 75% efficacy over two years – equally 

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weighted with P. falciparum in an era of renewed political will to move towards malaria 
elimination and eradication. 
However, unlike P. falciparum, to date there has been relatively little research into this malaria 
species and one of the limiting factors has been the inability to culture P. vivax parasites in vitro 
over a prolonged period of time. More recently, research into vivax malaria has increased with 
candidate vaccines being developed and taken forward to clinical trial [13, 14]. However, none of 
these vaccines have progressed past a Phase IIa trial, and options for assessing the immune 
responses to new candidates are extremely limited.  
3.2 
Lifecycle of the Plasmodium vivax malaria parasite 
The lifecycle of P. vivax is complex, involving both the vector (the Anopheles mosquito) and the 
human host (Figure 1). Sporozoites are injected into the human host when the female Anopheles 
mosquito takes a blood meal. From here they migrate within minutes to the liver, where they 
invade hepatocytes and the schizont develops (liver-stage). The mature schizont ruptures after 
around 7 days, releasing merozoites into the blood stream, which invade reticulocytes 
preferentially (unlike P. falciparum which invades all erythrocytes). As well as developing 
schizonts in the liver, P. vivax forms hypnozoites, a dormant stage which develop into schizonts 
weeks to years later, rupturing when mature and releasing merozoites to cause blood-stage 
malaria. It is this stage of the lifecycle that is responsible for relapse.  
Once merozoites have invaded reticulocytes they are termed trophozoites, and again mature 
into schizonts which release merozoites after 2 days. These merozoites go on to infect other 
reticulocytes, and the blood-stage cycle continues. A subset of trophozoites develop into 
gametocytes, the sexual-stage of the malaria lifecycle, rather than forming schizonts. This occurs 
early in blood-stage infection with P. vivax, unlike P. falciparum. These are taken up by the 
mosquito in a blood meal. The sporogonic cycle takes place within the mosquito. The 
microgametocyte (male) exflagellates and enters the macrogametocyte (female) to form a 
zygote within the mosquito midgut. Zygotes become motile (ookinetes) and penetrate the 
midgut wall where they develop into oocysts. These rupture, release sporozoites which migrate 
to the mosquito salivary glands ready to be injected when the mosquito takes another blood 
meal [15]. 
The liver-stage of infection is asymptomatic. Symptoms, and subsequent complications, develop 
during the blood-stage. 
 

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Figure 1: Lifecycle of P. vivax malaria 
 
3.3. 
 Challenges and directions in P. vivax vaccine development  
The unique characteristics of P. vivax pose some specific challenges in studying its biology and 
designing strategies to control it. For example, the lack of a method for continuous culture of P. 
vivax blood-stages makes it difficult to perform in vitro growth inhibition assays to identify 
synergistic combinations of blood-stage antigens that can be targeted to inhibit P. vivax blood- 
stage growth with high efficiency. Several groups have recently succeeded in establishing short-
term P. vivax culture for invasion assays using enriched reticulocytes from cord blood [16], but 
such methods are still dependent on access to fresh P. vivax isolates from malaria patients limiting 
the routine use of such assays to endemic regions. In the absence of a P. vivax blood-stage culture 
system, production of infected mosquitoes for sporozoite- or transmission-stage studies also 
requires access to P. vivax patients in endemic areas. In order to study hepatocyte invasion stages 
of P. falciparum as well as P. vivax, attempts have been made to establish in vitro liver models [17, 
18]. In addition, a reliable humanised mouse model for P. vivax liver-stages is now available [19]. 
Both approaches will help to identify and test novel drugs and vaccines targeting hepatocyte 
stages. Studies on expression of parasite proteins in hypnozoites will determine if these latent 
stages can also be targeted with novel drugs and vaccines.  
Most of the vaccines that are currently under development target individual stages of the 
parasite’s lifecycle and are based on single antigens (Table 1 [13]). The development of an effective 
vaccine will likely require combination of multiple antigens that provide synergy to achieve high 

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efficacy. For example, achieving high rates of blood-stage growth inhibition may require targeting 
a combination of key blood-stage antigens involved in reticulocyte invasion. In addition, 
combination of blood-stage antigens with liver-stage antigens may be needed to achieve high 
efficacy, and inclusion of antigens from sexual- and mosquito-stages will be needed to inhibit 
transmission. It is therefore necessary to initiate efforts to combine antigens both within and 
across developmental stages to achieve synergy and attain the goal of developing a vaccine for P. 
vivax malaria with high efficacy. 
 
3.4 
Rationale for the development of a blood-stage challenge model 
The development of a safe and reproducible P. vivax challenge model in malaria naïve humans 
could greatly accelerate clinical development of P. vivax vaccines, through selecting efficacious 
candidates for further clinical testing in more expensive and logistically challenging studies in the 
field. Herrera et al. have demonstrated the robustness and reproducibility of the P. vivax 
mosquito-bite delivered sporozoite challenge model in South America [20, 21], suitable for 
evaluation of efficacy of both pre-erythrocytic and blood-stage P. vivax vaccines. Within Europe, 
the safety and feasibility of a sporozoite P. vivax challenge, also delivered by mosquito bite, was 
demonstrated for the first time in April 2018 in Oxford within the ongoing VAC068 study 
(ClinicalTrials.gov Identifier: NCT03377296).  
Mosquito-bite CHMI most closely models natural infection, and since cryopreserved P. vivax 
sporozoites are yet to be developed for human trials, remains the mainstay for CHMI in assessment 
of pre-erythrocytic vaccines. However, vaccines and anti-malarials targeting the erythrocytic stage, 
and with optimization, likely transmission-blocking candidates, may be evaluated by a blood-stage 
challenge model, which offers a number of advantages over sporozoite CHMI.  
Infection of mosquitos requires a ready supply of blood containing gametocytes, from an infected 
patient. Without the possibility of long-term P. vivax ex-vivo culture, supply is limited to endemic 
areas. For CHMI studies conducted in non-endemic zones, this necessitates infection and 
importation of mosquitos from an appropriate area, with suitable entomological facilities and 
expertise, which may be logistically demanding, particularly in cases of vaccine efficacy studies, 
where timing of challenge may be critical.  
Blood stage CHMI also permits both better characterization and standardization of the parasite 
inoculum. Parasite density of the cryopreserved isolates can be established, increasing 
homogeneity of initial parasite inoculum density, when compared to sporozoites delivered by 
mosquito bite, and allowing the possibility of controlled variations of the administered parasite 
load. Full genome sequencing, including screening for mutations known to be associated with drug 
resistance can also be performed prior to challenge. In addition, safety data can be accrued 
through multiple uses of the same inoculum. Together, standardisation of strain and density 
permit for better comparison between candidate vaccines, when evaluated through challenge 
using the same inoculum source.  
Since parasitic load introduced via intravenous injection is smaller than that released following 
schizogony at the liver-stage, as in natural infection and sporozoite challenge, there is a longer 
period of blood-stage multiplication. This provides an extended period for assessment of parasite 
multiplication rate (PMR), before the development of clinical symptoms or diagnostic criteria 
might be reached. In addition, comparison of PMR between infectivity controls from blood-stage 
CHMI performed in Oxford within the VAC054 trial, assessing efficacy of the P. falciparum apical 
membrane antigen 1 (AMA1) vaccine candidate, FMP2.1/AS01, and historic controls challenged by 
mosquito-bite, illustrated that although mean PMR was comparable, blood-stage challenge 

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produced a much narrower range of PMR (smaller standard deviation with tight 95% confidence 
intervals). This provided greater power to detect partial vaccine efficacy, making blood-stage 
challenge a more robust tool for evaluation of candidate blood-stage vaccines [22]. This was 
indeed borne out in the recent Phase IIa efficacy study of the candidate vaccine RH5.1/AS01B, 
where the blood-stage challenge model was able to reliably detect a 17% reduction in the PMR 
between vaccinees and infectivity controls, with a high degree of confidence (P<0.02) (VAC063, 
ClinicalTrials.gov Identifier: NCT02927145). 
Significantly, in addition to advantages seen with P. falciparum blood-stage CHMI, P. vivax blood-
stage challenge provides a mode of infection which by-passes the liver stage. This eliminates the 
possibility of hypnozoite formation and therefore, risk of relapse. Equally, there is no requirement 
for CYP2D6 phenotype screening and primaquine therapy. A P. vivax blood-stage challenge is 
therefore less restrictive and confers important safety benefits over sporozoite challenge.  
 
 

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Vaccine candidate 
Development Phase 
Lifecycle stage 
Antigen 
Delivery system 
VMP001 
Phase I/IIa END 
Liver-stage 
PvCSP 
Rec. protein-AS01B 
CSV-S,S 
Pre-clinical 
Liver-stage 
PvCSP 
HBsAg fusion-AS01B 
PvCSP-LSP 
Phase I END 
Liver-stage 
PvCSP 
Synthetic peptides-Montanide ISA 720 
ChAd63-PvTRAP/MVA-PvTRAP 
Pre-clinical 
Liver-stage 
PvTRAP 
Prime-boost, viral vectors 
PvDBPII 
Phase I 
Blood-stage 
PvDBP 
Rec. protein-GLA-SE 
PvDBPII-DEKnull 
Pre-clinical 
Blood-stage 
PvDBP 
Rec. protein 
ChAd63-PvDBPII/MVA-PvDBPII 
Phase I/IIa 
Blood-stage 
PvDBP 
Prime boost, viral vectors 
PvMSP119 
Pre-clinical 
Blood-stage 
PvMSP1 
Rec. protein-Montanide ISA720 
PvAMA1 
Pre-clinical 
Blood-stage 
PvAMA1 Rec. protein-adjuvant 
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 
Table 1: P. vivax vaccine candidates under development: taken from Ref [13]. 
 

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3.5 
Study rationale 
As outlined above, the available methods for preventing and treating infections with P. vivax in the 
field are inadequate. Advancing the development of such methods needs renewed emphasis on 
understanding the biology, pathogenesis and transmission of P. vivax. A blood-stage challenge 
model would provide insights into this and greatly help validate and down select P. vivax vaccine 
candidates for further clinical testing in more time consuming and expensive field trials against 
natural challenge in endemic areas.  
This study aims primarily to assess the safety and feasibility of controlled blood-stage human P. 
vivax malaria infection. This will be the first time that this source of P. vivax infected blood will be 
utilised and the first P. vivax blood-stage CHMI in Europe. If demonstrated to be safe and effective, 
it is intended that this parasitised blood bank be used in future trials to evaluate candidate 
vaccines. 
As a secondary aim the safety of secondary and tertiary challenge will be assessed. Natural 
immunity to P. vivax is acquired over time, following repeated exposure. Homologous re-challenge 
would improve understanding of acquisition of the human immune response and parasite growth 
dynamics, with second and third exposures.  
Only four controlled blood-stage P. vivax primary challenge studies have previously been 
conducted, of which only two have been published. These studies focused on safety and parasite 
growth dynamics and have not reported on the immunological response. No prior studies have 
evaluated repeated homologous blood-stage P. vivax CHMI. In this study, detailed immunological 
analyses, including gene expression studies of the parasite and host will be performed for primary, 
secondary and tertiary challenges. These approaches will provide new and more detailed insight 
into naturally acquired immunity than has been achieved before in any P. vivax blood-stage CHMI 
study, which could be crucial in guiding next-generation vaccine development. 
3.6 
Rationale for re-challenge with P. falciparum  
The original hypothesis on the effects of homologous re-challenge was that using the same clone 
of P. vivax would lead to rapid anti-parasite immunity and lower parasite multiplication rate 
during re-challenge compared to the first malaria infection. However after completion of 
VAC069A to D with a significant number of participants completing secondary re-challenge, no 
anti-parasite immunity has been observed in any participants. However, of great interest, signs 
of clinical immunity have been consistently noted in participants undergoing secondary 
challenge. This clinical immunity is evidenced by reduced symptoms and signs of malaria disease 
with reduced fever and lower incidence of lymphopenia, thrombocytopenia and transaminitis 
recorded during secondary infection. This finding is in contrast to homologous re-challenge 
studies with P. falciparum where volunteers remained susceptible to clinical disease on repeat 
infection (Sandoval et al., unpublished data) and is compatible with historical records that 
suggest P. vivax induces clinical immunity more quickly than P. falciparum [23-25]. 
The P. vivax CHMI model established in this study provides a unique opportunity to investigate 
the mechanisms of naturally acquired clinical immunity that minimise disease rather than 
pathogen load. Changing the malaria species in the final re-challenge from homologous to a 
heterologous challenge with P. falciparum will help determine whether P. vivax induces clinical 
immunity that is species-specific or species-transcending. The latter result would indicate that 
immunity is antigen independent and does not require adaptive immune control of 
inflammation. A mechanistic understanding of clinical immunity could aid the development of 
interventions that are designed to reduce the risk of disease in P. vivax infected patients. In 
addition, as P. falciparum and P. vivax are co-endemic in many areas, an understanding of the 

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effects of clinical immunity of P. vivax on P. falciparum would be of great importance in malaria 
control programmes.  
This change in final Plasmodium species challenge remains in line with our experimental 
objectives (e.g. to assess the immune response to primary, secondary and tertiary infection) but 
instead allows us to switch focus from anti-parasite immunity to clinical immunity. This is entirely 
in line with the original goals of our funded programme of work to develop a rechallenge model 
of P.vivax malaria and examine mechanisms of naturally acquired immunity. 
Given we have seen no evidence of anti-parasitic immunity after multiple P.vivax challenges and 
have acquired a clear understanding of the effect on clinical immunity (and now have the 
samples to explore immune mechanisms for this effect), there is no scientific gain from 
subjecting these participants to another P. vivax challenge, which makes the ethics of a re-
challenge with P. vivax also questionable. By contrast, there is much to be gained scientifically 
from a final P. falciparum challenge for the reasons described, and this also fulfils the 
participants’ expectations as they consented for and are expecting a secondary or tertiary 
malaria challenge.  
Finally and importantly, there is no perceived clinical risk in subjecting these individuals to a 
heterologous challenge. While this has not been done before experimentally, exposure to multiple 
controlled homologous challenges has been shown to be safe by this group and others 
(Themistocleous et al, unpublished data, [26]), and exposure to multiple species occurs in the field, 
either simultaneously or sequentially, throughout an individual’s lifetime. The net result appears 
to be accumulation of partial protection rather than association with any adverse outcome. 

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4 OBJECTIVES 
 
Primary Objectives  
To assess the safety and feasibility of controlled blood-stage P. vivax human malaria infection in 
healthy adult volunteers, through experimental injection of cryopreserved P. vivax infected 
erythrocytes  
To choose, based on the first phase of the study (VAC069A), the optimal inoculation dose of P. 
vivax for use in blood-stage CHMI studies. 
Secondary Objectives 
To assess safety and feasibility of secondary and tertiary homologous blood-stage controlled 
human P. vivax malaria infection. 
To assess the immune response to primary, secondary and tertiary homologous and 
heterologous Plasmodium infection, through experimental injection of P. vivax and P. 
falciparum-infected erythrocytes. 
To assess gametocytaemia following primary, secondary and tertiary P. vivax infection through 
experimental injection of P. vivax infected erythrocytes. 
To assess transmission of P. vivax gametocytes from infected volunteers to mosquito vectors  
 
 
 
 
 
 
 
 
 
 

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5 CONTROLLED HUMAN MALARIA INFECTION STUDIES 
5.1 
Microbial challenge studies of human volunteers 
The deliberate infection of human volunteers with micro-organisms has contributed uniquely to 
our understanding of the pathogenesis, immune responses and the treatment and prevention of 
numerous microbial diseases including P. falciparum malaria, influenza, cholera, typhoid and 
hepatitis [27]. A review by the UK Academy of Medical Sciences on microbial challenge studies 
recognised that such studies are desirable for providing proof of concept for prophylactic and 
therapeutic interventions and can significantly accelerate progress to Phase III studies [27, 28].  
5.2  
Controlled human malaria infection with Plasmodium falciparum 
Plasmodium falciparum is a microbe particularly well-suited to “challenge” studies. It has a 
relatively short asymptomatic period, a well-established diagnostic laboratory test (thick film 
microscopy), and rapid recovery with no long-term infectious state following appropriate and 
timely treatment. Studies involving controlled human malaria infection (CHMI) are a powerful 
tool for investigating malaria vaccine and prophylactic drug efficacy [29]. With an increasing 
number of candidate malaria vaccines being developed, the number of centres conducting CHMI 
studies is expanding in developed countries and now Africa [29].  
Following the development of protocols for the continuous culture of P. falciparum in 1976 [30] 
and the generation of mature P. falciparum gametocytes in vitro in 1981 [31], it became possible 
to produce laboratory-reared infectious mosquitoes, meaning that CHMI trials could be 
performed more routinely [32]. 
The first well-documented CHMI study with laboratory-reared infectious mosquitoes was carried 
out in 1986 at the US Walter Reed Army Institute of Research (WRAIR), the US Naval Medical 
Research Institute (NMRI) and the US National Institutes of Health (NIH) [33]. The following year, 
the efficacies of the first recombinant protein and synthetic peptide P. falciparum vaccines were 
reported for experimentally infected volunteers [34, 35].  
CHMI has now become established as a key tool to assess the efficacy of novel malaria vaccines 
and drugs; a total of 1,343 volunteers were experimentally infected with P. falciparum between 
1985 and 2009 [29, 36]. As CHMI trials are carried out in a controlled environment, they allow 
detailed evaluation of parasite growth and immunological responses, providing key information 
for vaccine and drug development [29].  
5.3  
Controlled human malaria infection with Plasmodium vivax  
In contrast to P. falciparum, there is much less experience with P. vivax in the modern CHMI era, 
with only a small handful of studies reported in the last few years. A key bottleneck has been the 
inability to culture P. vivax long-term in vitro. Sporozoite challenge with P. vivax also carries the 
risk of relapse weeks to years after infection if hypnozoites are not cleared from the liver (there 
is no dormant liver form in the P. falciparum lifecycle). In only two of the studies published to 
date has efficacy of immunisation been assessed (Table 2 – taken from Payne et al., Trends in 
Parasitology, 2017 [37]).  
However, despite lack of modern CHMI, there is an extensive history of deliberate infection with 
P. vivax; most notably in malariotherapy, which was carried out for the treatment of 
neurosyphilis almost a century ago. The Austrian psychiatrist Julius Wagner-Jauregg later 
received a Nobel Prize for his work with this treatment [38] and the practice was widely adopted 
as the only effective treatment available at the time. Malariotherapy provided a wealth of 
information about P. vivax infection, which has been reviewed previously [39]. Deliberate 

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infection with P. vivax was also conducted in the USA from the 1940s to 1970s in prisoners 
involved in the Malaria Research Project at the Illinois State Penitentiary. The studies mainly 
examined compounds for their potential use as antimalarials [40]. Similar studies were also 
carried out at the United States Penitentiary, Atlanta. Key discoveries of the biology of P. vivax 
were made during this period including, for example, the association between Duffy negativity 
and resistance to P. vivax infection [41].  
Following on from the studies of P. vivax infection conducted in Illinois, CHMI experiments were 
carried out to see if prior exposure to irradiated mosquitoes could confer protection by 
immunization. Rieckmann et al. [42] reported no protection against CHMI in three participants 
previously exposed to P. vivax-infected irradiated mosquitoes on four occasions at intervals of 2 
– 4 weeks (total of <200 mosquitoes). During the 1970s, in Maryland, USA, three further studies 
assessed the ability to immunise following exposure to infected irradiated mosquitos, followed 
by challenge with non-radiated infected Anopheles. In these studies, there was no cross-species 
protection to the P. vivax Chesson strain, after exposure to P. falciparum, but exposure to X-
irradiated Chesson and El Salvador P. vivax strains did confer protection against P. vivax CHMI 
following some of the ‘immunisation phases’, but not all and was achieved for a short duration 
(3-5 months), following exposure to hundreds of X-irradiated mosquitoes [22–24]. Recent data 
emerging from the field of P. falciparum sporozoite immunization indicate that the magnitude of 
protection, longevity of protection, and the ability to protect against heterologous strains 
requires increasing doses of sporozoites. These experiments in Maryland demonstrated that 
CHMI with both P. falciparum and P. vivax could be successfully carried out, but the studies were 
very small.  
5.4 
P. vivax sporozoite challenge in the modern era 
More recent mosquito-bite delivered sporozoite CHMI trials have taken place in Cali, Colombia 
and at the Walter Reed Army Institute of Research (WRAIR), Maryland, USA. A total of four 
sporozoite challenge trials have been conducted in Cali. The first trial involved eighteen healthy 
volunteers, exposed to the bites of 2-10 P. vivax infected Anopheles albimanus mosquitoes, of 
which seventeen developed infection. Authors speculated that the volunteer who did not 
develop malaria had surreptitiously taken anti-malarial medication but this was never confirmed 
[20]. There were no serious adverse events (SAEs) in this trial, but seven volunteers required fluid 
therapy due to nausea and vomiting, and five developed blurred vision lasting 2-3 days after 
treatment initiation with chloroquine. The second P. vivax CHMI trial was carried out by the same 
group in Colombia, aiming to demonstrate the reproducibility of this method of infection using 
three different A. albimanus mosquito lots fed on blood from three P. vivax-infected donors [21]. 
Seventeen individuals whose red blood cells were positive for the Duffy antigen/chemokine 
receptor (DARC, “Duffy positive”) and five Duffy negative controls were enrolled then randomly 
assigned to three groups (with six Duffy positive individuals in two of the groups and five in the 
third group). Following 2-4 bites from infected mosquitoes, all Duffy positive participants (and 
none of the Duffy negative participants) developed blood-stage malaria. A third P. vivax CHMI 
trial was carried out in Cali among both ‘semi-immune’ (previously-exposed; n=9) and malaria-
naïve adult volunteers (n=7) [43]. Symptoms were significantly worse among the malaria-naïve 
subjects but there were no SAEs.  
The most recent CHMI trial from the Cali group assessed “immunisation” through repeated 
exposure to radiation-attenuated sporozoites, delivered by mosquito bite, followed by challenge. 
Both Duffy positive and Duffy negative volunteers were recruited. Twelve Duffy positive 
participants and five Duffy negative controls completed the seven “immunisations” followed by 
CHMI, alongside two Duffy positive negative controls, exposed to non-irradiated, non-infected 

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mosquitoes. Two weeks after the final immunisation participants were treated with chloroquine 
and primaquine to clear any malaria infections that may have developed during the 
immunisation phase. CHMI was carried out at 8 weeks after final “immunisation”, with each 
individual receiving 2 – 4  P. vivax-infected mosquito bites. There were no reported SAEs related 
to immunisation, although one volunteer developed severe elevation of hepatic transaminases 
(>10 times the upper limit of normal [x ULN]) with associated abdominal pain and vomiting 
following CHMI, with no alternative cause found. These symptoms resolved spontaneously. The 
protective efficacy of the immunisation schedule was 42% (five out of twelve Duffy positive 
participants protected). Interestingly, all of the volunteers protected in this trial were female 
[44]. 
Only one CHMI trial assessing a P. vivax vaccine has been published to date. The VMP001/AS01B 
vaccine was tested in healthy malaria-naïve adults at the WRAIR in the USA [45]. VMP001 is a 
soluble recombinant protein vaccine [46], encoding the P. vivax circumsporozoite protein (PvCSP) 
administered with the AS01B adjuvant (GlaxoSmithKline). The vaccine was administered to 30 
volunteers in three cohorts (10 in each) at doses of 15 µg, 30 µg and 60 µg, given three times at a 
4 week interval between the first and second dose; the third dose was given 8 (15 µg cohort), 6 
(30 µg cohort) or 4 (60 µg cohort) weeks after the second. Twenty-nine volunteers completed 
the vaccination phase, with twenty-seven proceeding to sporozoite CHMI, delivered by mosquito 
bite, two weeks after final vaccination, alongside 6 malaria-naïve controls. Vaccine protective 
efficacy was 0%; all volunteers had developed thick blood film-detectable parasitaemia by day 
13. The median pre-patent period for all immunised participants was 11.9 days versus 10.7 days 
for infectivity controls. The challenge was well tolerated with no untoward reactogenicity 
following mosquito bites and no untoward SAEs in challenge and treatment phases. Participants 
were treated with standard chloroquine and primaquine therapy with rapid clearance of 
infection. However, two volunteers went on to have multiple relapses. One participant 
experienced two relapses (at weeks 8 and 18 after CHMI), while the other experienced three (at 
weeks 11, 20 and 48 after CHMI) [9]. By study completion the participants had been followed up 
for 5 years, and had not had any further relapses [45]. Exploratory genotyping for the 
cytochrome P450 (CYP) allele CYP2D6 was undertaken in 25 of the 33 volunteers. The volunteers 
with relapses were found to have either an intermediate-metaboliser phenotype or poor-
metaboliser phenotype. These phenotypes were associated with significantly lower levels of 
primaquine clearance 24 hours after dosing [9].  
The first P. vivax sporozoite challenge in Europe was recently conducted in April 2018, in Oxford 
within the VAC068 study (ClinicalTrials.gov Identifier: NCT03377296). Two malaria-naïve adult 
volunteers were exposed to five infectious bites (bites by mosquitos with >10 sporozoites as 
detected on microscopic examination of salivary glands post-feeding) by Anopheles dirus 
mosquitoes. Infection in the mosquitoes was established by direct membrane feeding on the 
blood from a P. vivax infected source patient, in Songkhala, Southern Thailand, before transport 
of the infected mosquitoes to the UK. Both volunteers were successfully infected, with parasitic 
DNA being detectable from day 9 after challenge by qPCR in both subjects. Symptoms consistent 
with malaria infection, including fatigue and subjective feverishness, chills, headache, loss of 
appetite, nausea and malaise, were reported by one volunteer from day 11 after challenge and 
on day 14 by the second volunteer. Fever was detected in both subjects on day 14. Thick film 
microscopy was positive (≥2 morphologically normal asexual parasites seen in 200 high-power 
(1000x) fields) in the asymptomatic volunteer on the evening of day 13 and at day 14 in the more 
symptomatic volunteer, leading to diagnosis on day 14 in both volunteers. A 250mL blood 
donation was made by each volunteer, immediately prior to initiation of anti-malarial treatment, 
without complication, for cryopreservation and future use as a parasitised blood inoculum for 

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blood-stage challenge. After 48 hours’ anti-malarial treatment, thick film microscopy was 
negative and parasitaemia by qPCR had substantially reduced on two consecutive samples. Both 
volunteers completed the clinical follow-up period, including a two-week course of primaquine 
for radical cure of hypnozoites, until 90 days post-challenge, without safety concerns. To-date 
(more than 8 months post completion of primaquine therapy) there has been no report of 
symptoms to suggest relapse in either volunteer (email questionnaire follow-up is being 
continued for 5 years). 
  

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Trial Site 
Number of volunteers 
Pre-patent period 
(days)a 
Number of infected 
mosquitoes OR inoculum 
Number of volunteers with 
patent parasitaemia 
References 
Sporozoite (mosquito-bite) CHMI studies 
 
Cali, Columbia 
18 
9 – 13 
2 - 10 
17/18 
[20] 
Cali, Columbia 
17 Duffy positive 
5 Duffy negative 
9 – 16 
2 - 4 
17/17 (Duffy positive) 
0/5 (Duffy negative) 
[21] 
Cali, Columbia 
7 malaria-naïve 
9 semi-immune 
11 – 13 
2 - 4 
16/16 
[43] 
WRAIR, USA 
27 vaccinees  
6 infectivity controls 
10 – 13 
10 – 11 
5 
 
[45] 
Cali, Columbia 
12 Duffy positive vaccinees 
2 Duffy positive controls 
5 Duffy negative controls 
12 – 13 
2 - 4 
7/12 vaccinees 
2/2 Duffy positive controls 
0/5 Duffy negative controls 
[44] 
Blood-stage CHMI studies  
 
QIMRB, Australia 
2 
8-9 
13,000 genome equivalents 
2/2 
[47] 
QIMRB, 
Australia 
6 
8-9 
31,786 (± 11,947) genome 
equivalents (= 15 ± 5 viable 
P. vivax parasites) 
6/6 
[48] 
Table 2: Overview of Published Plasmodium vivax CHMI Studies. aThe pre-patent period refers to the period before malaria diagnosis: by blood film 
(sporozoite) or qPCR (blood-stage).

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5.5 
Modern blood-stage controlled human P. vivax infection 
There have been four studies of blood-stage P. vivax CHMI, of which two are published, all 
conducted at the Queensland Institute of Medical Research (QIMR), Brisbane Australia. Both 
published studies used a cryopreserved source of parasitised erythrocytes obtained from a 
returned traveller with blood group A, Rhesus negative, who was infected with P. vivax during a 
trip to the Solomon Islands [47, 48].  
In the first study, two volunteers were inoculated with 270µL of reconstituted parasitised red 
cells, containing approximately 13,000 genome equivalents. Both subjects were successfully 
infected, with PCR becoming positive on day 8 or 9. Clinical symptoms consistent with malaria 
infection were present from day 11 in one volunteer, who reported myalgia, headache and 
malaise and day 14 in the other, with both experiencing chills and sweats at day 14. Treatment 
with artemether/lumefantrine was started at day 14 and volunteers were admitted for ongoing 
monitoring. Both volunteers experienced worsening of symptoms over the initial 12 hours post-
treatment, including nausea and vomiting, headache, sweats and rigors in one volunteer. Pyrexia 
above 39oC was detected in both volunteers. After 24 hours of anti-malarials, all symptoms had 
resolved, although non-tender clinically palpable mild splenomegaly was detected in one subject 
at day 15. By day 28, splenomegaly had resolved fully and no other adverse events were 
reported by either subject in the period since discharge. Whole genome sequencing was 
performed, demonstrating a genetic sequence most closely related to Cambodia strains, of the 
publically available strains. Gametocytaemia was measured by quantitative reverse-transcriptase 
PCR for the pvs25 transcript, a mature gametocyte marker. Pvs25 transcripts were detectable 
from day 11 or 12 and peaked at day 14 in both volunteers, coinciding with peak quantitative 
parasite PCR, prior to the initiation of anti-malarial treatment [47].  
A second study was conducted to demonstrate reproducibility of the blood-stage controlled 
infection method and assess transmissibility of infection to Anopheles stephensi mosquitoes. Six 
volunteers were infected after receiving an inoculum containing approximately 31,786 (+/- 
11947) genome equivalents, estimated by investigators using linear regression growth modelling 
to equate to 15+/-5 viable parasites. Evolution of clinical symptoms was similar to the first study, 
with volunteers developing symptoms in keeping with malaria infection on days 11-13 (mean 
12.2). There were no serious adverse events during the course of either study, but for 4 of 6 
volunteers, liver enzymes became significantly elevated, above 5 times the upper limit of normal. 
Mild bilirubinaemia was also detected in three subjects, however increases were transient and 
did not exceed 2 times the upper limit of normal. No clinical manifestations were associated with 
biochemical abnormalities. No other cause for this deranged liver function was found and liver 
ultrasound was normal but abnormalities subsequently resolved spontaneously without 
sequelae [48]. Transmissibility of infection was assessed by both direct feeding assay (DFA) and 
membrane feeding assay (MFA), carried out 3 days prior to the anticipated peak parasitaemia 
and initiation of treatment on day 14, as predicted from the first study. A total of 16 DFAs and 32 
MFAs were performed, with rates of successful feeding above 90% across the two methods, 
without significant difference between the two. Overall, 1801 mosquitoes were dissected for 
examination of oocysts 7-9 days after feeding, of which 1.8% (n=32) were demonstrated to be 
infected, although the rate of infectivity was five times higher for DFA when compared to MFA. 
Other atypical ovoid structures were also detected by investigators on the mosquito midgut, 
which could not be identified by PCR as either microsporidia or fungal pathogens, known to 
infect mosquitoes. For standardisation of the method, if used for drug or vaccine efficacy 
evaluation, other methods for confirmation of oocysts will be required. The reason for low 
infectivity could not be determined, however, a possible incompatibility of the Solomon Island 

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derived parasite strain and the laboratory-reared A. stephensi colony, which had originally been 
sourced from India, was cited by the authors as a potential contributory factor. While small, 
these studies demonstrate the safety, feasibility and reproducibility of a P. vivax blood-stage 
challenge model and show the potential for use as a transmission model for evaluation of 
transmission-blocking vaccine candidates, although the methodology is yet to be optimised.  
5.6 
 Re-challenge in controlled human malaria infection 
Re-challenge is well established in the sporozoite controlled infection model, with both mosquito 
bite delivery or more recently, inoculation of cryopreserved sporozoites. This has primarily been 
in assessment of durability of pre-erythrocytic vaccine-induced responses, where sterile 
protection has been established at primary challenge. However, repeated sporozoite challenge 
has also been investigated as a form of “immunisation”. There is much less experience with 
repeated controlled blood-stage malaria infection for both P. falciparum and P. vivax, although, 
historically, re-challenge was routinely performed as part of deliberate malaria infection in 
malariotherapy for neurosyphilis patients. In repeated infection, malariotherapy patients had a 
reduced number of fever episodes and a log reduction in the maximum mean parasite count 
after a homologous P. vivax re-challenge, suggesting conferred protection from a prior challenge. 
However, with little experience in human blood-stage re-challenge in the modern era, the level 
of immunity afforded remains largely unknown.  
There have been no previous studies assessing repeated P. vivax blood-stage CHMI, and only two 
studies which have assessed P. falciparum blood-stage re-challenge. A group at the Queensland 
Institute of Medical Research, Australia, assessed repeated exposure to small doses of 
parasitised erythrocytes, followed by administration of anti-malarial treatment at sub-clinical 
infection, in five malaria-naïve healthy volunteers. After three rounds of low-dose challenge and 
cure, three out of the four volunteers who completed the study did not develop infection. 
However, with very small numbers, lack of a control group and detection of residual atovaquone, 
which may have caused this protective effect, the interpretation of these results is difficult. 
The first P. falciparum blood-stage re-challenge in volunteers who had reached criteria for 
malaria diagnosis, before initiation of treatment, was conducted in a recent study at Oxford 
(VAC063, ClinicalTrials.gov Identifier: NCT02927145). Nine volunteers vaccinated with the 
RH5.1/AS01 candidate, and eight unvaccinated controls received a second blood-stage challenge, 
alongside six malaria naïve controls receiving a primary challenge. Vaccinees were re-boosted 
two weeks before secondary challenge (at a timepoint four months after primary challenge). 
After secondary challenge, higher vaccine-induced antibody responses and a greater significant 
reduction of parasite multiplication rate was demonstrated in vaccinated volunteers compared 
to the first CHMI, indicating an enhanced partially protective effect upon second challenge. 
However, remarkably a significant reduction in parasite multiplication rates compared to primary 
CHMI was also seen in two of eight re-challenged control volunteers. This was particularly 
striking in one participant who had no detectable infection by qPCR until day 18, a significant 
delay as compared to initial PCR patency at day 3-5 in other volunteers. Findings lend support to 
the development of protective immunity, following a prior blood-stage exposure, underlying the 
need for adequate controls in assessing vaccine efficacy in re-challenge studies. Furthermore, 
these preliminary results also suggest potential for re-challenge studies to contribute to 
understanding of acquisition of blood-stage immunity.  
5.6.1 Safety of re-challenge in controlled human malaria infection 
Safety of re-challenge is indicated by the natural history of induced protection from previous 
malaria exposure in endemic areas, as well as the wealth of data from repeated infection 

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administered as malariotherapy, which strongly indicates that exposing a previously malaria-
exposed volunteer to a second or third challenge would not confer any additional risk over that 
of a first challenge. Indeed, it is highly possible that this may in fact confer some protective 
immunity, as in the natural exposure setting, where asymptomatic infections and tolerance of 
high levels of parasitaemia without symptoms or clinical signs of malaria are seen. Repeated 
challenge may therefore result in a milder infection and/or delayed time to diagnosis. 
Alternatively, if there is no immunity from the previous challenge or challenges, then the 
expected course of a second or third challenge would be comparable to the primary CHMI, with 
no change in risk.  
Hundreds of healthy volunteers have previously been re-challenged with P. falciparum in 
sporozoite challenge, delivered either by mosquito bite, or infected sporozoite, in the UK, 
Netherlands and the USA. No safety concerns were reported in any of these trials  [49-52] In the 
only previous study assessing a second homologous P. falciparum blood-stage challenge in 
volunteers reaching criteria for malaria diagnosis, recently conducted in Oxford (VAC063, 
ClinicalTrials.gov Identifier: NCT02927145), preliminary data is also supportive of a level of risk of 
re-challenge, which is the same as primary controlled malaria infection. During the second 
challenge in this study, solicited haematological and biochemical severe adverse events occurred 
at the same frequency as among controls undergoing a primary challenge and there were no 
serious adverse events (SAEs). All solicited and laboratory adverse events were self-limiting. 
Unsolicited AEs have yet to be peer reviewed, however, there were no events of medical concern 
in either vaccinated or control volunteers receiving a second challenge.  
5.7 
Source and preparation of the P. vivax cryopreserved inoculum 
The cryopreserved P. vivax parasitized red blood cells that will be used were produced by Dr 
Angela Minassian, Prof Simon Draper and colleagues at the University of Oxford in April 2018, as 
part of the VAC068 study (ClinicalTrials.gov Identifier: NCT03377296), approved by the South 
Central - Oxford A Research Ethics Committee.  
5.7.1 Collection of P. vivax infected erythrocytes  
Parasitised erythrocytes were collected from two P. vivax-infected volunteers (otherwise 
healthy), who donated blood following sporozoite P. vivax CHMI, delivered by mosquito bite. 
Volunteers were infected via five infectious bites (defined as bites by mosquitoes with >10 
sporozoites as detected on microscopic examination of salivary glands post-feeding) from 
infected laboratory-reared Anopheles dirus mosquitoes, under controlled conditions. Infected 
mosquitoes were provided by, and imported from, The Mahidol University, Bangkok. The colony 
is maintained through feeding on rigorously screened human blood (provided by the Red Cross) 
to induce egg-stimulation, animal blood is never used. For infection of mosquitoes, a source 
patient was recruited from a medical clinic in one of Thailand’s Southern endemic areas – 
Songkhala (~7 hours’ drive from the Vivax Research Unit in Bangkok). Microscopic diagnosis of 
the source patient was established at both the field site and in the Bangkok reference laboratory, 
where PCR analysis confirmed P. vivax mono-infection. Molecular speciation by PCR (research-
grade laboratory assay) was verified at the Jenner Institute Laboratories and clonality of the 
infection was established by the Wellcome Trust Sanger Institute in Cambridge, UK, on a 2mL 
whole blood sample, sent directly by courier from Thailand to Oxford. Mosquitoes were infected 
via direct membrane feeding with blood from the source patient, and infectivity was confirmed 
at 5-7 days post-feeding through oocyst count on dissection of the midgut. 
Both UK volunteers (“Donors”) were followed from days 1-6 post-challenge by telephone and 
from the evening of day 6 at twice-daily clinic visits. Blood sampling was performed at each visit 

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for thick film microscopy and PCR. Clinical symptoms consistent with malarial infection were 
reported from day 11.5 post-challenge in Donor 1 and from day 14 in Donor 2, with both 
becoming febrile at day 14. Thick film positivity was defined as the detection of at least two 
morphologically normal malaria parasites seen in 200 high-power (1000x) fields. Thick film 
microscopy was positive at day 14.5 in volunteer 1 and at day 13.5 in volunteer 2, corresponding 
to 31,010 and 16,717 genome copies/mL by PCR, respectively. Blood donation was performed in 
each volunteer immediately prior to initiation of treatment on the morning or afternoon of day 
14. A 250mL blood sample was collected using aseptic technique, via a whole blood donation kit 
(Leuokotrap WB, Haemonetics Corp), containing an in-line leukodepletion filter. For 
anonymization of the blood donor, blood collected for cryopreservation was labelled with either 
“Donor 1” or “Donor 2”. Traceability of the blood donor is however maintained within a 
confidential clinical record, which may be accessed by the Chief Investigator, or by key members 
of the clinical team, to whom responsibility is delegated, if requested by the sponsor or other 
external bodies. After blood collection, treatment with artemether/lumefantrine (Riamet®) was 
administered, followed by a two week course of primaquine. No supportive treatment or 
admission was required. Follow-up (clinic visits) until 90 days post-challenge was completed, with 
no safety concerns. Ongoing follow-up by email will continue for 5 years in order to monitor for 
relapse. 
 
5.7.2 Cryopreservation and storage of blood bank 
After collection, blood was maintained at 37oC and transported immediately to the Jenner 
Institute Laboratory, University of Oxford. All laboratory processing was conducted under GMP-
like conditions, under QP and QA oversight. Procedures were performed within fumigated 
microbiological safety cabinets, under precautions in compliance with Containment Level 3 Code 
of Practice and with sterile technique.  
First, the red cells were separated from plasma by centrifugation of the leukodepleted blood 
before mixing with 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 1.5mL cryovials and frozen at -80oC for one night 
before transfer to a dedicated liquid nitrogen tank. After 6 days, samples were transferred to 
Thermo Fisher Bishop’s Stortford temperature-monitored liquid nitrogen facility, Hertfordshire, 
UK, where samples are stored on behalf of the University of Oxford. This process was performed 
under conditions similar to those used for blood thawing in previous CHMI trials at Oxford and 
according to the Jenner Institute Laboratory SOPs. 
5.7.3 Infectivity and characterisation of parasite stock 
Confirmation of parasitic density within the blood collected for cryopreservation was performed 
via microscopy and quantitative PCR on the leukodepleted blood samples. Thick film microscopy 
demonstrated 11 asexual parasites per 1µL leukodepleted blood for Donor 1, and 5 asexual 
parasites per 1µL leukodepleted blood for Donor 2. Quantitative PCR confirmed the presence of 
23,566 genome copies/mL in Donor 1, and 14,078 genome copies/mL in Donor 2. This suggested 
minimal parasitic loss through the leukodepletion process when compared to the diagnostic PCR 
values of 31,010 and 16,717 genome copies/mL, respectively. 
Viability has also been demonstrated in an ex-vivo short-term culture of thawed infected red 
blood cells in enriched McCoy 5A medium. Parasitic growth was detectable by light microscopy, 
quantitative PCR and flow cytometry through an initial 40-hour growth cycle in samples collected 

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from Donor 1, with normal progression of normal morphology as seen on Giemsa stained thick 
and thin films. However, parasite growth was sub-microscopic in samples obtained from Donor 
2. Therefore, we intend to proceed with further testing of cryopreserved samples from Donor 1, 
and will use this as the challenge inoculum in this and future studies. Further testing will include 
whole genome sequencing to further characterize the parasitic strain, with particular scrutiny for 
SNVs present in the five putative drug resistant genes pvdhfr, pvdhps, pvcrt, pvmdr and pvmrp.  
5.7.4 Testing of source patient and blood donors for blood-borne and mosquito-borne 
infections 
Initial testing for blood-borne infections and mosquito-borne disease was carried out on blood 
samples from the original source patient in Thailand, taken at the time of P. vivax diagnosis. 
Since Anopheles is a known vector of Wucherichia bancrofti, the main causative agent of 
lymphatic filariasis, blood was screened for filarial disease via rapid diagnostic test for IgG4 
antibodies to the W. bancrofti Wb123 antigen (SD BIOLINE Lymphatic Filariasis IgG4) at the Thai 
field site. In addition, although there is no evidence that blood-borne infections can be 
transmitted by mosquito bite, for maximal assurance of safety, serology for HIV-1 and HIV-2, 
HTLV, Hepatitis B and C and syphilis were performed on a serum sample from the Thai source 
patient, at Oxford University Hospitals NHS Trust. Due to anecdotal reports of Japanese B 
encephalitis and Chikungunya in Anopheles species, blood from the source patient was also 
screened for both arboviral infections by PCR, as performed by the WRAIR unit, who used the 
same mosquito source for a CHMI trial that assessed the efficacy of the VMP001/AS01 P. vivax 
circumsporozoite recombinant protein vaccine[45]. As a further precautionary measure, 
although Anopheles species are not known to be vectors of Dengue, Zika or West Nile virus, on 
the independent advice of clinical Tropical Medicine specialist, Prof. Nicholas Day, Professor of 
Tropical Medicine and Director of the Mahidol Oxford Tropical Medicine Research Unit in 
Thailand, PCR for these infections was performed on the source patient’s blood. In summary, 
molecular analysis (PCR) for Chikungunya, Japanese B encephalitis, West Nile, Dengue and Zika 
viruses was performed on whole blood at the Rare Imported Pathogens Laboratory (RIPL) in the 
UK. All source patient infection screen tests were negative.  
Both UK volunteers (“donors”) were subjected to screening for blood-borne infections, in line 
with the Joint UK Blood Transfusion and Tissue Transplantation Services Professional Advisory 
Committee guidelines https://www.transfusionguidelines.org/red-book/chapter-9-microbiology-
tests-for-donors-and-donations-general-specifications-for-laboratory-test-procedures/9-1-
general-requirements. Testing comprised serological tests for HIV-1 and HIV-2, hepatitis B and C, 
syphilis (anti-treponemal Ab) and HTLV-1 and HTLV-2 at screening, and nucleic acid amplification 
tests for HIV-1 and HIV-2 and hepatitis B and C, as well as repeat serological tests for HTLV-1 and 
HTLV-2 and syphilis 7 days before sporozoite challenge. Neither volunteer had travelled to any 
area which represented a risk of exposure to any other form of blood-borne infection. All tests 
were negative. Volunteers also underwent repeat serological testing for HIV, hepatitis B and C, 
syphilis, HITLV-1 and HTLV-2, 90 days after challenge was undertaken, to ensure that no 
seroconversion from a recently-acquired infection (that may have been undetectable around the 
time of challenge) had occurred since the challenge period. All tests remained negative.  
In addition, volunteers were screened for Epstein-Barr virus (EBV) and Cytomegalovirus (CMV) 
prior to enrolment. Both volunteers were IgG seropositive for both infections. However, 
leukodepletion has been shown to be effective in preventing transmission of both EBV and CMV 
[53] therefore, EBV-CMV concordant serostatus would not be considered a requirement for 
recipients of this donated blood in this study, unless PCR testing of the blood-bank is positive for 
either infection. This is in line with conduct of previous P. falciparum CHMI studies at Oxford, 

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which have utilised a cryopreserved source of infected erythrocytes from a donor who was 
seropositive for both EBV and CMV. Initial studies using this inoculum had initially required that 
participants in CHMI trials be seropositive for both infections. However, following the 
development of PCR assays for both infections, molecular analysis of the inoculum was negative 
for both EBV and CMV. Following a risk-assessment QIMR, Australia, subsequently removed 
seropositivity to these viruses as a criterion for participation as a recipient of the inoculum. In a 
previous P. falciparum challenge study (VAC054) at Oxford, 63% of volunteers were seronegative 
for CMV and 11% were seronegative for EBV pre-CHMI, and no cases of seroconversion were 
recorded post-CHMI [22]. Volunteers will however be informed of the theoretical risk of 
transmission of infection and this will be included within the consent form. Serostatus will be 
tested prior to CHMI at screening for volunteers as part of this protocol, however, this solely 
relates to interest in the effect on the immunological response. Serum samples collected after 
each CHMI will be tested for for serostatus analysis to HIV, Hepatitis B, Hepatitis C, EBV and CMV 
to confirm lack of transmission of blood-bourne viruses. Serum samples post-CHMI will also be 
stored in case further analysis of serostatus is required or requested by the Chief Investigator 
subsequently.   
5.7.5 Sterility and screening for blood-borne infections of cryopreserved blood bank 
Blood collected for cryopreservation was tested for bacterial contamination using validated GMP 
or GLP laboratory techniques. These included tests of sterility by direct inoculation, mycoplasma 
specific culture and detection and quantification of bacterial endotoxin by kinetic chromogenic 
limulus amoebocyte lysate assay. All of these assays were performed by SGS Vitriology, Glasgow, 
UK, using assays compliant with harmonised European Pharmacopeia and United States 
Pharmacopeia standards.   
A further screen for blood-borne infections is being conducted on the plasma, derived directly 
from the blood bank (separated from the red cells prior to freezing down), in line with testing 
procedures performed by the NHS Blood Transfusion service. DNA PCR for HIV-1, hepatitis B, 
hepatitis C, EBV, CMV, serology for HIV-2 and serology for HTLV-1, HTLV-2, and Treponema 
pallidum (RPR) will be performed on thawed plasma samples at Kings College Hospital NHS 
Foundation Trust laboratory.  
We intend to proceed to human challenge if all the above screens for contamination and 
infection are negative, with the exception of PCR for CMV or EBV. In the event that DNA PCR for 
CMV or EBV is positive, volunteers for this study will only be enrolled if they are also seropositive 
for these infections, i.e. are seroconcordant.   
 
5.8 
Source and preparation of the P. falciparum cryopreserved inoculum 
The P. falciparum infectious inocula to be used in VAC069E were produced by Drs Gregor 
Lawrence, Allan Saul and colleagues at QIMR in Brisbane, Australia in 1994 [54]. The protocol for 
the study was reviewed and approved by the QIMR Ethics Committee and the Healthy Volunteer 
Studies Research Ethics Subcommittee, Lothian Health Board (Edinburgh). Procedures were 
designed to minimise the risk of other infectious agents in the cryopreserved samples. 
5.8.1 Collection and cryopreservation of inoculum 
Laboratory-reared Anopheles stephensi mosquitoes were infected with the P. falciparum clone 
3D7 (a chloroquine-sensitive strain) by membrane feeding on a blood meal containing 
gametocytes. Ten and fourteen days later, the mosquitoes were fed on two volunteers. 
Parasitaemia in the volunteers was followed by daily microscopy from day 4 after infection. Blood 

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was taken and frozen in aliquots from the volunteers 6 hours after they developed fever, when 
both were microscopically parasite positive. The volunteers were treated with chloroquine soon 
after blood was drawn with complete recovery. 
Initial development of the blood inoculum to be used in this study and its use in five volunteers is 
described by Cheng et al. 1997 [54]. The inoculum used for all volunteers comes from one of the 
donors described above. This is because the second donor (whose blood has not been used) had 
a much lower parasitaemia [55]. Blood was collected at the Australian Red Cross Blood Bank in an 
aseptic manner using standard blood bank equipment. The leukocytes were removed with a 
leukocytic filter. The thawing and washing of the cells reduced the amount of serum transferred 
with the red cells by a factor of 1000, compared to injecting the same volume of blood. The volume 
of inoculum to be given to each volunteer contains a very small volume of red blood cells, 
equivalent to only 1.5 to 4 microlitres of blood.  
The red cells were cryopreserved using a protocol from the American Association of Blood Banks 
Technical Manual that is normally employed for freezing blood from patients and donors with rare 
blood groups. Blood from both volunteers was group O and Rhesus negative.  
Viability of the inoculum was confirmed by culture in a trial in Nijmegen in 2011 [56]. For each vial 
of inoculum used, a small amount will be cultured after inoculations are complete to confirm 
viability and the estimated number of parasitised red blood cells inoculated. 
Between 1994 and 2003 the cryopreserved samples to be used in this trial were stored in dedicated 
liquid nitrogen cylinders in a secure facility at QIMR. The liquid nitrogen containers were kept 
locked and accessible only to approved staff. In 2003 the samples were transferred to Biotec 
Distribution Ltd., Bridgend, UK and then to Thermo Fisher Bishop’s Stortford, Hertfordshire, UK, in 
2007 where they have been stored on behalf of Oxford University in temperature-monitored liquid 
nitrogen. 
5.8.2 Previous Studies 
The blood-stage P. falciparum 3D7 inoculum has to date been used in over 400 volunteers in 32 
studies.  
Oxford University has previously undertaken six malaria challenges of healthy human volunteers 
using this inoculum. The first was a pilot study in 2003 in which 5 volunteers received the inoculum. 
As expected, the results showed less variation in rate of rise of parasitaemia than is typically 
experienced with a liver-stage (or sporozoite) malaria challenge. The trial was successful, with all 
volunteers being diagnosed with malaria between the morning of day 7 and the morning of day 9 
after inoculation. The symptoms of malaria experienced by volunteers at and around the point of 
diagnosis were as expected. No SAEs were reported. All volunteers were clinically well after 
antimalarial treatment when reviewed in clinic at day 42 after inoculation. During the trial one 
volunteer became mildly anaemic but this was transient, with a normal haemoglobin on repeat 
testing one week later; and one volunteer had a raised bilirubin of 35 µmol/L on two occasions. All 
volunteers successfully completed follow-up [57]. 
Following this a Phase IIa study of the Safety, Immunogenicity and Parasite Growth Inhibitory 
Activity of AMA1-C1/Alhydrogel® + CPG 7909 was conducted (VAC035). This involved 8 healthy 
volunteers (5 vaccinees and 3 controls) receiving the blood-stage CHMI with the same inoculum. 
All individuals challenged developed malaria (confirmed by positive blood film and positive qPCR), 
although only 2 of the 8 subjects developed clinical malaria symptoms pre-diagnosis [58]. A 
significant inverse relationship between vaccine-induced growth inhibitory activity (GIA) and PMR 
was seen in vaccinated subjects, but no impact on overall PMRs was observed across the 

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vaccinated and control groups. However, the sample size in this study was very small, limiting the 
statistical power to assess differences in PMR between the groups [58]. 
VAC054 next looked at the safety, immunogenicity and efficacy of the AMA1 vaccine candidate 
FMP2.1 given with the AS01 Adjuvant System. In this trial 12 vaccinated volunteers and 15 
infectivity controls received the blood-stage CHMI inoculum, which was homologous to the 
vaccine. The vaccine induced functional antibodies which were able to inhibit growth in vitro in a 
growth inhibition activity (GIA) assay, but demonstrated no efficacy with all vaccinees and controls 
developing blood-stage malaria. There was no effect on PMR in vaccinees compared with the 
infectivity control group. However, this trial demonstrated the reproducibility of the blood-stage 
CHMI model, with much larger group sizes than used in previous studies. The study also 
demonstrated its utility for measuring modest reductions in PMR in comparison to mosquito-bite 
CHMI where analysis of historical data showed a higher dispersion of data in the infectivity controls 
[22]. 
VAC063 looked at the safety, immunogenicity and efficacy of the blood-stage vaccine candidate 
RH5.1 given with the AS01 Adjuvant System. Fifty healthy adult volunteers were enrolled into the 
trial and the RH5.1/AS01B formulation was administered using a range of RH5.1 protein vaccine 
doses (2, 10, and 50 μg) and was found to be safe and well tolerated. A regimen using a delayed 
and fractional third dose, in contrast to three doses given at monthly intervals, led to significantly 
improved antibody response longevity over ∼2 years of follow-up. Following primary and 
secondary CHMI of vaccinees with blood-stage P. falciparum, a significant reduction in parasite 
growth rate was observed, defining a milestone for the blood-stage malaria vaccine field. The trial 
also demonstrated that growth inhibition activity (GIA) measured in vitro using purified 
immunoglobulin G (IgG) antibody strongly correlates with in vivo reduction of the parasite growth 
rate [59]. 
VAC063C was a follow-on study to VAC063 to assess repeat challenge with P. falciparum. 6 
volunteers previously enrolled in VAC063 underwent tertiary challenge, 2 volunteers previously 
enrolled in VAC063 underwent secondary challenge and 3 volunteers underwent primary 
challenge. No SAEs related to the malaria challenge occurred during the trial. Re-challenge resulted 
in no anti-parasitic immunity and no obvious change in the clinical symptom profile of participants. 
Two volunteers undergoing primary challenge developed transaminitis, one of whom had severe 
elevation of ALT and GGT and was mildly symptomatic. Coagulation and bilirubin remained normal 
and the transaminitis resolved spontaneously within 4 weeks. However, there was barely any 
deviation from baseline in liver function tests in those participants undergoing a secondary or 
tertiary infection, and this was independent of parasite load. This may indicate that the risk of 
tissue damage and injury can be reduced in the absence of parasitic control (Sandoval et al., 
unpublished data; Themistocleous et al., unpublished data).   
5.9 
Conduct of CHMI trials 
Following a collaborative consensus process involving investigators from the USMMVP, Sanaria, 
University of Maryland, University of Oxford, RUNMC, The Seattle Biomedical Research Institute 
and the KEMRI-Wellcome Kilifi Research Programme, a consensus document; “Standardization of 
Design and Conduct of P. falciparum Sporozoite Challenge Trials” was developed, and provides a 
comprehensive guide to the appropriate conduct of sporozoite CHMI studies [36]. There is no 
equivalent document for controlled blood-stage infection or for P. vivax challenge, however, this 
study will be conducted in line with the following key points.  

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 All volunteers should have a medical assessment no longer than 48 hours before 
challenge, including an interim medical history, directed physical examination, pregnancy 
test for female volunteers. 
 Volunteers should be questioned about the occurrence of adverse events and use of 
medication at each follow-up visit. 
 In the event that a volunteer does not attend a scheduled follow-up visit it is imperative 
that Investigators find that volunteer as quickly as possible and assess them for patent 
parasitaemia and clinical malaria. Should the volunteer withdraw consent from further 
follow-up prior to receipt of antimalarial drugs, it may be appropriate to withdraw the 
volunteer from the trial protocol and administer a course of antimalarial chemotherapy 
under close supervision.  
 Grading and reporting of adverse events should be performed using international and 
local guidelines. It should be noted that the occurrence of a low frequency of grade 3 
severe adverse events, of short duration, and with no long-term sequelae, is not 
unexpected in CHMI studies. A minority of those challenged are known to experience 
grade 3 systemic adverse events and this fact should be included in the informed consent 
form. 
 Vital signs should be recorded at least once daily from day 6 post-CHMI (or sooner if any 
clinical concerns) and at any subsequent visits for medical attention. Directed physical 
examination should be performed when necessary.  
 It is critical that every volunteer must receive every dose of anti-malarial therapy. In some 
settings fully directly observed treatment will be essential. Where directly observed 
treatment is not used, Investigators must follow volunteers closely to ensure compliance 
with the treatment regimen.  
 After challenge, all volunteers should be followed until they have completely finished 
anti-malaria treatment.  
 Volunteers should be evaluated at least two weeks after finishing treatment. 
 A local safety monitor and an independent safety monitoring committee should be 
established to act as independent experts in evaluating adverse events. The safety 
monitor or monitoring committee may advise the Investigators on initiating anti-malarial 
treatment for a specific volunteer or volunteer group. While safety monitoring 
committees are not a requirement for Phase I trials, they should be considered a 
requirement for CHMI trials which have an efficacy/human challenge component and 
which have major potential safety concerns. 
5.9.1 Clinical presentation post-CHMI 
Nearly all unvaccinated volunteers in CHMI studies develop symptoms of clinical malaria 
infection; based on data predominately from P. falciparum challenge studies approximately one-
fifth of volunteers temporarily develop symptoms graded as severe (symptoms that prevent daily 
activities), but severe or life-threatening malaria has never occurred [60]. The expected time-
course, symptoms and management of clinical malaria are outlined in Section 9.  
Routine laboratory checks generally show a moderate decrease in leukocyte and platelet 
numbers during infection, with no change in haemoglobin concentration [61]. Bleeding or 
thrombogenic complications have never been described [60, 61]. Abnormalities of liver enzymes 
have been observed, but these abnormalities have rarely resulted in clinical manifestations (just 
one volunteer with raised ALT associated with abdominal pain and vomiting in the recent RCT 
from Cali [44]) and they resolved after a few days.  

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As of 2011, human malaria challenge infections have been conducted with P. falciparum in over 
1,300 volunteers [60-62]. In 2009, safety concerns were raised when young volunteer suffered a 
cardiac event shortly after treatment for diagnosed malaria, following a P. falciparum challenge. 
This was diagnosed as probable myopericarditis, although ischaemia could not be ruled out. 
Although a definite relationship between the cardiac event, which resolved fully and rapidly, and 
the experimental malaria infection was not established [63], it has been generally agreed that 
volunteers with an increased risk of cardiac disease should be excluded from such trials [36]. A 
further case of myopericarditis has since been identified in a recent P. falciparum CHMI study, 
also at the Nijmegen, Netherlands centre, but in this case the individual was also diagnosed with 
an intercurrent rhinovirus infection so that the relation to malaria infection is again uncertain. 
There was a brief episode of clinical chest pain and the volunteer made a full recovery [64]. 
5.9.2 Ethical considerations of CHMI trials 
Participants in CHMI trials are healthy volunteers who do not obtain direct health benefit from 
participation. Challenge trial investigators must exercise all possible safeguards for volunteer 
safety to ensure that trial participation is of minimal risk. Investigators must also ensure that 
maximal scientific benefit accrues from each challenge trial. Key ethical considerations agreed by 
consensus of the field are outlined in a review by Moorthy from 2011, and include; [36]   
1. Volunteer safety is the paramount consideration in conduct of CHMI trials.  
2. Adherence to both international and local guidelines with respect to ethical 
considerations and in accordance with the Declaration of Helsinki and local regulatory 
and ethics committee requirements.  
3. CHMI trials should be conducted according to ICH and/or WHO Good Clinical Practice 
Guidelines with the aim of maximising scientific benefit whilst minimising risk. 
4. The raw data (both microscopy and PCR where available) from challenge trial datasets 
should be made publicly available to facilitate scientific benefit to the community.  
5. If an unexpected SAE which is possibly related to CHMI occurs at a challenge trial centre, 
recognising legal restrictions, every effort should be made to communicate information 
on this SAE to the community of challenge trial centres within 90 days of the occurrence 
of the SAE.  
5.9.3 Oxford’s experience conducting CHMI trials 
The University of Oxford has been conducting CHMI studies (with P. falciparum) for the last 14 
years. To date, more than 400 volunteers have undergone CHMI in studies conducted by the 
University of Oxford, including more than 100 unvaccinated control volunteers. An analysis has 
shown that the symptoms of malaria experienced by volunteers undergoing sporozoite challenge 
by mosquito bite in Oxford are broadly similar to those experienced by volunteers challenged by 
mosquito bite at Radboud University Nijmegen Medical Center (RUNMC) and the US Military 
Malaria Vaccine Program (USMMVP) [65]. In Oxford, four volunteers amongst >400 individual 
challengees have required admission to hospital for observation following challenge (SAE related 
to CHMI). All of these subjects made a complete recovery. Oxford has never before conducted a 
blood-stage P. vivax challenge study, but we have based our study design on that of the previous 
P. vivax blood-stage challenge studies in Australia, combined with our own extensive experience 
of P. falciparum sporozoite challenge studies and our recent VAC068 P. vivax sporozoite 
challenge study.  
In one Phase I/IIa sporozoite challenge study assessing the efficacy of viral vectored malaria 
vaccines in Oxford (VAC039; Clinicaltrials.gov reference: NCT01142765), a volunteer who 
underwent sporozoite challenge on 1st October 2010 failed to attend his next scheduled study 

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visit on 7th October 2010 [66]. The police were immediately informed and began a nationwide 
search for the individual. All volunteers had been informed at screening that the police would be 
notified should they go missing following CHMI if they had not completed a full course of an 
appropriate anti-malarial treatment. The volunteer was found in the Netherlands by the local 
police 17 days following CHMI. He then had very mild malaria symptoms. He was admitted to a 
local hospital where he received appropriate treatment for P. falciparum. He had no signs of 
severe malaria but showed an altered mental state considered unrelated to malaria with 
apparent memory loss and suicidal ideation. He was therefore transferred for in-patient 
psychiatric assessment and discharged a few days later to his GP’s care. He has subsequently 
been reportedly diagnosed as schizophrenic. 
It emerged that from 2nd October 2010, the day after CHMI, the volunteer had experienced an 
alteration in his expected behaviour following his arrest the previous evening by the police 
relating to their investigation of a serious crime, of which he was later convicted. This arrest 
appeared to trigger his leaving home and disappearance and appears relevant to the subsequent 
finding of memory loss and suicidal ideation. It emerged on subsequent investigation that the 
volunteer actually had a history of some psychiatric morbidity pre-dating his involvement in the 
study by many years, which was not disclosed at screening by the volunteer or his GP. Of note, 
the volunteer had attended 9 clinic visits prior to challenge and appeared a reliable and 
appropriate volunteer.  
This event was extensively discussed with investigators, colleagues and appropriate authorities 
and non-study related causality agreed. Given the exceptional circumstances relating to this case, 
it seems very unlikely that a similar event would happen again in the future. Management of the 
event was extensively reviewed by the trial’s sponsor, the MHRA and ethical committee who felt 
that appropriate and timely action was taken. Follow-up procedures following CHMI have been 
reviewed locally, and it has been decided that for future studies volunteers should be contacted 
daily on days 1-5 post sporozoite challenge in order to make sure they are contactable and well. 
Blood-stage challenge model volunteers are seen from Day 1 post-challenge and therefore 
followed even more closely initially.      
5.9.4 Conducting CHMI in the context of the COVID-19 pandemic 
Due to the COVID-19 pandemic this trial was temporarily halted in March 2020. The incidence of 
COVID-19 continues to change in England and some ongoing transmission is likely to remain 
when this trial restarts in 2021 [67]. Each phase of this malaria challenge trial will only proceed, 
when rates of COVID-19 infections in England are low and the risk of contracting COVID-19 
during malaria challenge is small, however the risk cannot be eliminated. There is currently no 
evidence for the possible effects of co-infection with malaria and COVID-19. As the risk of co-
infection cannot be quantified, participants will be tested for COVID-19 prior to malaria challenge 
and any participant that tests positive for COVID-19 will not proceed with malaria challenge.  
Participants who test positive for COVID-19 will be advised to self-isolate as per UKHSA guidance 
and directed to local NHS services for further care if required. Positive COVID-19 PCR results will 
be reported as a notifiable disease as per UKHSA guidance. 
Fevers which occur following malaria challenge may pose diagnostic difficulties as concurrent 
COVID-19 infection will likely remain an ongoing possibility in England. Data from the 12 primary 
and 4 secondary CHMI with P. vivax conducted in Oxford to date, show that no participants 
developed symptoms that are consistent with malaria within the first week post challenge. 
Pyrexia or subjective feelings of feverishness occurred in only half of the participants prior to 
diagnosis and treatment of malaria. However the majority of remaining participants who were 
initially apyrexial, did subsequently develop fever after commencing malaria treatment, although 

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this was short lived and resolved within 2 days. Following malaria challenge, participants first 
developed pyrexia or subjective feelings of feverishness between days 10 to 20 post challenge 
(median 14 days), with corresponding malaria qPCR levels of 100 to 9600 genome copies/mL 
(median 2700 genome copies/mL). No recorded pyrexia of >37.8°C occurred at a malaria qPCR 
below 1000 genome copies/mL in any participant. Malaria diagnosis was made in participants 
within a few days following the onset of symptoms with some participants reporting no 
symptoms prior to laboratory diagnosis. Participants were diagnosed with malaria based on a 
combination of clinical symptoms, malaria qPCR and thick film microscopy between days 12 and 
21 post challenge (median 15.5 days).  
Fevers within the first week after malaria challenge are therefore unlikely to be due to malaria. 
After the first week post challenge, malaria as the cause of fever becomes increasingly likely with 
increasing time since challenge. However, as concurrent COVID-19 infection will likely remain a 
possibility, testing for COVID-19 has been added in the post-challenge period for participants 
who become febrile. For participant safety, if a participant tests positive for COVID-19 prior to 
malaria diagnosis, they will be treated for malaria regardless of if they have reached the criteria 
for malaria diagnosis.  
There is no evidence for any detrimental effect of anti-malarial medication on the outcome of 
concurrent COVID-19 infection. This is in the context of widespread antimalarial use during the 
COVID-19 pandemic in Africa, where no safety signal has emerged. Recent randomised controlled 
trials have shown no benefit of Hydroxychloroquine in COVID-19 [68], but also no harm, and 
Chloroquine will not be used for malaria treatment in this trial. The anti-malarial medications 
Riamet and Malarone are not expected to have any effects on the outcome of COVID-19 disease. 
Social distancing requirements to control COVID-19 will likely remain in place to some extent in 
England. To abide with these, participants’ attendances at study clinics will be carefully planned 
to adhere to social distancing guidelines. Participant flow will be arranged so that contact with 
other people is minimised and level 1 Personal Protective Equipment (PPE - compromising apron, 
gloves, surgical mask and eye protection) will be worn by members of clinical staff for all 
participant visits. This will follow an infection control SOP that covers all PPE requirements for 
clinical trials at the Oxford Vaccine Centre in the era of the COVID-19 pandemic. 
As the national rollout of COVID-19 vaccines proceeds, some participants may be offered a 
COVID-19 vaccine around the time of planned malaria challenge. It is unknown what effect 
concomitant COVID-19 vaccine administration around the time of CHMI may have on the 
immune response to malaria infection, which is being studied in this trial. In addition 
reactogenicity from COVID-19 vaccination may be difficult to distinguish from symptoms of 
malaria and may lead to earlier diagnosis and treatment of malaria after challenge. Therefore 
concomitant COVID-19 vaccination around the time of CHMI has been added as an exclusion 
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6 DESCRIPTION AND JUSTIFICATION OF STUDY DESIGN  
6.1 
Overview 
This is a blood-stage P. vivax human challenge study with the primary aim of assessing the safety 
and feasibility of the challenge model using this bank of cryopreserved P. vivax infected 
erythrocytes for the first time. This will be assessed in VAC069A at three different doses of 
inoculum, by injection at three dilutions, with a view to identifying the lowest concentration 
producing a reliable infection within a practicable timeframe. A secondary objective is to 
determine the safety and feasibility of secondary and tertiary homologous controlled blood-
stage P. vivax infection. If safe and feasible, this study will serve as the basis for a challenge 
model for future P. vivax candidate vaccine efficacy studies, as well as re-challenge studies 
without vaccination.  
Healthy, malaria-naïve adults aged between 18 and 50 years will be recruited at the CCVTM, 
Oxford. CHMI will be induced by injection of P. vivax infected erythrocytes and all follow-up in 
the post-challenge period will be performed at the CCVTM in Oxford. 
Volunteers will have blood taken at regular intervals post-CHMI to assess the immune response 
to primary, secondary and tertiary P. vivax infection, as well as parasite growth dynamics and 
gametocytaemia. Depending on the observed gametocytaemia and availability of laboratory 
strains of the appropriate Anopheline mosquito vectors, we will also assess transmission of P. 
vivax gametocytes from the infected volunteers to mosquitos. If performed, this will be done by 
taking blood for Direct Membrane Feeding Assays (DMFA) and/or by direct feeding of the 
mosquitos on the volunteers’ skin at, or as close as possible to, peak gametocytaemia. 
Close monitoring will continue until volunteers meet criteria for treatment or until 28 days after 
challenge, when treatment will be started empirically.  
Therapy will be with either a standard 60-hour course of oral artemether-lumefantrine (Riamet®) 
or 3 day course of oral atovaquone-proguanil (Malarone). 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 need for radical cure with primaquine therapy.  
Follow-up will be for up to 96 days after the last challenge via clinic visits.  
In the final phase of the study VAC069E, volunteers who have previously undergone primary or 
secondary challenge with P. vivax will undergo a repeat challenge with P. falciparum. The aim of 
this is to study if the development of clinical immunity following primary infection with P. vivax 
also extends to another Plasmodium species ie P. falciparum.  
6.2 
Study groups 
Up to twenty-four volunteers will be recruited through the five phases of the study VAC069A-E 
(see Table 4). Each stage of study (VAC069A-E) comprises a CHMI, involving up to three groups, 
and will take place sequentially at intervals of five to eighteen months, as described below. A 
new group of volunteers will be recruited at each stage (VAC069A-D) to undergo primary CHMI in 
parallel with secondary or tertiary CHMI of volunteers already enrolled in the study. VAC069E will 
not recruit new volunteers. Instead, volunteers who completed at least one previous CHMI with 
P. vivax during VAC069D will be re-enrolled. Recruitment for VAC069A-D is summarised as 
follows:  
VAC069A: 6 volunteers recruited into Groups 1-3 (plus two back-up volunteers) 
VAC069B: 2-3 new volunteers recruited into Group 6 (plus one back-up volunteer) 
VAC069C: 4-8 new volunteers recruited into Group 9 (plus up to four back-up volunteers) 

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VAC069D: 4-8 new volunteers recruited into Group 12 (plus up to four back-up volunteers) 
Back-up volunteers may take the place of another volunteer, should they withdraw or become 
ineligible prior to challenge.  
When first enrolled in the study, volunteers will be recruited into either Groups 1-3, 6, 9 or 12 
and will undergo primary CHMI. Following primary CHMI, enrolled volunteers will enter a new 
group and partake in a new phase of the study, as follows: volunteers initially in Groups 1-3, will 
enter Group 4 (secondary CHMI) and subsequently, Group 5 (tertiary CHMI); volunteers recruited 
into Group 6 will enter Group 7 (secondary CHMI) and then Group 8 (tertiary CHMI); volunteers 
recruited into Group 9, will subsequently enter Groups 10 (secondary CHMI). Volunteers in 
Groups 10 and 12 who completed at least one CHMI with P. vivax during VAC069D will be re-
enrolled into Group 13 to undergo a heterologous re-challenge with P. falciparum. 
During phases VAC069B-D, recruitment will only be open to one new group per phase of the 
study, therefore, volunteers will not be able to choose the group to which they are allocated.  
In March 2020 due the COVID-19 pandemic, this trial was temporarily halted. At the time of trial 
halt VAC069A and VAC069B had been completed. Participants in Groups 1-3 had completed their 
primary CHMI as part of VAC069A and these participants formed Group 4 who underwent 
secondary CHMI as part of VAC069B. Participants in Group 6 had completed primary CHMI as 
part of VAC069B.  
On restarting the trial no new participants will be recruited into the groups from VAC069A and B, 
only those participants who have already undergone primary or secondary CHMI as part of 
VAC069A and VAC069B will continue to undergo secondary and tertiary CHMI respectively. 
VAC069C will be delayed by about twelve months. VAC069C Group 9 will be reduced in size for 
logistical reasons to ensure compliance with social distancing. To compensate numbers and 
ensure we have enough volunteers going through three successive challenges, VAC069D Group 
12 has been increased in size and this group will now subsequently undergo secondary and 
tertiary CHMI, in addition to the originally planned primary CHMI. However, the increased size of 
this group will not affect the total number of participants undergoing challenge at the same time 
as the VAC069D phase of the study because the number of vaccinees from VAC071 and VAC079 
studies, who will be undergoing challenge in parallel to VAC069D, will be reduced.  
Following completion of the CHMI in VAC069D in October 2021, results from phases of the study 
completed to date indicated that no anti-parasitic immunity develops upon re-challenge with the 
same clonal P. vivax inoculum. However, clinical immunity developed after the first infection and 
upon homologous secondary challenge, volunteers had reduced clinical symptoms and signs of 
malaria. In order to study if the clinical immunity that develops following primary infection with 
P. vivax also extends to other Plasmodium species, repeat challenge will be conducted with P. 
falciparum. In the next phase VAC069E, volunteers who completed at least one CHMI with P. 
vivax in VAC069D will be re-enrolled and undergo a final CHMI with P. falciparum. 
6.2.1 VAC069A Initial challenge and dose-finding: Groups 1 (n=2), 2 (n=2) and 3 (n=2). 
Six volunteers will be recruited into three groups, with two back-up volunteers who may take the 
place of a volunteer in any group, should they withdraw or become ineligible prior to challenge. 
Should they wish, volunteers will be able to choose the group to which they are allocated, 
provided the group has not yet been filled. Otherwise, volunteers will be allocated to groups on a 
sequential basis, from Group1 to Group 3. 
Each group will comprise two volunteers. All groups will receive an inoculum of parasitised red 
blood cells, reconstituted in 0.9% normal saline, to a total volume of 5mL. Group 1 will receive a 

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whole vial’s worth of infected erythrocytes (starting total volume 1.5mL, containing 0.5mL 
packed red blood cells and 1mL Glycerolyte – these cells are thawed and extensively washed to 
remove Glycerolyte prior to resuspension in saline). Group 2 will receive one fifth of the dose of 
parasitised erythrocytes, at a 1:5 dilution in 0.9% normal saline, and Group 3 will be inoculated 
with one twentieth of the dose, at a 1:20 dilution in 0.9% normal saline. This will constitute the 
primary blood-stage challenge. Later challenges will be conducted at the lowest concentration 
proving potency and feasibility within this first phase of the study (see Tables 3 and 4).   
6.2.2 VAC069B Secondary re-challenge with new primary controls: Groups 4 (n=6) and 6 (n=2-
3). 
Approximately eight months (and up to nine months) later, the six volunteers from Groups 1, 2 
and 3 in VAC069A will undergo secondary challenge using the optimal inoculum as determined in 
that first phase of the study. They will constitute ‘Group 4’ in this phase of the study. Three new 
malaria naïve volunteers, as well as a one volunteer who will act as a back-up, will be recruited 
(Group 6) to undergo primary challenge and act as controls to Group 4. The three malaria-naïve 
volunteers in Group 6 will also act as the non-vaccinated control group for three vaccinated 
volunteers in a parallel vaccine efficacy trial, known as VAC071.   
 VAC071 will be a Phase IIa study to assess efficacy of the chimpanzee adenovirus serotype 63 
(ChAd63)/modified vaccinia virus Ankara (MVA) viral-vectored vaccine candidate encoding the P. 
vivax Duffy-binding protein region II (PvDBP_RII), in a heterologous prime-boost regimen. The 
ChAd63-PvDBP/MVA-PvDBP vaccine candidate has proven safety and tolerability, as well as 
induction of strain-transcending antibodies in an open-label dose-escalation Phase Ia study [37].  
6.2.3 VAC069C Tertiary (n=6) and secondary (n=2) re-challenges with new primary challenge 
controls (n=4-8): Groups 5, 7 and 9. 
Approximately sixteen to twenty months later any volunteers remaining in the study from Group 
4 in VAC069B will undergo tertiary challenge, again using the optimal inoculum as determined in 
VAC069A and will constitute Group 5 in this third phase of the study. Any remaining volunteers 
who underwent primary challenge in Group 6 will now form Group 7 and undergo secondary 
challenge. Four to eight new malaria-naïve volunteers will be recruited (Group 9) and act as 
controls for Groups 5 and 7 and for vaccinated volunteers undergoing primary CHMI as part of 
VAC071 and VAC079. In addition, up to four volunteers will be recruited to act as back-ups for 
Group 9. 
VAC079 will be a Phase IIa study to assess the safety and efficacy of the protein subunit vaccine 
PvDBP_RII administered with the adjuvant Matrix M. The safety and immunogenicity of 
PvDBP_RII has previously been shown with adjuvant GLA-SE (made by IDRI, Seattle, USA) in a 
Phase Ia clinical trial in India, and induces strain-transcending functional antibodies [69]. The 
safety profile of Matrix M with other malaria protein subunit vaccines is excellent, as shown in a 
previous clinical trial [70] but it has not yet been tested in combination with PvDBP_RII in 
humans. Challenge of vaccinated volunteers in the VAC071 and VAC079 studies will occur in 
parallel for comparative assessment of vaccine efficacy. Where consent is obtained, the data 
generated from the challenge of malaria-naïve controls within the VAC069 study will be utilised 
on an anonymous basis, for comparative analysis with the challenge of vaccinated volunteers in 
VAC071 and VAC079.  
Conduct of secondary and tertiary CHMI in parallel with these control groups, will permit direct 
comparison across the two, or three, groups respectively, allowing adequate assessment of 
safety and feasibility of secondary and tertiary challenge, as well as immune responses and 
gametocytaemia during secondary and tertiary CHMI.  

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6.2.4 VAC069D Tertiary (n=2) and secondary (n=4-8) re-challenges with new primary challenge 
controls (n=4-8): Groups 8, 10 and 12. 
Between five to ten months later the remaining volunteers from Group 7 in VAC069C will 
undergo tertiary challenge and constitute Group 8 in this fourth phase of the study.  The four to 
eight volunteers from Group 9 in VAC069C will undergo secondary challenge and form Group 10 
in this phase of the study. Four to eight new malaria-naïve volunteers will be recruited to form 
Group 12 and undergo primary CHMI. They will act as naïve controls for Groups 8 and 10 and as 
controls for vaccinated volunteers undergoing primary CHMI as part of VAC071 and VAC079. Up 
to four back-up volunteer will also be recruited for Group 12. 
6.2.5 VAC069E Re-challenge with P. falciparum: Group 13. 
In this final phase of the study scheduled for eight to fourteen months after VAC069D, volunteers 
in Groups 10 and 12 who completed at least one P. vivax CHMI in VAC069D will be re-enrolled 
into Group 13 to undergo repeat CHMI with P. falciparum instead. There will be no volunteers 
undergoing primary challenge in this phase. 
 
 

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Table 3: Study groups. CHMI = controlled human malaria infection. Parasitised red blood cells will be 
reconstituted to a total volume of 5mL normal saline (0.9%) (one inoculum vial contains approximately 
0.5mL cryopreserved packed red blood cells). Secondary and tertiary CHMI will take place between 
approximately 5 to 14 months after the preceding challenge. CHMI during phases B, C and D will take 
place in parallel with primary challenge of malaria naïve vaccinated volunteers recruited as part of the 
VAC071 and VAC079 studies. 
 
Month (approx.) 
0 
9 
26 
34 
40 
Months between CHMI 
0 
9 
16-24 
5-10 
8-14 
Phase of Study 
A 
B 
C 
D 
E 
Group 
Group size 
Group 1  
2 
1
o CHMI: 1 vial 
  
  
  
  
Group 2  
2 
1
o CHMI: 1:5 
  
  
  
  
Group 3  
2 
1
o CHMI: 1:20 
  
  
  
  
Group 4  
(previously groups 1-3) 
3 
  
2
o CHMI 
  
  
  
Group 5  
(previously group 4)  
1-2 
  
  
3
o CHMI 
  
  
Group 6   
2 
  
1
o CHMI 
  
  
  
Group 7   
(previously group 6) 
1-2 
  
  
2
o CHMI 
  
  
Group 8  
 (previously group 7) 
1-2 
  
  
  
3
o CHMI 
  
Group 9   
4-8 
  
  
1
o CHMI 
  
  
Group 10  
(previously group 9) 
4-8 
  
  
  
2
o CHMI 
  
Group 12  
4-8 
  
  
  
1
o CHMI 
 
Group 13  
(previously groups 10 
and 12) 
4-12 
 
 
 
 
P. falciparum 
CHMI  

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Table 4: Study phases. CHMI = controlled human malaria infection. Repeat CHMI for Groups 4-13 
will take place approximately 5 to 14 months after the preceding challenge. 
 
6.3 
Duration of volunteer participation 
The duration of involvement in the study, from enrolment until the end of the follow-up 
following the third CHMI, will be approximately 30 months for Groups 1-8. This has been 
extended by approximately 12 months because of the temporary trial halt due to the COVID-19 
pandemic. Duration of involvement for participation in groups 9-12 who are undergoing three 
CHMI will be approximately 18 months. Follow-up for participants in group 13 who will undergo 
repeat CHMI with P. falciparum will be 3 months. All volunteers will be followed for 3 months 
after their last challenge. 
6.3.1 Definition of the start and end of the trial  
The start of the trial is defined as the date of the site initiation visit (SIV). The end of the trial is 
the date of the last follow-up of the last volunteer. 
 
6.4 
Potential risks for volunteers 
6.4.1 Phlebotomy  
The maximum volume of blood drawn over the study period should not compromise these 
otherwise healthy volunteers. There may be minor bruising, local tenderness or pre-syncopal 
symptoms associated with venepuncture, which will not be documented as AEs if they occur. The 
total volume of blood taken over the study period will relate to the number of challenges 
Phase of 
study 
Month 
(approx.) 
Max Total 
participants 
Groups  
(previous Group) 
Group 
size 
Intervention 
VAC069A 
0 
6 
1 
2 
1
o CHMI 
2 
2 
1
o CHMI 
3 
2 
1
o CHMI 
VAC069B 
9 
9 
4  
(previously 1-3) 
6 
2
o CHMI 
6 
2-3 
1
o CHMI 
VAC069C 
26 
12 
5 
(previously 4) 
1-6 
3
o CHMI 
7 
(previously 6) 
1-2 
2
o CHMI 
9 
4-8 
1
o CHMI 
VAC069D 
34 
18 
8  
(previously 7) 
1-2 
3
o CHMI 
10  
(previously 9) 
4-8 
2
o CHMI 
12 
4-8 
1
o CHMI 
VAC069E 
45 
12 
13  
(previously 10 and 12) 
4-12 
Repeat CHMI 
with P. 
falciparum 

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participated in by the volunteer. Volunteers will have a maximum of 1042 mL taken for each 
challenge taken over the challenge period (minimum 96 days). At each further challenge, a 
maximum of 1042mL would be taken, beginning at least five months after the preceding 
challenge. Male regular blood donors may donate one unit (470mL) every 12 weeks and females 
every 18 weeks. However, a recent multicentre study by NHS Blood and Transplant (in Oxford 
and Cambridge) compared outcomes in 45,000 regular blood donors who were randomised to 
different intervals between blood donations (as regularly as giving 470 mL every 8 weeks in male 
participant group and every 12 week in females) over 2 years[71]. There were no significant 
differences were observed in quality of life, physical activity, or cognitive function although there 
were more symptoms related to blood donation, in groups donating more frequently, including 
tiredness, breathlessness, feeling faint, dizziness and restless legs. Symptoms were most 
increased among male participants.  Lower mean haemoglobin and ferritin concentrations were 
also detected.  
In this study, participants will never donate 470mL of blood in one sitting; the maximum volume 
they will donate in a single visit is 149mL so we would not expect them to report a high 
frequency of symptoms at the time of or just after blood donation. They will be closely 
monitored at all times of blood donation, and haemoglobin will be checked regularly, as 
described in the study procedures. Any abnormal result will be re-checked and referred to the GP 
for further investigation and management, as deemed clinically appropriate by the Investigator.  
The maximum total blood volume drawn in this study will be 2971mL over approximately 18 
months. For male participants, this is 1259mL less than the 8-week interval group in the NHSBT 
study and for females, 151mL more than the 12-week interval group, over the 18 month period.  
However, this maximum volume assumes that each participant is not treated until C+28 for each 
of the three challenges they participate in. In practice, given volunteers are malaria-naïve at 
enrolment they are likely to be diagnosed between C+14 and C+16 in their primary challenge. 
This means the blood volume scheduled to be collected between C+17 and C+28 (192mL) is 
unlikely to be drawn. Therefore, we are confident that the actual volume of blood collected 
during the study period should not compromise our volunteers. However, the additional effect of 
malarial infection on potential anaemia is acknowledged. For this reason, the full blood count will 
be closely monitored throughout the study period. If a participant develops anaemia or there is 
any clinical concern, they will be reviewed by a physician and iron studies may be performed. 
Participant safety will be of paramount concern, and if there is any concern that a volunteer 
cannot safely tolerate the planned blood draws, the volume of blood collected from them may 
be reduced or the participant withdrawn from the study.  
6.4.2 Risk of Infection with Blood Borne Organisms  
This controlled blood-stage challenge will involve a blood transfusion, albeit of very small volume 
relative to standard blood transfusion. As for any transfusion, the risk of transmission of a blood 
borne infection cannot be completely eliminated. However, in order to minimise the risk, 
extensive screening for blood borne infections has been performed. 
Regarding the P. vivax blood bank, both direct blood donors, as well as the initial source patient 
in Thailand, who provided blood for the mosquito infection by direct membrane feeding have 
undergone testing for blood borne infections. For viral infections and syphilis, testing was either 
serological or by nucleic acid amplification methods, in accordance with the Joint UK Blood 
Transfusion and Tissue Transplantation Services Professional Advisory Committee guidelines. The 
source patient was screened for antibodies to W. bancrofti at the Thai field site.  
In addition, the blood bank has been subjected to direct screening and tested negative for 
bacterial contamination, sterility testing, endotoxin screen and mycoplasma specific culture. 

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Plasma derived from the bank has also been screened for blood borne infections, as detailed in 
Section 5.8.4.and has tested negative for all blood-borne infection screens by PCR.  Further to 
this, as outlined in section 5.7.4, volunteers also underwent repeat serological testing for HIV, 
hepatitis B and C, syphilis, HITLV-1 and HTLV-2 testing was undertaken 90 days after challenge, to 
ensure that no seroconversion from a recently-acquired infection (that may have been 
undetectable around the time of challenge and therefore undetectable in the bank) had occurred 
since the challenge period. All tests remained negative. Since both donors have been resident in 
the UK or continental Europe in countries where Bovine Spongiform Encephalopathy (BSE) has 
been reported, there is a theoretical risk of transmission of variant Creutzfeldt Jacob Disease 
(vCJD) from the inoculum. This risk is reduced by leukodepletion, as is practised in UK blood 
donation, according to the Joint UK Blood Transfusion and Tissue Transplantation Services 
Professional Advisory Committee guidelines. Since universal leukodepletion was introduced in 
the UK in 1999, no cases of transmission of vCJD by transfusion have been recorded [72]. The risk 
of infection is further reduced by the very small size of the inoculum.  
Regarding the P. falciparum blood bank, the donor was screened for a range of blood borne 
infections prior to the inoculum being collected, and has remained well since the first use of the 
inoculum in 1997 [55]. The donor tested negative for HIV, Hepatitis A, B and C, Syphilis, HTLV1 and 
Ross River virus. The donor was seropositive for Epstein Barr virus (EBV) and Cytomegalovirus 
(CMV), indicating previous infection. The blood was initially frozen for a year whilst the donor was 
monitored and retested. Over 430 volunteers have received the inoculum since 1997 and there 
have been no serious adverse events, and no cases of blood borne infection associated with this 
[73]. There were two individuals who were infected with P. falciparum malaria with the purpose 
of preparing the inoculum but all challenge inocula have since come from one of these donors as 
the other donor had a much lower parasitaemia when blood was collected. 
More recently, an ampoule of the parasite seed stock from the donor was thawed and cultured 
within the Queensland Institute of Medical Research (QIMR) laboratory. Culture supernatant tested 
negative for Mycoplasma by PCR. The blood was also tested for CMV and EBV by PCR using 
methodologies not available at the time of the initial cryopreservation, and tested negative. 
Although the donor remains antibody positive for EBV and CMV, it is apparent that the risk of 
transmission of both CMV and EBV has been greatly reduced by leukodepletion of the donor blood 
(both EBV and CMV cause latent infection of leukocytes) and washing of the blood with clinical 
grade saline prior to use. Initially only CMV and EBV seropositive volunteers were enrolled due to 
a concern about the risk of transmission of these viruses, but the risk has since been deemed 
extremely low. The requirement for CMV and EBV seronegativity was therefore waived by QIMR 
and in the subsequent blood-stage CHMI trials carried out in both Nijmegen [56] and Oxford 
(VAC054), serostatus data were collected but not used as inclusion/exclusion criteria. None of the 
seronegative volunteers in VAC054 or the Nijmegen trial seroconverted during follow-up after 
CHMI. Subsequent Oxford CHMI studies have since stopped excluding volunteers based on EBV 
and/or CMV seronegativity.  
The risk of transmission of variant Creutzfeldt Jacob Disease (vCJD) from the inoculum appears 
remote. The two donors are Australian residents, where no cases of either Bovine Spongiform 
Encephalopathy (BSE) in cattle or vCJD in humans have been reported to date. 
The volume to be transfused for this study will be at least nine hundred times smaller than the 
amount received in typical blood transfusion (at the highest concentration). Serum from 
volunteers will be collected before and after challenge for storage.  

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6.4.3 Plasmodium vivax and falciparum infection  
 
Volunteers are likely to develop symptomatic malaria infection following CHMI. Symptoms and 
signs will include feverishness, fever, tachycardia, hypotension, chills, rigors, sweats, headache, 
anorexia, nausea, vomiting, diarrhoea, myalgia, arthralgia, low back pain, thrombocytopenia and 
lymphopenia [65]. Unmonitored and untreated, P. vivax and P. falciparum infection can be 
serious and rarely fatal and, for this reason, volunteers will be followed up closely post-challenge 
and only enrolled in the study if they are deemed reliable and capable of complying with the 
intensive follow-up schedule. A very small proportion of volunteers in previous P. vivax blood-
stage and sporozoite-stage CHMI studies have temporarily required intravenous fluid therapy for 
nausea and vomiting prior to treatment [44, 48]. If the Investigator judges the volunteer to be 
unwell enough to require intravenous fluids or continuous medical input, they shall be 
transferred to the John Radcliffe Hospital under the care of the NHS Infectious Diseases team 
until they are well enough to be discharged home.  
With regards to the above, it is relevant to note that safety and parasitological data from 128 
malaria-naïve subjects participating in CHMI studies in Oxford and the Netherlands were 
analysed and compared to a report from the US Military Malaria Vaccine Program. The authors 
found that cohorts with a longer prepatent period or a higher peak parasitaemia did not 
consistently show a higher frequency of adverse events [74]. However, these data were on P. 
falciparum challenge, and there is a lot less experience with P. vivax challenge.   
6.4.4 Medications dispensed to volunteers in course of trial  
(a) Treatment of Plasmodium vivax and falciparum Infection 
P. vivax and P. falciparum malaria infection will be treated with either oral Riamet or oral 
Malarone. 
Riamet contains 20mg of artemether and 120mg lumefantrine per tablet. 6 doses of 4 tablets 
will be given; the first dose is followed by additional doses after 8, 24, 36, 48 and 60 hours. If 
there is a contraindication to Riamet, oral Malarone will be used (see SmPC for side effects and 
contraindications to Riamet and Malarone). At a minimum, two doses (the first and third dose) of 
Riamet will be directly observed. The first dose will be observed in clinic and the volunteers will 
not return home until they are seen to be tolerating the medication.  
Riamet is generally well tolerated, but may cause some side effects. Common side effects include 
headache, dizziness, abdominal pain and loss of appetite, sleeping problems, palpitations, 
nausea, vomiting, diarrhoea, pruritus, skin rash, cough, muscle or joint pain and fatigue. 
Volunteers will be counselled that certain side effects, for example dizziness, may impact on the 
performance of skilled tasks such as driving.  
As Riamet may increase the QT interval, Riamet will not be administered to volunteers at risk for 
QT prolongation. Exclusion criteria for this study will include prolonged QT on baseline ECG, a 
history of long QT syndrome, a family history of congenital QT prolongation or sudden death, 
cardiac arrhythmias, severe heart disease, and a history of hypokalaemia or hypomagnesaemia. 
Volunteers will be advised to avoid grapefruit juice whilst taking Riamet as it can affect the 
bioavailability of the artemether [75]. Riamet is also contraindicated in volunteers using 
concomitant medications affecting the QT interval, and will not be given to these volunteers. 
Concomitant use of Riamet may decrease effectiveness of hormonal contraceptives.  
Women using hormonal contraceptives will be advised to use an effective additional and/or 
alternative method of contraception whilst on Riamet treatment, until the start of the next 
menstruation after treatment. 

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Malarone contains 100 mg proguanil hydrochloride and 250 mg atovaquone per tablet. 4 tablets 
will be given once daily for 3 days. The first and second out of the three doses of Malarone will 
be directly observed in clinic. Prior to starting Malarone, volunteers will be screened for drug 
interactions and contraindications (including a serum β-hCG test in female volunteers). Malarone 
is generally well tolerated but may cause some side effects, most commonly headache, 
diarrhoea, nausea, vomiting, stomach pain, dizziness rash, fever, low mood, reduced appetite, 
cough or sleep disturbance.   
(b) Treatment of Symptoms Associated with Malaria 
Volunteers will be dispensed cyclizine and paracetamol for the treatment of symptoms 
associated with malaria unless there are contraindications to these medications. Cyclizine is 
generally well tolerated however there is a risk of an allergic reaction. Other side effects include 
skin rashes or itching, drowsiness, headache, dry mouth nose or throat, blurred vision, 
palpitations, difficulty urinating, constipation, anxiety, insomnia or hallucinations. Rare side 
effects include hypotonia, seizures, dizziness, hypertension, paraesthesia, jaundice, hepatitis, 
confusion or dyskinesias. Participants may be dispensed an appropriate, licensed alternative anti-
emetic to cyclizine if they are unable to take cyclizine. Paracetamol is generally well tolerated 
however it can cause an allergic reaction or rarely pancytopaenia. 
6.4.5 Risk of reaction to the blood sample  
The donor of the blood used in the P. vivax inoculum is Blood Group O, Rhesus (Rh) and Kell 
antigen negative. People with Blood Group O negative blood are considered ‘universal donors’, 
as recipients of their blood are unlikely to develop red cell alloantibodies even when given much 
larger volumes of blood than is envisaged here. The Kell antigen is relatively rare in Caucasian 
populations, but if Kell positive red blood cells were infused into a Kell negative female 
volunteer, there is a theoretical risk that anti-Kell antibodies may induce foetal haemolysis in a 
future pregnancy. For this reason, and to avoid this risk, we ascertained that the donor blood 
used in these studies is Kell negative.  
The donor of the blood used in the P. falciparum inoculum is also Blood Group O, Rhesus and Kell 
negative. 
The maximum volume of blood to be used in this study (0.5mL packed red cells) is much smaller 
than that given in transfusion of one unit packed red cells (470mL). The risk of a transfusion 
reaction or development of antibodies to donor red cells, which may make blood transfusion 
more difficult in the future, cannot be completely excluded but is considered highly unlikely both 
because of the small volume of blood used and because the blood has been leukodepleted. 
Nevertheless, the volunteers will be monitored for this possibility in the period immediately after 
the administration of the malaria parasite dose. A baseline set of observations (respiratory rate, 
heart rate, temperature and blood pressure) will be taken before the administration of the 
inoculum. Volunteers will be reviewed 15 minutes (+/- 5 minutes) and 1 hour (+/- 20 minutes) 
after receiving the inoculum, with a repeat set of observations at each time point, before they 
are able to leave the CCVTM. The serum collected for storage (as described above) could be used 
to test for antibodies at a later date if deemed necessary. 
6.4.6 Mosquito bites (for volunteers partaking in direct feeding assays only) 
Mosquito bites may cause local inflammatory reactions with redness, itching, swelling, scaling 
and/or tenderness. Topical anti-histamine cream for use twice daily for 3 days post-mosquito 
bite will be dispensed to both volunteers on the day of mosquito feeding, according to SOP 
VC021: Handling, Storage and Dispensing of Non-IMP Medication. Serious allergic reactions 

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including anaphylaxis are rare, but may occur and for this reason volunteers will be take part in 
mosquito feeds in an area where Advanced Life Support trained physicians and defibrillator are 
immediately available. In sporozoite CHMI studies, delivered by mosquito bite, no serious allergic 
reactions have been seen to date. Data regarding itching and use of antihistamine cream will be 
collected but, as this is an expected part of the procedure, it will not be classed as an adverse 
event in the immediate post-challenge period. 
6.5 
Potential benefits for volunteers 
Volunteers will not benefit directly from participation in this study. However, it is hoped that this 
proof-of-concept study will provide a basis for future P. vivax challenge studies and evaluation of 
candidate vaccines. Exploratory immunology performed on the volunteers’ blood samples may 
also inform on future vaccine design. Volunteers will also receive information about their general 
health status. 

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7 RECRUITMENT AND WITHDRAWAL FOR TRIAL VOLUNTEERS 
Volunteers may be recruited by use of an advertisement +/- registration form formally approved 
by the ethics committee(s) and distributed or posted in the following places: 
- 
In public places, including buses and trains, with the agreement of the owner / proprietor. 
- 
In newspapers or other literature for circulation. 
- 
On a website operated by the Investigators’ clinical trials group or with the agreement of 
the owner or operator (including on-line recruitment through our website).  
- 
As a post on a Twitter or Facebook or other similar account owned and operated by the 
Investigators’ clinical trials group. 
- 
By e-mail distribution to a group or list only with the express agreement of the network 
administrator or with equivalent authorisation.    
- 
By email distribution to individuals who have already expressed an interest in taking part 
in any clinical trial at the Oxford Vaccine Centre. 
- 
On stalls or stands at exhibitions or fairs. 
- 
Via presentations (e.g. presentations at lectures or invited seminars). 
- 
Oxford Vaccine Centre databases: We may contact individuals from databases of groups 
within the CCVTM (including the Oxford Vaccine Centre database) of previous trial 
participants who have expressed an interest in receiving information about all future 
studies for which they may be eligible. 
7.1 
Informed consent 
7.1.1 Informed consent 
The information sheet will be made available to the volunteer at least 24 hours prior to the 
screening visit. At the screening visit, the volunteer will be fully informed of all aspects of the 
trial, the potential risks and their obligations. The following will be emphasised: 
 Participation in the study is entirely voluntary. 
 Refusal to participate involves no penalty or loss of medical benefits. 
 The volunteer may withdraw from the study at any time. However if the volunteer has 
undergone CHMI and not completed a course of appropriate antimalarial therapy then 
the volunteer will need to maintain contact with the Investigators for monitoring and 
treatment. 
 The volunteer is free to ask questions at any time to allow him or her to understand the 
purpose of the study and the procedures involved. 
 There is no direct benefit from participating. 
 The volunteer’s GP will be contacted to corroborate their medical history and confirm 
that the volunteer is eligible to take part in the study. Volunteers will only be enrolled in 
the study if written information regarding the volunteer’s medical history is obtained 
from the GP. 
 The volunteer will be registered on the TOPS database (The Overvolunteering Prevention 
System; www.tops.org.uk).  
 Blood samples will be taken and any leftover samples stored indefinitely for use in other, 
ethically approved research.  
The aims of the study and all tests to be carried out will be explained. The volunteer will be given 
the opportunity to ask about details of the trial, and will then have time to consider whether or 
not to participate. If they do decide to participate, volunteers will be asked to complete a 
questionnaire testing their understanding of the trial [36]. This helps to ensure that individuals 

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understand the trial sufficiently to give informed consent. Provided the volunteer answers all 
questions in the questionnaire correctly, they will be asked to sign a consent form.  A photocopy 
of the signed consent form will be taken and given to the participant to take away and keep and 
the original will be stored in the CRF. 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. Provided 
they subsequently answer all questions in the quiz correctly they may then complete the consent 
form and be screened for the trial.  
Initial informed consent will include consent for participation in up to three blood-stage CHMIs. 
On-going consent for participation in the study will routinely be assessed verbally at each clinical 
review.  Given the significant time interval between challenges, prior to each of the second and 
third challenge, in order to identify new medical events, a re-screening procedure will be 
followed. At the re-screening visit, the volunteer will be reminded about all aspects of the trial, 
the potential risks and their obligations, with emphasis on the above listed points. The volunteer 
will be given the opportunity to ask about details of the trial, and will have time to consider 
whether or not to continue their participation in the trial.  
7.2  
Inclusion and exclusion criteria  
7.2.1 Inclusion criteria 
The volunteer must satisfy all the following criteria to be eligible for the study: 
 Healthy adult aged 18 to 50 years.  
 Red blood cells positive for the Duffy antigen/chemokine receptor (DARC). 
 Normal serum levels of Glucose-6-phosphate dehydrogenase (G6PDH). 
 Negative haemoglobinopathy screen 
 Able and willing (in the Investigator’s opinion) to comply with all study requirements. 
 Willing to allow the Investigators to discuss the volunteer’s medical history with their 
General Practitioner. 
 Volunteers with the potential to become pregnant only: Must practice continuous 
effective contraception* for the duration of the clinic visits (first 3 months post-CHMI). 
 Agreement to permanently refrain from blood donation 
 Written informed consent to participate in the trial. 
 Reachable (24/7) by mobile phone during the period between CHMI and completion of all 
antimalarial treatment.  
 Willing to take a curative anti-malarial regimen following CHMI.  
 Willing to reside in Oxford for the duration of the study, until antimalarials have been 
completed. 
 Answer all questions on the informed consent quiz correctly.  
* Female volunteers are required to use an effective form of contraception during the course of 
the study as malaria challenge could pose a serious risk to both maternal health and the unborn 
foetus. 
Acceptable forms of contraception for female volunteers include: 
 Established use of oral, injected or implanted hormonal methods of contraception (an 
additional form of contraception will be required when taking the antimalarial 
medication, as this can interfere with the efficacy of hormonal contraception). 
 Placement of an intrauterine device (IUD) or intrauterine system (IUS). 
 Total abdominal hysterectomy. 

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 Barrier methods of contraception (condom or occlusive cap with spermicide). 
 Male sterilisation, if the vasectomised partner is the sole partner for the subject. 
 True abstinence, when this is in line with the preferred and usual lifestyle of the subject 
(periodic abstinence and withdrawal are not acceptable methods of contraception). 
 
7.2.2 Exclusion criteria 
The volunteer may not enter the study if any of the following apply: 
 History of clinical malaria (any species). 
 Travel to a clearly malaria endemic locality during the study period or within the 
preceding six months. 
 Use of systemic antibiotics with known antimalarial activity within 30 days of CHMI (e.g. 
trimethoprim-sulfamethoxazole, doxycycline, tetracycline, clindamycin, erythromycin, 
fluoroquinolones and azithromycin). 
 Haemoglobin <120 g/L for a female volunteer or <130 g/L for a male volunteer prior to 
primary CHMI. (However, for enrolment into secondary and tertiary CHMIs slightly lower 
haemoglobin values (≤0.5 g/L) will be permitted at the discretion of the Investigator, to 
account for the blood volume donated during the previous CHMI). 
 Receipt of immunoglobulins within the three months prior to enrolment.  
 Receipt of blood transfusion at any time in the past.  
 Peripheral venous access unlikely to allow twice daily blood testing (as determined by the 
Investigator). 
 Receipt of an investigational product in the 30 days preceding enrolment, or planned 
receipt during the study period. 
 Prior receipt of an investigational vaccine likely to impact on interpretation of the trial 
data or the P. vivax parasite as assessed by the Investigator.  
 Planned receipt of a COVID-19 vaccine between 2 weeks before the day of CHMI until 
completion of antimalarial treatment 
 Any confirmed or suspected immunosuppressive or immunodeficient state, including HIV 
infection; asplenia; recurrent, severe infections and chronic (more than 14 days) 
immunosuppressant medication within the past 6 months (inhaled and topical steroids 
are allowed). 
 History of allergic disease or reactions likely to be exacerbated by malaria infection. 
 Pregnancy, lactation or intention to become pregnant during the study. 
 Use of medications known to cause prolongation of the QT interval and existing 
contraindication to the use of Malarone. 
 Use of medications known to have a potentially clinically significant interaction with 
Riamet and Malarone. 
 Any clinical condition known to prolong the QT interval. 
 History of cardiac arrhythmia, including clinically relevant bradycardia. 
 Disturbances of electrolyte balance, e.g. hypokalaemia or hypomagnesaemia. 
 Family history of congenital QT prolongation or sudden death. 
 Contraindications to the use of both of the proposed anti-malarial medications Riamet 
and Malarone. 
 History of cancer (except basal cell carcinoma of the skin and cervical carcinoma in situ). 
 History of serious psychiatric condition that may affect participation in the study. 
 Any other serious chronic illness requiring hospital specialist supervision. 

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 Suspected or known current alcohol abuse as defined by an alcohol intake of greater than 
25 standard UK units every week. 
 Suspected or known injecting drug abuse in the 5 years preceding enrolment. 
 Hepatitis B surface antigen (HBsAg) detected in serum. 
 Seropositive for hepatitis C virus (antibodies to HCV) at screening or at C-7 (unless has 
taken part in a prior hepatitis C vaccine study with confirmed negative HCV antibodies 
prior to participation in that study, and negative HCV RNA PCR at screening for this 
study). 
 Positive family history in both 1st AND 2nd degree relatives < 50 years old for cardiac 
disease. 
 Volunteers unable to be closely followed for social, geographic or psychological reasons. 
 Any clinically significant abnormal finding on biochemistry or haematology blood tests, 
urinalysis or clinical examination. In the event of abnormal test results, confirmatory 
repeat tests will be requested. Procedures for identifying laboratory values meeting 
exclusion criteria are shown in Appendix A.  
 Any other significant disease, disorder, or finding which may significantly increase the risk 
to the volunteer because of participation in the study, affect the ability of the volunteer 
to participate in the study or impair interpretation of the study data. 
 Inability of the study team to contact the volunteer’s GP to confirm medical history and 
safety to participate.  
Additional exclusion criteria for Groups 6-13 
 Body weight <50Kg, as measured at screening 
7.2.3 Prevention of ‘Over Volunteering’ 
Volunteers will be excluded from the study if they are concurrently involved in another trial. In 
order to check this, volunteers will be asked to provide their National Insurance or Passport 
number (if they are not entitled to a NI number) and will be registered on a national database of 
participants in clinical trials (www.tops.org.uk). 
7.2.4 Exclusion criteria on day of CHMI 
The following constitute absolute contraindications to CHMI: 
 Acute disease, defined as moderate or severe illness with or without fever. 
 Current COVID-19 infection, defined as ongoing symptoms with positive COVID-19 PCR 
swab test taken during current illness or positive COVID-19 PCR swab test within 
preceding 7 days without symptoms. 
 Pregnancy. 
Potential volunteers who are due to have a COVID-19 vaccine in the period between 2 weeks 
before the day of malaria challenge and expected time of completion of malaria treatment will 
not be enrolled (estimate up to 3 weeks after day of challenge based on experience in this study 
to date). If a volunteer receives an appointment for COVID-19 vaccination after they have 
undergone malaria challenge but before reaching malaria diagnosis criteria, they will be advised 
to delay their COVID-19 vaccination until after reaching malaria diagnostic criteria and 
completion of antimalarial treatment. If a participant does not wish to delay their COVID-19 
vaccination, they will be treated with antimalarial treatment at that time point and withdrawn 
from the study but will be encouraged to complete follow-up for safety. 
 

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7.3 
Withdrawal of volunteers 
In accordance with the principles of the most recent revision of the Declaration of Helsinki (2013) 
and any other applicable regulations, a volunteer has the right to withdraw from the study at any 
time and for any reason, and is not obliged to give his or her reasons for doing so. In addition the 
volunteer may withdraw/be withdrawn from further study procedures at any time in the 
interests of the volunteer’s health and well-being, or for any of the following reasons: 
 Administrative decision by the Investigator. 
 Ineligibility (either arising during the study or retrospectively, having been overlooked at 
screening). 
 Significant protocol deviation. 
 Volunteer non-compliance with study requirements. 
 Incorrect answers to screening questionnaire at the second attempt upon re-screening 
for either second or third CHMI 
 An AE, which requires discontinuation of the study involvement or results in inability to 
continue to comply with study procedures. 
The Local Safety Monitor (LSM) may recommend withdrawal of volunteers. The reason for 
withdrawal from further study procedures will be recorded in the CRF. If a volunteer withdraws 
after having completed a course of antimalarials, as much continued safety data collection as 
possible, including procedures such as safety bloods, will be continued, with agreement of the 
volunteer. For all AEs, appropriate follow-up visits or medical care will be arranged, with the 
agreement of the volunteer, until the AE has resolved, stabilised or a non-trial related causality 
has been assigned.  
Any volunteer who fails to attend for two or more follow-up visits will be deemed to have 
withdrawn from the study. If a volunteer withdraws from the study after CHMI but before 
reaching the criterion for malaria diagnosis, a complete, appropriate, curative course of anti-
malarial therapy must be completed. The importance of this will be emphasised to volunteers at 
screening. If a volunteer refuses to take anti-malarial therapy after malaria diagnosis, a rapid 
assessment of mental state and capacity will be undertaken, with the involvement of NHS 
psychiatric and infectious diseases services. If necessary the volunteer may be detained under 
section 4 of the UK Mental Health Act until this assessment can be carried out. 
If a volunteer withdraws from the study, blood samples collected before their withdrawal from 
the trial will be used/stored unless the volunteer specifically requests otherwise. Similarly, all 
data collected up to the point of withdrawal will be stored, unless they specifically request for it 
to be destroyed. Volunteers are free to request that their blood samples be destroyed anytime 
during or after the study.  
In all cases of subject withdrawal, excepting those of complete consent withdrawal, safety data 
collection will continue until 96 days after challenge (C+96), if subjects have undergone CHMI. 
7.4 
Pregnancy 
Should a volunteer become pregnant during the trial, she will be treated with antimalarials 
immediately and will be withdrawn from the study. We will not routinely perform venepuncture 
on such volunteers, other than blood films to check that the parasitaemia has been cleared by 
the antimalarials. Additionally, if a volunteer who had been negative for CMV IgG antibodies at 
screening should become pregnant during the study, given the theoretical risk of CMV infection 
from exposure to the inoculum, venepuncture would also be performed to check CMV 
serostatus. With the volunteer’s permission she shall be followed up until pregnancy outcome. 

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The management of any volunteer found to be pregnant at any time after challenge up to the 
point of malaria treatment will be discussed with the on-call infectious diseases consultant at the 
Oxford University Hospitals NHS Foundation Trust, including advice on antimalarial drug choice. 
Should a volunteer become pregnant after receiving antimalarial treatment (but prior to the end 
of the study), they shall be withdrawn from the study as soon as we have confirmed that their 
parasitaemia has cleared. With the volunteer’s permission, she shall then be followed up until 
pregnancy outcome. 

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8 CONTROLLED BLOOD-STAGE MALARIA INFECTION INOCULUM 
8.1 
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 standard operating procedures used in 
previous studies at Oxford [57, 58]. Work will be carried out in the derogated category III 
laboratory at the Jenner Institute, Old Road Campus Research Building (ORCRB) or New 
Biochemistry Building. Sample manipulations will be performed within a safety cabinet that has 
been fumigated, sterilised and dedicated for this purpose.  
Thawing of the blood inoculum and preparation of the syringes will be carried out in accordance 
with local standard operating procedures.  
8.2 
Administration of the inoculum 
The inoculation will take place in Oxford at the CCVTM. The inoculum will be administered by 
intravenous injection into an indwelling intravenous cannula.  
In VAC069A, the first P. vivax CHMI, either one whole vial, containing approximately 0.5mL of red 
blood cells, one fifth of a vial, or one twentieth of a vial will be reconstituted in 0.9% saline, to a 
total volume of 5mL. The reconstituted inoculum will be injected via an indwelling cannula, 
preceded and followed by a saline flush. One volunteer from each group (Groups 1-3), receiving 
one of the three concentrations, will be administered the inoculum first. The inoculum will 
subsequently be administered to each of the second three volunteers from each group. This is in 
case the time between administration of the inoculum affects the viability of the parasites. 
In subsequent phases of the study using P. vivax VAC069B to D, the inoculum dose will be the 
same for all volunteers and based on the optimal dose as determined in VAC069A. Results from 
VAC069A showed that all volunteers were successfully infected, with a mean day of diagnosis of 
15 days after CHMI (range 12.5-16.5). The two volunteers receiving the highest inoculum dose 
were diagnosed at days 12.5 and 15.5 respectively, those receiving a 1:5 dilution were both 
diagnosed at day 15 and those receiving the 1:20 dilution were diagnosed at days 15.5 and 16.5. 
All three dilutions were therefore demonstrated to be reliably infective. Based on these 
infectivity data, and accounting for possible variation in number of parasitised erythrocytes 
between thawed cryopreserved vials, an inoculum dilution of 1:10 (prepared as per Jenner 
laboratory SOP) has been chosen for all subsequent CHMI.  
In the final phase of the study VAC069E, the P. falciparum inoculum 3D7 will be used for CHMI. 
Each vial of the 3D7 inoculum is estimated to contain between ~3.9×106 to 5.2×108 erythrocytes, 
of which ~1 in 40 will be parasite-infected erythrocytes. The inoculum will be diluted with saline 
to achieve a final dose of approximately 1,000 infected red blood cells in a total volume of 5 mL 
of normal saline, to be injected intravenously. 
For all phases of the study, all volunteers will receive the inoculum within a maximum of 4 hours 
of removal from frozen storage. Subjects will be observed for a minimum of one hour after the 
inoculation before being discharged from the clinic. 
 

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9 TREATMENT OF TRIAL VOLUNTEERS 
This section describes the clinical procedures for evaluating study participants and follow-up 
after CHMI. All participants (including back-up volunteers) will be required to attend all visits up 
to the point of malaria challenge. Back-up volunteers also must be available on the morning of 
the malaria challenge, in case one of the volunteers withdraws at the last minute. This means 
that confirmation of whether or not a back-up volunteer will be needed for the challenge will not 
be made until the day of challenge (one of the Investigators will call the back-up volunteer that 
morning to confirm either way).  
9.1 
Trial sites 
Volunteers will be recruited and undergo screening visits, and clinic visits post-CHMI at the 
CCVTM, Oxford. From day of challenge until completion of anti-malarial therapy, all volunteers 
will reside in Oxford or surrounding areas and attend follow-up visits at the CCVTM, Oxford. Post-
challenge follow up visits will also take place in Oxford. Mosquito-bite transmission studies will 
take place at the Old Road Campus Research Building, Oxford. 
9.2 
Study procedures 
Procedures will be performed at the time points indicated in the schedule of procedures (Tables 
5-14). Additional procedures or laboratory tests may be performed, at the discretion of the 
Investigators if clinically necessary (e.g. urine microscopy in the event of positive urinalysis).  
Study procedures for the first, second and third challenge will be the same, with the exception of 
differences at re-screening, as compared to initial screening, prior to the second and third 
challenges, as described below. 
9.2.1 Observations 
Pulse, blood pressure and temperature will be measured at the time points indicated in the 
schedule of procedures. Weight and height will be measure at screening and weight shall be re-
measured at C-1 (C-2 for VAC069C onwards). 
9.2.2 Blood tests 
Blood will be drawn at the time points indicated in the schedule of procedures below and the 
following laboratory assays performed: 
At Oxford University Hospitals NHS Foundation Trust, using NHS standard procedures 
 Haematology; Full blood count, to include reticulocyte count at C-2 visit. Duffy 
antigen/chemokine receptor (DARC) gene positivity, and glucose-6-phosphate 
dehydrogenase (G6PDH) levels and a haemoglobinopathy screen will be performed at 
screening only. Stored serum or blood will be tested for red blood cell alloantibodies in a 
sample taken at C-1/C-2 of the volunteer’s 1st CHMI and at C+90/96 following each CHMI. 
 Biochemistry; Sodium, potassium, urea, creatinine, albumin, liver function tests, 
magnesium and calcium. Magnesium and calcium will only be measured at screening and 
re-screening. Serum will also be tested for beta-human chorionic gonadotrophin (β-HCG) 
at C-1 or C-2, C+7, C+14, C+21 and C+28/day of malaria diagnosis. 
 Diagnostic serology; HBsAg, HCV antibodies, HIV antibodies, CMV antibodies and EBV 
antibodies will be performed at screening, re-screening and C+90 or C+96; and HTLV 
antibodies at screening. If a volunteer is seropositive for EBV or CMV at screening, further 
serological testing for EBV or CMV will not be conducted post-CHMI.  
 Immunology; Human Leukocyte Antigen (HLA) typing at C-1 or C-2. 

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At the University of Oxford research laboratories (at the Jenner Institute and Department of 
Biochemistry) 
 Diagnostic Tests: Blood films for malaria parasites and PCR for P. vivax and P. falciparum 
DNA and gametocytes. 
 Immunology: Immunological responses to P. vivax and P. falciparum infection will be 
assessed by a variety of established and exploratory assays. This may include: 
o Antibodies and B cell responses to whole parasites or red blood cell invasion 
ligands by array, ELISA and flow cytometry, including total IgG, isotypes and 
avidity; 
o T cell responses to parasite antigens by ex-vivo ELISpot and flow cytometry assays; 
o High dimensional cell phenotyping by mass cytometry 
o Serum cytokine analysis. 
o B cells, plasma and/or serum may be analysed and used to produce human 
monoclonal antibodies against P. vivax or P. falciparum malaria. 
o Neutralising in vitro antibody assays. 
 Genetic tests: DNA analysis of genetic polymorphisms potentially relevant to 
immunological responses and gene expression studies of the host and the parasite 
amongst others may be performed at the discretion of the Investigators. 
 COVID-19 serology: Antibody to SARS-COV-2 will be measured at screening, re-screening 
and at the final study visit following the final CHMI for each participant (+/- at interim 
time points retrospectively) 
 Samples may be sent to collaborating laboratories for other immunological assays or 
studies of malaria. 
 Samples may also be sent to collaborating laboratories within and outside the UK for 
immunomonitoring and/or harmonisation of key immunological assays and/or studies of 
malaria. 
Immunological assays will be conducted according to the procedures established in the test 
laboratories. Specific details of which immunological tests will be performed at each time-point 
are detailed in the separate immunomonitoring plan – this will also clearly state which assays are 
conducted to meet the trial’s primary objectives and which are exploratory. With the volunteers’ 
informed consent, any leftover cells, plasma, serum, whole blood (or their purified components) 
will be registered under University of Oxford HTA licence 12217 and stored indefinitely for future 
analysis of human malaria infection. This may include human DNA and RNA analysis. If a subject 
elects not to permit this, all of that subject’s leftover samples will be discarded after the required 
period of storage to meet Good Clinical Practice (GCP) and regulatory requirements. 
Depending on observed gametocytaemia and availability of appropriate laboratory–reared 
Anopheline mosquito vectors, blood may also be drawn for Direct Membrane Feeding Assays 
(DMFA) to assess transmission to mosquitos. This will involve a 10ml blood draw on day 14 post-
challenge and/or on day of diagnosis, whichever is sooner.   
 
9.2.3  Urinalysis 
Urine will be tested for the presence of clinically significant proteinuria, glycosuria or haematuria 
(as defined in Appendix A) at the screening and re-screening visits. For female volunteers only, 
urine will be tested for beta-human chorionic gonadotrophin (β-HCG) at screening, re-screening 
and at diagnosis, prior to initiation of treatment.  

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9.2.4  Electrocardiogram 
An electrocardiogram will be performed at the initial screening visit for detection of any 
conduction abnormalities, including long QT syndrome.  
9.2.5 SARS-COV-2 testing 
Whilst the COVID-19 pandemic is ongoing, a SARS-COV-2 antigen test will be undertaken within 2 
days of the day of malaria challenge in all volunteers. This can either be a lateral flow test or a 
PCR test and can be self-administered. Asymptomatic COVID-19 infection will be a contra-
indication to proceeding to CHMI. Any participant who develops symptoms, which may be 
consistent with COVID-19 disease after undergoing malaria challenge and prior to completing 
malaria treatment, may also undergo testing for COVID-19 by PCR swab or lateral flow test or 
asked to self-administer a test at home. If a volunteer tests positive on a lateral flow test, it will 
be at the Investigator’s discretion to perform a confirmatory PCR swab test.  
If a participant develops symptoms consistent with COVID-19 after completing malaria 
treatment, they will be referred to local community testing for COVID-19.  
 
9.3 
Clinical reviews 
All clinical reviews and procedures will be undertaken by one of the clinical team. The procedures 
to be included in each visit are documented below. Each review is assigned a time point and a 
window period within which the review will be conducted. 
9.3.1 Screening visits  
Where possible, and where agreement to contact by telephone has been provided by email, 
individuals may be phoned by a member of the clinical team to discuss the study, prior to the 
screening visit. This pre-screening telephone call will provide an opportunity for interested 
individuals to discuss the study, eligibility criteria and study requirements. Where a pre-screening 
call takes place, if individuals continue to express an interest in taking part in the trial following 
the pre-screening call, a screening visit will be scheduled.  
Screening visits may take place up to 90 days prior to enrolment. Informed consent will be 
gained at screening. If consent is given, the screening procedures indicated in the schedule of 
procedures (Table 5) will be undertaken, including testing for Duffy antigen/chemokine 
receptor (DARC) positivity, G6PDH levels and haemoglobinopathy screen.  
The subject’s general practitioner will be contacted with the written permission of the subject 
after screening to ascertain any significant medical history and as notification that the subject 
has volunteered for the study. During the screening the volunteers will be asked to provide their 
National Insurance or passport number so that this can be entered on to a national database 
which helps prevent volunteers from participating in more than one clinical trial simultaneously 
or over-volunteering for clinical trials (TOPS). 
Abnormal clinical findings from the medical history, physical examination, urine or blood tests at 
any point in the study will be assessed according to the scales in Table 15 and site specific 
laboratory adverse event grading tables kept in the trial master file (ref: SOP VC027). If a test is 
deemed clinically significant it may be repeated to ensure it is not a single occurrence. If an 
abnormal finding is deemed to be clinically significant, the volunteer will be informed and 
appropriate medical care arranged with the permission of the volunteer. Exclusion of the 

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volunteer from enrolling in the trial or withdrawal of a volunteer from the trial will be at the 
discretion of the Investigator. 
9.3.2 Re-screening visits for participants not yet enrolled in the study 
Where more than 90 days has lapsed between the initial screening visit and date of enrolment, a 
re-screening visit will be conducted to ascertain if there have been any changes in the medical 
history, medical examination findings or urinalysis, or blood tests for; haematology, 
biochemistry, liver function tests or blood borne virus serology (HIV, Hepatitis B, Hepatitis C, EBV 
and CMV). Other blood tests and ECG will not be repeated routinely. In addition, where a reply 
from the subject’s GP has already been received, the GP will not be re-contacted unless there is 
any need to obtain further information where appropriate (e.g. regarding a new medical event or 
finding) and at the discretion of the Investigator.  
9.3.3 Re-screening visits prior to 2nd and 3rd Challenges 
A re-screening visit will take place prior to each subsequent challenge. Re-screening visits may 
take place up to one month and no less than 1 week prior to re-enrolment. Re-screening visits 
prior to the P. falciparum CHMI in VAC069E may take place up to 90 days and no less than 1 
week prior to re-enrolment. This will be to identify any new medical events. A new informed 
consent will be obtained at the re-screening visit. If consent is given, study specific procedures at 
the visit will be the same as for the screening visit, with the exception that testing for Duffy 
antigen/chemokine receptor (DARC) positivity, G6PDH levels or haemoglobinopathy screen will 
not be repeated (as described in Table 5). In addition, the volunteer’s general practitioner will 
not be re-contacted to corroborate their medical history, unless the Investigators have any 
concerns, e.g., if any new medical events are declared. However, the general practitioner will be 
informed of the volunteer’s intention to participate in subsequent challenges.  
9.3.3 Two days before CHMI (C-2)  
Any new medical issues or symptoms that have arisen will be assessed. Physical observations, 
serum β-HCG test in female volunteers and venepuncture for immunology, malaria PCR and 
safety bloods will be undertaken according to Table 5. A SARS-COV-2 test will also be taken either 
by the study team or self-administered by the participant. Results of safety bloods and the SARS-
COV-2 test taken for this visit must be available and reviewed prior to challenge. Any participant 
with a positive SARS-COV-2 test will not proceed to CHMI. 
9.3.4 Day of CHMI (C0)  
CHMI will be administered, according local standard operating procedure (SOP), this will include 
 Interim history and examination of the injection site and any body systems felt to be 
necessary by the Investigator and verify continuing eligibility / contraindications. 
 Baseline physical observations (respiratory rate, pulse, blood pressure and temperature). 
 Intravenous access via cannulation in forearm vein, flushed with normal saline. 
 For each volunteer the inoculum must be injected within 4 hours of the inoculum being 
thawed, preceded and followed by further normal saline flushes. 
 Physical observations will be repeated at 15 minutes (+/- 5 mins) post-administration of 
inoculum and again at 1 hour (+/- 20 mins), in order to assess for immediate adverse 
reactions, with regular visual observation throughout [76]. If the volunteer should show 
signs or symptoms of a transfusion reaction, observations will be repeated and the 
volunteer will be medically reviewed by a physician. 

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 Volunteers 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. 
Before leaving CCVTM, volunteers will be informed about the unlikely possibility of a delayed 
transfusion reaction and will be provided with the 24 hour emergency mobile telephone number, 
to enable them to contact a physician in the event of concern. 
A Medic-Alert type card will be issued to each volunteer. This will contain the study physician 
1contact details and a request that the research team be contacted immediately in the event of 
illness/accident. Each subject will also be issued with an accurate oral thermometer. If the 
subject does not have their own mobile telephone they will be issued with one for the duration 
of the study, and counselled about the importance of keeping it switched on or checking the 
messages regularly. In addition full contact details for each subject will be documented, including 
home and work addresses, home and work landline telephone numbers where available and 
next-of-kin address and telephone numbers. Mobile telephone numbers will be verified prior to 
challenge to ensure the volunteers are easily contactable. Subjects must also provide the 
Investigators with the name and 24 hour telephone number of a close friend, relative or 
housemate who will be kept informed of their whereabouts for the duration of the study.  
Volunteers must be resident in Oxford / the surrounding area until they have two consecutive 
qPCR results with substantial reduction in genome copies/mL after commencing antimalarial 
treatment. If volunteers do not reach malaria diagnosis, they must remain in the Oxford area 
until they have completed anti-malarial treatment. 
Volunteers will be counselled that should they fail to return for treatment having been infected 
with P. vivax or P. falciparum malaria they could become very unwell and potentially die. They 
will be informed that should they fail to attend a scheduled clinic visit post-challenge, their 
nominated contact, next of kin and the police may be informed and a search started.  
9.3.5 Days 1-28 post-CHMI (C+1 – C+28)  
VAC069A  
Volunteers will be reviewed in clinic once in the morning of day 1 after challenge (C+1), then 
twice daily from day 2 until day 12 post-challenge inclusive (C+2-C-12.5). From day 13 to day 20 
(C+13-20.5), visits may either be once or twice daily depending on the qPCR result. Once a qPCR 
threshold of 1000 genome copies/µL is reached, visits will continue twice daily. If qPCR has not 
reached this threshold, visits will reduce to once daily only. 
VAC069B  
Volunteers will be contacted by phone daily from the day following challenge (C+1) until day 5 
post–challenge (C+5). From days 6-9 (C+6 to C+9) they will attend clinic once daily for medical 
review and assessment of parasitaemia, by both qPCR and smear microscopy. From day 10 
onwards they will attend clinic twice daily, in the early morning and late afternoon, for medical 
review and monitoring of parasitaemia, by qPCR and smear microscopy. 
From day 21 after challenge, visits may be reduced to once daily at the discretion of the 
Investigator (as agreed by the Chief Investigator) e.g., if qPCR is very low or undetectable, and 
volunteer is completely asymptomatic (qPCR results may be unblinded for this purpose). 
VAC069C  

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Volunteers will be contacted by phone daily from the day following challenge (C+1) until day 6 
post–challenge (C+6). From days 7-9 (C+7 to C+9) they will attend clinic once daily for medical 
review and assessment of parasitaemia. From day 10 onwards they will attend clinic twice daily, 
in the early morning and late afternoon, for medical review and monitoring of parasitaemia by 
qPCR. 
From day 21 after challenge, visits may be reduced to once daily at the discretion of the 
Investigator (as agreed by the Chief Investigator) e.g., if qPCR is very low or undetectable, and 
volunteer is completely asymptomatic (qPCR results may be unblinded for this purpose). 
VAC069D 
Following review of the results from the CHMI in VAC069C and the VAC079 vaccine and challenge 
trial running in parallel, the number of visits in the post challenge period has been reduced for 
subsequent challenges to minimise the number of bleeds and burden on volunteers.  
Review of the qPCR results and parasite multiplication rate (PMR) modelling from the CHMI in 
VAC069C and VAC079 has shown that sufficient data can be obtained for accurate PMR 
modelling if bleeds for qPCR continue once a day until qPCR reaches ≥1000 genome copies/ml, 
instead of starting twice a day visits from C+10 onwards. Once qPCR levels reach ≥1000 genome 
copies/ml, then visits will increase to twice a day in order to not delay diagnosis as qPCR may 
reach the diagnostic threshold of ≥5000 genome copies/ml with malaria symptoms within about 
half a day. In volunteers in the VAC079 study, who were diagnosed between days 19 to 27 post 
challenge, PMR modelling would also be minimally affected if participants are treated at C+21, 
instead of C+28, even if they have not reached malaria diagnostic criteria at that timepoint. 
Changing both parameters, shortening time to treatment from C+28 to C+21 and reducing 
frequency of bleeds from twice to once daily until >1000 genome copies/mL threshold is 
reached, did not significantly change the PMRs and also did not change the significance of the 
comparison between the vaccine and the control groups. In VAC069C, volunteers reached 
diagnostic criteria between C+14 to C+19 with median of 15 days post challenge, so the changes 
will have less impact on the number of visits in control volunteers in VAC069. However the post-
challenge visits will be standardized between the two vaccine trials (VAC071 and VAC079) and 
VAC069 to allow direct comparison between controls and vaccinees. 
Volunteers in phase VAC069D to F will therefore be contacted by phone daily from the day 
following challenge (C+1) until day 6 post-challenge (C+6). From days 7 to 9 (C+7 to C+9) they will 
attend clinic once daily for medical review and assessment of parasitaemia. From day 10 
onwards they will attend visits daily, until their malaria qPCR count reaches ≥1000 genome 
copies/ml, when visits will increase to twice a day. Visits will continue twice a day until diagnosis, 
or if diagnosis criteria are not met, until day 21 post-challenge inclusive (C+21). 
VAC069E 
Volunteers will be contacted by phone daily from the day following challenge (C+1) until day 4 
post-challenge (C+4). On days 5 to 6 (C+5 to C+6) they will attend clinic once daily for medical 
review and assessment of parasitaemia. From day 7 onwards volunteers will attend visits daily, 
until their malaria qPCR count reaches ≥1000 genome copies/ml, when visits will increase to 
twice a day until diagnosis. If diagnosis criteria are not met, visits will continue, once or twice 
daily depending on the qPCR count, until day 21 post-challenge inclusive (C+21). 
All volunteers will be seen by one of the clinical study team at each visit. The team will consist of 
the Investigators who are physicians (with experience in acute medicine and infectious diseases 
and familiarity assessing patients with malaria) and nurses.  
At each follow-up visit: 

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 Physical observations will be performed. Venepuncture will be performed as per schedule 
of attendance (Table 6-10), including blood for assessing parasite growth rate, immune 
response and level of gametocytaemia.  
 Volunteers will be questioned as to whether they have; 
o Experienced any of the foreseeable symptoms of malaria. 
o Experienced any other symptoms.  
o Taken any medications including over the counter medications. 
Full physical examination will be performed if deemed necessary by the Investigators. Subjects 
will be encouraged to contact one of the Investigators on the 24 hour emergency mobile 
telephone number if they develop symptoms of malaria or concerning AEs between the regular 
clinic reviews. The severity of symptoms will be assessed using grading criteria summarised in 
Tables 12 & 13. 
Diagnosis will be made according to criteria described below. In VAC069A, if volunteers reach day 
21 post-challenge (C+21) without reaching diagnostic criteria, they shall be started on 
antimalarials. In VAC069B and C, if diagnosis criteria have not yet been met, antimalarial 
treatment shall be initiated at day 28 after challenge (C+28). For VAC069D to E, if diagnosis 
criteria have not yet been met, antimalarial treatment shall be initiated at C+21. Subjects will be 
reviewed in clinic in the subsequent days after starting anti-malarial therapy, as described in 
section 9.5.   
If a volunteer is unwell and unable to attend the CCVTM for a visit they will be reviewed on the 
phone and appropriate arrangement made for clinical review as appropriate.  
9.3.6 Direct feeding of mosquitos 
Direct feeding of mosquitos on the volunteers’ skin may also be performed if the volunteers 
additionally consent to this. This is to assess whether the sexual form of the parasite (the 
gametocyte) can be transmitted back to, and develop within, the mosquito host. This will take 
place at the Old Road Campus Research Building (ORCRB), just a short walk from the CCVTM. The 
procedure will involve laboratory-reared Anopheline mosquitos in a gauze-covered paper cup 
being placed on the volunteer’s exposed skin and allowed to bite. As the biting behaviour of the 
mosquitos is variable, this procedure may take up to a few hours and volunteers will be 
compensated accordingly. Volunteers will be offered a topical antihistamine to reduce itching 
post-mosquito bite. 
 
9.4 
Algorithm for initiation of treatment  
9.4.1 Algorithm for initiation of treatment in Groups VAC069A&B 
Diagnostic criteria will be based on symptoms and signs in-keeping with malarial infection, thick 
film microscopy results and real-time quantitative polymerase chain reaction (qPCR), 
summarised in the algorithm in Figure 2. 
Symptoms or signs that will be considered as indicative of symptomatic malarial infection will be 
fever >37.8oC, rigors or other severe symptoms or signs related to malarial infection, including, 
but not limited to, sweats, headache, anorexia, nausea or vomiting. 
Microscopy will be performed by one or more experienced microscopists and a positive 
microscopy result will be defined as the detection of at least two morphologically normal malaria 
parasites seen in 200 high-power (1000x) fields. This will be diagnostic in the presence of 
symptoms in-keeping with malaria infection. If the volunteer is asymptomatic, diagnosis will be 

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based on a combination of microscopy and qPCR results, as detailed below. Further microscopy, 
e.g. thin film microscopy, may also be performed in order to obtain further information on 
parasite life-cycle stage, but these results will not be considered for diagnosis and treatment 
decisions.  
Asexual blood-stage parasitaemia will be measured by qPCR for the P. vivax 18S rRNA gene. In 
the presence of symptoms, >5,000 genome copies/mL will be considered diagnostic but a qPCR 
result >10,000 copies/mL will be considered positive, even in the absence of symptoms.  
The Investigators are able to treat any volunteer for malaria regardless of the thick film 
microscopy or qPCR result if they are clinically concerned (and have discussed the case with the 
Chief Investigator), or a volunteer wishes to withdraw from the study. If necessary, volunteers 
can be discussed with the LSM and/or the infectious diseases consultant on call at the John 
Radcliffe Hospital for further management under the care of the infectious diseases team, 
inclusive of parenteral anti-malarial therapy if deemed appropriate.  

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Figure 2: Algorithm for diagnosis and initiation of treatment VAC069A&B. 
Negative 
diagnosis, 
continue 
monitoring 
≥2 parasites 
in 200 fields 
<2 parasites 
in 200 fields 
Positive 
diagnosis, 
begin 
treatment 
Positive 
diagnosis, 
begin 
treatment 
Symptomatic 
≥2 parasites 
in 200 fields 
Asymptomatic 
qPCR 
≥5,000 
genome 
copies/mL 
qPCR 
<5,000 
genome 
copies/mL 
qPCR 
<10,000 
genome 
copies/mL 
qPCR 
≥10,000 
genome 
copies/mL 
Negative 
diagnosis, 
continue 
monitoring 
Positive 
diagnosis, 
begin 
treatment 
<2 parasites 
in 200 fields 
qPCR 
≥5,000 
genome 
copies/mL 
qPCR 
<5,000 
genome 
copies/mL 
Positive 
diagnosis, 
begin 
treatment 

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9.4.2 Algorithm for initiation of treatment in Groups VAC069C-E 
It is now well established that nucleic acid amplification-based diagnostics have greater 
sensitivity than microscopic methods for detecting malaria infection both in clinical infection and 
in the P. falciparum challenge model. In the latter, qPCR targeting the 18S ribosome DNA target 
has been used as the diagnostic tool, reliably detecting parasitaemia earlier than thick film blood 
smear[77-80].  
The qPCR assay that we have used in numerous Oxford-led CHMI P. falciparum challenge studies 
is now qualified and highly sensitive, reliably detecting parasitaemias as low as 20 parasites per 
mL of blood (0.02 parasites per μL), long before clinical symptoms manifest. The qPCR assay has 
also performed well in an international External Quality Assurance (EQA) exercise [77]. 
It is also important to note that other trial centres in the USA routinely use PCR as a diagnostic 
tool in CHMI studies. NCT020-15091 was a multi-institution, Phase I, open-label, dose-escalation 
trial with CHMI, designed to assess the safety, immunogenicity, and protective efficacy of PfSPZ 
Vaccine, and this used PCR as the primary diagnostic [81]. 
For both P. vivax and P. falciparum, we use a qPCR assay targeting the pan-plasmodium 18S 
ribosome DNA target. The only other centre worldwide that routinely performs blood-stage 
challenge studies for P. vivax (QIMR, Australia), uses PCR as the sole diagnostic tool in CHMI 
studies, and our qPCR assay is based on this, utilising identical primer and probe sequences. In all 
other aspects, apart from the QIMR derived detection sequences and reaction mixture 
employed, the extraction of DNA and qPCR is identical to the existing and formally validated 
P.falciparum qPCR method employed as sole, objective, diagnostic tool in the two P.falciparum 
trials described below. This pan-plasmodium qPCR is in the process of undergoing a similar 
formal validation and work completed so far, demonstrates that this is similarly fit for purpose. 
As examples of qualification parameters that contribute to validation, the assay has excellent 
specificity (within the context of a P.viax only CHMI study), linearity, acceptable accuracy (based 
on reference samples of microscopically quantified P.vivax infected blood), precision and 
reproducibility (based on several inter-operator tests with acceptable %CV values when assessing 
prepared samples at the described diagnostic thresholds in >20 replicates). The assay has an 
acceptable detection and reliable quantitation limit of 50-100 genomes per ml (well below 
reproducible microscopic detection levels. While detection is possible down to approximately 25 
genomes per ml as indicated below, it is less reproducible at this level). The assay is also 
extremely robust, with both EDTA whole blood samples, extracted DNA and standards remaining 
stable and giving reproducible qPCR scores at 4°C storage for many days (which is beyond the 
intended use for real time qPCR follow up). The range of the qPCR is similar to that of the 
previously validated P.falciparum assay, with at least a 4 log range from 100 genomes per ml to 
1,000,000 for the standards employed (actual range used in assays is from 25 to 1,000,000 
genome copies per ml). These criteria assessments are in accordance with the ICH Harmonised 
Tripartite Guideline Part II: Validation of Analytical Procedures: Methodology, (European 
Medicines Agency, 2006). This is further supported by data from the lab’s participation in the 
UKNEQAS external quality assurance scheme since mid-2018, with 5 rounds of samples assayed 
in both P.falciparum and P.vivax assays completed to date, with a 100% success rate. 
To date, comparative qPCR and microscopy data are available for two blood-stage P. vivax CHMI 
studies conducted with the PvW1 blood inoculum in Oxford; VAC069A&B (n=11 including 3 
participants who underwent CHMI twice) (NCT03797989) and CHMI of VAC071 Group 1 
participants (n=3) (NCT04009096). Here, the greater sensitivity of qPCR compared to thick film 
microscopy has been consistently demonstrated with 3/14 participants diagnosed by qPCR prior 
to microscopic patency and, where infection was microscopically patent (11/14 participants), 

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qPCR detected parasitaemia (>25 genome copies/mL) at least 6 days prior to thick film positivity 
(as defined as ≥2 asexual forms seen in 200 high-power fields). At the pre-specified qPCR 
thresholds for diagnosis, as described in Figure 2, 3/14 participants did have a positive thick film 
result prior to reaching diagnostic criteria (≥5,000 genome copies/mL), however, none of these 
three participants had experienced symptoms consistent with the clinical threshold for diagnosis 
at the time of thick smear positivity and were later diagnosed within 1.5 days of microscopic 
patency, without complication. 
Positive thick smear results may result from chance findings, since following blood-stage 
challenge, parasites may be seen on light microscopy at any time-point. Where this occurs, and 
premature diagnosis results, this limits the qPCR dataset available for PMR modelling. Removing 
this possibility, therefore, increases the data available for analysis of the primary outcome 
measure. In addition, use of qPCR as the sole diagnostic tool leads to greater standardisation of 
diagnosis across study groups, permitting improved inter-group as well as inter-trial comparison. 
In Oxford, we have now safely conducted two P. falciparum blood-stage CHMI studies, where 
qPCR is the sole diagnostic method (Clinicaltrials.gov identifiers NCT03906474 and 
NCT02927145). Analysis of the parasitaemia at diagnosis in one of these studies, VAC063B, 
indicates that removal of microscopy as a diagnostic measure, reduced the range over which 
diagnosis was made compared to two prior P. falciparum blood-stage CHMI studies, where 
microscopy was utilised (see Figure 3).  
As microscopy findings are not considered within the primary efficacy analysis for this study, 
microscopy for Group 1 participants was principally utilised as an additional diagnostic tool for 
safety reasons. The data from the two previous P. vivax blood-stage CHMI studies were therefore 
analysed to apply the revised diagnostic criteria retrospectively, to determine if timing of 
diagnosis would have been altered. Using the revised criteria, the majority of diagnoses would 
not have been delayed, although it is estimated 36% of volunteers would have had a small delay 
to diagnosis. However, since clinical criteria remain integral to the diagnostic algorithm, this 
small delay should not result in any increased risk to volunteers, and the ability for investigators 
to treat any participant in the event of clinical concern, following discussion with the CI, 
regardless of parasitaemia, will ensure participant safety.  
 
 
VAC054 
(n=15)
VAC063A 
(n=15)
VAC063B 
(n=6)
(qPCR only)
100
101
102
103
104
105
106
Parasites/mL

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Figure 3: Parasitaemia at the diagnosis timepoint in unvaccinated control volunteers in three 
prior P. falciparum blood-stage CHMI studies. In both the VAC054 and VAC063 CHMI studies, 
where microscopy was used as a diagnostic measure alongside qPCR and clinical assessment, 
diagnosis was made over a wider range of parasitaemia compared to the VAC063B study, where 
thick blood smear microscopy was not performed. Diagnostic parasitaemia ranged between 
1440-273,247 parsites/mL in the VAC054 study, 815- 124063 parasites/mL in VAC063A and 7137- 
100946 parasites/mL in the VAC063B CHMI. Individual and median data points are shown[22].  
On this basis, for VAC069C-E, diagnostic criteria will be based on symptoms in keeping with 
malarial infection results and real-time quantitative polymerase chain reaction (qPCR) only, i.e. 
thick film microscopy will not be used as a diagnostic measure, as summarised in the algorithm in 
Figure 4. Symptoms or signs that will be considered as indicative of symptomatic malarial 
infection will be fever >37.8oC, rigors or other severe symptoms or signs related to malarial 
infection, including, but not limited to, subjective feverishness, sweats, headache, myalgia, 
arthralgia, nausea or vomiting. 
Asexual blood-stage parasitaemia will be measured by qPCR for the pan-plasmodium 18S DNA 
gene. In the presence of symptoms or signs indicative of malarial infection, ≥5,000 genome 
copies/mL will be considered diagnostic but a PCR result ≥10,000 copies/mL will be considered 
positive, regardless of symptoms. All Investigators (except the Principal Laboratory Investigator) 
will be blinded to the results. Blinding of PCR operators to the group of a volunteer will be 
maintained by assigning a new, unique identifier to the volunteer for PCR samples from the C-1 
visit. If, as judged by the investigator, a volunteer develops symptoms in-keeping with malaria, 
the volunteer will be discussed with the Chief Investigator. If deemed appropriate by the Chief 
Investigator, the qPCR results for this volunteer will be unblinded by the Principal Laboratory 
Investigator and the volunteer treated only if any available qPCR result is ≥5000 genome 
copies/mL. At the point that a volunteer has a qPCR result exceeding 10,000 genome copies/mL, 
the Principal Laboratory Investigator will unblind the Chief Investigator to the result and the 
volunteer will be treated, regardless of the presence of symptoms.  
Importantly, in the unlikely event of a technical failure with the qPCR machine, we also have a 
working back-up qPCR machine that will be available for use 24/7 if required. 
The Investigators are able to treat any volunteer for malaria regardless of the qPCR result if they 
are clinically concerned (and have discussed the case with the Chief Investigator), or a volunteer 
wishes to withdraw from the study. If necessary, volunteers can be discussed with the LSM 
and/or the Infectious Diseases Consultant on call at the John Radcliffe Hospital for further 
management under the care of the infectious diseases team, inclusive of parenteral anti-malarial 
therapy if deemed appropriate. 
 
 

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Figure 4: Algorithm for diagnosis and initiation of treatment in VAC069C-E. N.B. In VAC069C, if 
day 28 after challenge is reached without meeting any diagnostic criteria; or day 21 in VAC069D 
and E, treatment will be initiated regardless of qPCR results, i.e. even if parasitaemia is 
undetectable. 
 
9.4.3 Participants with fever post malaria challenge and COVID-19 testing 
In the first week following CHMI, if a participant becomes unwell with a febrile illness, it is highly 
unlikely that the cause of this is malaria. If a participant develops a fever between days C+1 to C+6 
and their symptoms are consistent with possible COVID-19 disease the participant will be advised 
to follow UKHSA guidelines regarding staying at home and will be advised to take a COVID-19 test 
with the kit provided by the study team. If the SARS-COV-2 PCR test is negative, the participant can 
continue with study visits as per protocol. 
From C+7 onwards, if a participant develops a fever of ≥37.8°C or other symptoms consistent 
with possible COVID-19 disease, they will be advised to inform the study team before attending 
their clinic visit. The participant’s clinic visit will be conducted in an isolated clinic room with 
study staff wearing appropriate PPE. If a participant fulfils current COVID-19 testing criteria, the 
study team will either advise the participant to perform a home COVID-19 rapid antigen test 
(provided) or a COVID-19 test will be taken by the study team in addition to the study specified 
malaria blood tests.  
If a participant has a persistent fever or study clinicians are clinically concerned for COVID-19 
disease, then a SARS-COV-2 test can be repeated at the discretion of the Investigator, if the first 
test was negative. 
If following malaria challenge and prior to malaria diagnosis and treatment, a participant is found 
to have a positive SARS-COV-2 test from any source, they will be commenced on malaria 
treatment, irrespective of the severity of COVID-19 disease and irrespective of malaria symptoms 
or malaria qPCR result at that time point. 
 
 
qPCR 
≥5,000 
genome 
copies/mL 
qPCR 
<5,000 
genome 
copies/mL 
Negative 
diagnosis, 
continue 
monitoring 
Positive 
diagnosis, 
begin 
treatment 
qPCR 
<10,000 
genome 
copies/mL 
qPCR 
>10,000 
genome 
copies/mL 
Negative 
diagnosis, 
continue 
monitoring 
Positive 
diagnosis, 
begin 
treatment 
Symptomatic 
Asymptomatic/ 
clinical criteria for diagnosis not met 

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9.5 
Malaria management 
Volunteers will be treated with a standard oral course of either artemether-lumefantrine 
(Riamet®) or atavoquone/proguanil (Malarone). Both are recommended treatment of P. vivax 
and P. falciparum infection in the UK, although use for non-falciparum malaria is an unlicensed 
use for both medications. The decision on which medication will be used, will be at the 
Investigator’s discretion and depends on factors including if there are any contraindications to 
either medication. 
If a volunteer withdraws/is withdrawn from the study after administration of CHMI but before 
reaching the criteria for malaria treatment, then a complete, appropriate, curative course of anti-
malarial therapy must be completed. The importance of this will be emphasised to volunteers at 
screening. 
9.5.1 Malaria management – Riamet  
Artemisinin-based combination therapies (ACTs) containing mefloquine, piperaquine or 
lumefantrine are now the recommended treatment for P. vivax in areas where there might be 
chloroquine resistance (WHO 2015 guidelines). Although chloroquine remains effective in most 
recent studies in Thailand, there are some signs that low level chloroquine resistance may be 
becoming established along the Thai-Myanmar border.  
Riamet® is a combination drug consisting of 20mg artemether and 120mg lumefantrine per 
tablet. The treatment regime will consist of 6 doses of total 80mg artemether/480mg 
lumefantrine (4 tablets) – to clear the blood-stage infection. The first dose will be directly 
observed, at treatment initiation, followed by additional doses after 8, 24, 36, 48 and 60 hours 
(window period +/- 1 hour for each dose). The 24 hour dose will also be directly observed, and 
volunteers will record the time that intervening doses were taken at home in a medication diary.  
Prior to starting Riamet®, volunteers will be screened for drug interactions and contraindications. 
This includes checking for pregnancy by urine pregnancy test (serum β-hCG test in female 
volunteers at C+7 and C+14) and prolonged QT (on pre-challenge ECG). Volunteers will be 
reminded of the potential side effects and given the patient information sheet for Riamet® and a 
card outlining when their doses of Riamet should be taken. Volunteers will be advised to avoid 
grapefruit juice. Tablets should be taken together with a fatty meal (a light snack will provided 
when doses are observed in clinic). At least two doses for each volunteer will be directly 
observed in clinic.  
9.5.1 Malaria management – Malarone  
Malarone (see SmPc for Malarone) is a combination drug consisting of proguanil hydrochloride 
and atovaquone. A treatment course of Malarone consists of 4 ‘standard tablets’ of Malarone 
(proguanil hydrochloride 100 mg, atovaquone 250 mg) once daily, orally for 3 days. Two out of 
three doses of Malarone will be directly observed in clinic. Prior to starting Malarone, volunteers 
will be screened for drug interactions and contraindications (including a serum β-hCG test in 
female volunteers). Volunteers will be reminded of the potential side effects of Malarone and 
given the patient information sheet for Malarone. 
9.5.2 Malaria management – supportive medications  
Provided there are no contraindications, volunteers will be provided with both paracetamol (1 g 
orally up to four times a day) and a course of cyclizine (50 mg orally three times a day) for 
symptomatic relief. (See SmPC for Paracetamol & Cyclizine). Participants may be provided with 
an appropriate, licensed alternative anti-emetic to cyclizine if they are unable to take cyclizine. 

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Volunteers will be given the patient information sheet for these medications and advised how 
frequently they can take doses. Volunteers will be issued with a medication diary card on which 
they will be asked to document all doses of medications taken post-CHMI.   
All medications used in the trial will be handled and dispensed according to SOP VC021: 
Handling, Storage and Dispensing of Non-IMP Medication.  
9.5.3 Follow-up after commencing malaria treatment 
In VAC069A-C: at each visit at 24 hours and 48 hours after initiation of malaria treatment, 
procedures will be performed according to Tables 10a to 10c, including physical observations, 
assessment of AEs and venepuncture for blood tests including measurement of malaria qPCR. 
Once daily malaria qPCR blood tests will continue until the volunteer has two consecutive qPCR 
readings with substantial reduction in genome copies per mL. After completing the course of 
anti-malarials, on day 3 after initiation of treatment, participants will be reviewed again in clinic 
and undergo physical observations, assessment for AEs and venepuncture. 
For VAC069D-E: post treatment visits will occur at 24 hours and 72 hours after initiation of 
treatment. During VAC069C a number of volunteers had an additional qPCR test on T+3 as the 
malaria parasite count at T+2 was still positive for some participants. All T+3 malaria qPCRs 
results were significantly reduced. In order to minimise the number of visits, the T+2 visit has 
been removed. Procedures will be performed according to Table 10d to 10e, including physical 
observations, assessment of AEs and venepuncture for blood tests including measurement of 
malaria qPCR. Malaria qPCR blood tests can be repeated if qPCR readings are not substantially 
reduced by T+3. 
 
9.5.3 Criteria for inpatient transfer to the NHS  
If any of the following criteria are met, admission under the care of the Infectious Diseases Team, 
Oxford University Hospitals NHS Foundation Trust will be considered: 
 Failure of symptoms to improve within 48 hours of starting anti-malarial therapy. 
 Unable to tolerate oral antimalarial therapy. 
 Dehydration requiring intravenous fluid therapy. 
 Signs or symptoms suggestive of pulmonary oedema.  
 Signs or symptoms of neurological dysfunction including altered consciousness. 
 Signs, symptoms or laboratory evidence of significant renal dysfunction. 
 Unanticipated concern about subject’s home circumstances. 
 Any other significant finding which the Investigators feels warrant inpatient admission - 
this includes symptoms or signs of suspected worsening COVID-19 disease e.g. respiratory 
failure, in the unlikely event of a participant testing PCR positive for COVID-19 during the 
post-challenge period.  
Ultimately, the decision regarding admission will be taken by the Investigators in conjunction 
with the Infectious Diseases Consultant on call.  
 
9.6 
Safety measures for conduct of CHMI 
Volunteer safety is of paramount importance. The following measures are in place to safeguard 
volunteer safety: 

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 Volunteers will only be enrolled in the study if both Investigators and the volunteers’ GP 
feel this is appropriate. 
 Volunteers’ understanding of the trial information will be tested by means of a 
questionnaire at screening. This provides further confidence that fully informed consent 
has been obtained. 
 If the subject does not have their own mobile telephone they will be issued with one for 
the duration of the study and counselled about the importance of keeping it switched on 
or checking the messages regularly.  
 Before challenge, full contact details for each subject will be documented, including home 
address and mobile telephone numbers. Mobile telephone numbers will be verified prior 
to challenge to ensure the volunteers are easily contactable. Home and work landline 
telephone numbers where available and next-of-kin address and telephone numbers will 
also be documented. Subjects must also provide the Investigators with the name and 24 
hour telephone number of a close friend, relative or housemate who lives nearby and will 
be kept informed of their whereabouts for the duration of the study. 
 On the day of challenge volunteers will be provided with a medic alert card containing 
contact details for the study team and brief details of the study including the optimal 
treatment for P. vivax or P. falciparum. 
 Volunteers will be able to contact a medically qualified member of the study team 24 
hours a day throughout the study period and will be instructed to contact the Investigator 
immediately should they manifest any signs or symptoms they perceive as serious. 
 If necessary, the study team will visit volunteers in their own homes if they are unable to 
attend clinic for review.  
 At least two doses of Riamet® for each volunteer will be observed by the study team. (For 
volunteers taking Malarone, two out of three doses will be observed). 
 Volunteers will be counselled that should they fail to return for treatment having been 
infected with malaria they could become very unwell and potentially die. They will be 
instructed to remain in Oxford and the immediate surrounding area for the duration of 
the intensive follow-up schedule (days 0-21 post-challenge). They will be informed that 
should they fail to attend a scheduled clinic visit post-challenge, their nominated contact, 
next of kin and the police may be informed and a search started.  
9.6.1 Measures to be taken if a volunteer goes missing post-CHMI 
In the unlikely event that a volunteer should (a) fail to attend for a scheduled clinical visit or (b) 
be un-contactable by telephone after challenge and before completion of an appropriate course 
of anti-malarial therapy, the following stakeholders will be informed; 
 All Investigators.  
 The volunteer’s nominated contact and next of kin. 
 The trial Sponsor. 
 The local safety committee. 
 The ethical committee(s). 
 Relevant hospital trust R&D departments. 
 The local police department. 
 Local Accident and Emergency departments. 
Active efforts will be made to locate the volunteer by the police. While all parties will aim to 
preserve the volunteer’s confidentiality, if necessary details of the volunteer’s identity and 
participation in the study may be passed to the national media in order to help locate the missing 
individual. Volunteers will be informed of this during screening. 

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9.7 
Follow-up post-treatment  
9.7.1 Six days after initiation of treatment (T+6) 
Physical observations will be performed and AEs assessed. Venepuncture will be performed 
(Tables 10). Where applicable, medication diary cards and malaria symptom diary cards will be 
collected from volunteers. 
9.7.2 Nine days after initiation of treatment (for VAC069C-D) (T+9 (+2)) 
Volunteers will be reviewed by telephone to check for full resolution of malaria symptoms and 
any side effects from malaria medication. 
9.7.3 Ten days after initiation of treatment (for VAC069E) (T+10 (±2)) 
Physical observations will be performed and AEs assessed. Venepuncture will be performed (as 
per Table 10e). Where applicable, medication diary cards, malaria symptom diary cards and 
unused, dispensed medications will be collected from volunteers.  
9.7.4 Days 28, 45 and 90 post CHMI (for VAC069A)Physical observations will be performed and 
AEs assessed. Venepuncture will be performed (as per Table 10a). Where applicable, medication 
diary cards, malaria symptom diary cards and unused, dispensed medications will be collected 
from volunteers.  
9.7.5 Days 56 and 96 post CHMI (for VAC069B-E) (C+56 (±14) and C+96 (±14)) 
Physical observations will be performed and AEs assessed. Venepuncture will be performed (as 
per Tables 10b-e). Where applicable, medication diary cards, malaria symptom diary cards and 
unused, dispensed medications will be collected from volunteers.  
Time windows for these visits have been increased to allow any potential participants who are 
self-isolating due to COVID-19, to attend the study visits once they have ended self-isolation.  
 
9.8 
Secondary and tertiary blood-stage CHMI 
All volunteers, except those in Groups 10 and 12, will receive a second and third blood-stage 
challenge, using a 1:10 dose of inoculum selected from VAC069A, approximately 5 to 10 months 
after the preceding CHMI. Following a re-screening visit, as detailed in Section 9.3.2, the 
procedures followed will be the same as for the initial challenge (as described in Sections 9.3.3-
9.7.3 and Tables 5-10 inclusive).  
Volunteers who underwent CHMI with P. vivax in Groups 10 and 12 in VAC069D will undergo a 
repeat (secondary or tertiary) blood-stage challenge with P. falciparum at approximately 8-14 
months after the preceding CHMI. Procedures will be as described above for VAC069E. 
 
 

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Day 
S/RS 
C-1 
Attendance number 
1 
2 
Timeline (days) (window) 
-90 (-90—14) 
-1 
Inclusion/Exclusion criteria 
X 
 
Informed consent 
X 
 
Informed consent questionnaire 
X 
 
Physical observations* 
X 
X 
Medical history/examination 
X 
(X) 
Electrocardiogram 
X 
 
Urinalysis 
X 
 
Review contraindications 
 
X 
Urine β-hCG (females only) 
X 
 
Serum βhCG (mL) (females only) 
 
5 
Haematology, G6PD*** and haemoglobinopathy 
screen*** (mL) 
4 
 
Haematology (mL) 
2 
2 
Biochemistry (mL)** 
3 
3 
HIV, Hepatitis B, Hepatitis C, EBV, CMV serology (mL)*** 
5 
 
HLA typing*** 
 
4 
Immunology plasma (mL) 
 
30 
Immunology serum (mL) 
 
40 
Safety serum (mL) 
 
4 
Plasma for exploratory studies (mL) 
 
3 
Gene expression profiling (mL) 
 
20 
qPCR & gametocyte PCR  
 
3 
Blood film (mL) 
 
1 
Visit Total Blood Volume (mL) 
14 
115 
Cumulative blood volume  (mL) 
14 
129 
Table 5a: Schedule of events for VAC069A&B pre-CHMI (timeline/window are in relation to the day 
of challenge). S = screening visit; RS = re-screening visit; (X) = If considered necessary, emphasising 
any acute complaints. *Physical observations include blood pressure, pulse and temperature, plus 
height and weight at screening/re-screening and C-1 visit for VAC069A&B. **Biochemistry will 
include sodium, potassium, urea, creatinine, albumin, liver function test, plus magnesium & calcium 
at re-screening/screening only. ***To be tested at initial screening visits only, i.e. will not be 
performed at re-screening prior to second and third challenges.  
 
 
 
 
 
 
 

VAC069: A study of blood-stage controlled human Plasmodium vivax malaria infection. IRAS project 
ID: 252499 
VAC069 Clinical Trial Protocol, V8.0, 14th June 2022, University of Oxford.  Page 81 of 122 
Day 
S/RS 
C-2 
Attendance number 
1 
2 
Timeline (days) (window) 
-90 (-90 to -7) 
-2 (-3 to -1) 
Inclusion/Exclusion criteria 
X 
 
Informed consent 
X 
 
Informed consent questionnaire 
X 
 
Physical observations* 
X 
X 
Medical history/examination 
X 
(X) 
Electrocardiogram 
X 
 
Urinalysis 
X 
 
Review contraindications 
 
X 
Urine β-hCG (females only) 
X 
 
Serum βhCG (mL) (females only) 
 
5 
Haematology#, G6PD***, haemoglobinopathy screen*** 
(mL) 
4 
2 
DARC (mL)*** 
2 
 
Biochemistry (mL)** 
3 
3 
HIV, Hepatitis B, Hepatitis C, EBV, CMV serology (mL) 
5 
 
HLA typing*** 
 
4 
Safety serum (mL) 
 
4 
Exploratory Assays (mL) 
 
85 
qPCR & gametocyte PCR  
 
2 
SARS-COV-2 test 
 
X 
Visit Total Blood Volume (mL) 
14 
105 
Cumulative blood volume  (mL) 
14 
119 
Table 5b: Schedule of events for VAC069C-E pre-CHMI (timeline/window are in relation to the day 
of challenge). S = screening visit; RS = re-screening visit; (X) = If considered necessary, emphasising 
any acute complaints. *Physical observations include blood pressure, pulse and temperature, plus 
height and weight at screening/re-screening and weight at C-2 visit for VAC069C-E. **Biochemistry 
will include sodium, potassium, urea, creatinine, albumin, liver function test, plus magnesium & 
calcium at re-screening/screening only. ***To be tested at initial screening visits only, i.e. will not be 
performed at re-screening prior to second and third challenges.  # FBC to include reticulocyte count 
at C-2 visit

VAC069: A study of blood-stage controlled human Plasmodium vivax malaria infection. IRAS project ID: 252499 
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Day 
C 
C+1 
C+2 
C+2.5 
C+3 
C+3.5 
C+4 
C+4.5 
C+5 
C+5.5 
C+6 
C+6.5 
C+7 
C+7.5 
Attendance number 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13 
14 
15 
16 
Timeline (days) (window) 
0 
1 
2 
2 
3 
3 
4 
4 
5 
5 
6 
6 
7 
7 
Physical observations* 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Medical history/examination 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
Review contraindications 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Review Aes & medications 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Medic alert card provided 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Diary card issued 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Serum βhCG (mL) (females only) 
 
 
 
 
 
 
 
 
 
 
 
 
5 
  
Haematology (mL) 
 
 
 
 
 
 
 
 
 
 
 
 
2 
  
Biochemistry (mL)** 
 
 
 
 
 
 
 
 
 
 
 
 
3 
  
Immunology  plasma (mL) 
 
 
 
 
 
 
 
 
 
 
 
 
30 
  
Immunology serum (mL) 
 
 
 
 
 
 
 
 
 
 
 
 
40 
  
Gene expression profiling (mL) 
 
 
 
 
 
 
 
 
 
  
15 
  
15 
  
qPCR & gametocyte PCR (mL) 
 
3 
3 
3 
3 
3 
3 
3 
3 
3 
3 
3 
3 
3 
Blood film(mL) 
 
1 
1 
1 
1 
1 
1 
1 
1 
1 
1 
1 
1 
1 
Visit Total Blood Volume (mL) 
 
4 
4 
4 
4 
4 
4 
4 
4 
4 
19 
4 
99 
4 
Cumulative blood volume  (mL) 
 
133 
137 
141 
145 
149 
153 
157 
161 
165 
184 
188 
287 
291 
Table 6a: Schedule of events for day of challenge to day 7 after challenge for volunteers in VAC069A (timeline/window are in relation to the day of 
challenge). C = challenge; (X) = If considered necessary. Visits continue according to schedule until diagnostic criteria are reached; when diagnostic criteria are 
met, venepuncture and procedures are followed according to Table 10a.*Physical observations include blood pressure, pulse and temperature. **Biochemistry 
comprises sodium, potassium, urea, creatinine, albumin, liver function tests.  
 
 
 

VAC069: A study of blood-stage controlled human Plasmodium vivax malaria infection. IRAS project ID: 252499 
VAC069 Clinical Trial Protocol, V8.0, 14th June 2022, University of Oxford.  Page 83 of 122 
Day 
C 
C+1 
C+2 
C+3 
C+4 
C+5 
C+6 
C+7 
Attendance number 
3 
- 
- 
- 
- 
- 
4 
5 
Timeline (days) (window) 
0 
1 
2 
3 
4 
5 
6 
7 
Physical observations* 
X 
 
 
 
 
 
X 
X 
Medical history/examination 
(X) 
 
 
 
 
 
X 
X 
Review contraindications 
X 
 
 
 
 
 
 
 
Daily phone call 
 
X 
X 
X 
X 
X 
 
 
Review AEs & medications 
 
X 
X 
X 
X 
X 
X 
X 
Medic alert card provided 
X 
 
 
 
 
 
 
 
Diary card issued 
X 
 
 
 
 
 
 
 
Serum βhCG (mL) (females only) 
 
 
 
 
 
 
 
5 
Haematology (mL) 
 
 
 
 
 
 
 
2 
Biochemistry (mL)** 
 
 
 
 
 
 
 
3 
Immunology  plasma (mL) 
 
 
 
 
 
 
 
30 
Immunology serum (mL) 
 
 
 
 
 
 
 
30 
Gene expression profiling EDTA sample (mL) 
 
 
 
 
 
 
9 
 
Gene expression profiling Li Heparin sample (mL) 
 
 
 
 
 
 
3 
 
qPCR & gametocyte PCR (mL) 
 
 
 
 
 
 
3 
3 
Blood film(mL) 
 
 
 
 
 
 
1 
1 
Visit Total Blood Volume (mL) 
- 
- 
- 
- 
- 
- 
16 
74 
Cumulative blood volume  (mL) 
129 
129 
129 
129 
129 
129 
145 
219 
Table 6b: Schedule of events for day of challenge – day 7 after challenge for volunteers in VAC069B(timeline/window are in relation to the day of 
challenge). C = challenge; (X) = If considered necessary. Visits continue according to schedule until diagnostic criteria are reached; when diagnostic criteria are 
met, venepuncture and procedures are followed according to Table 10b.*Physical observations include blood pressure, pulse and temperature. **Biochemistry 
comprises sodium, potassium, urea, creatinine, albumin, liver function tests.  
 
 

VAC069: A study of blood-stage controlled human Plasmodium vivax malaria infection. IRAS project ID: 252499 
VAC069 Clinical Trial Protocol, V8.0, 14th June 2022, University of Oxford.  Page 84 of 122 
Day 
C 
C+1 
C+2 
C+3 
C+4 
C+5 
C+6 
C+7 
Attendance number 
3 
- 
- 
- 
- 
- 
- 
4 
Timeline (days) (window) 
0 
1 
2 
3 
4 
5 
6 
7 
Physical observations* 
X 
 
 
 
 
 
 
X 
Medical history/examination 
(X) 
 
 
 
 
 
 
X 
Review contraindications 
X 
 
 
 
 
 
 
 
Daily phone call 
 
X 
X 
X 
X 
X 
X 
 
Review AEs & medications 
 
X 
X 
X 
X 
X 
X 
X 
Medic alert card provided 
X 
 
 
 
 
 
 
 
Diary card issued 
X 
 
 
 
 
 
 
 
Serum βhCG (mL) (females only) 
 
 
 
 
 
 
 
5 
Haematology (mL) 
 
 
 
 
 
 
 
2 
Biochemistry (mL)** 
 
 
 
 
 
 
 
3 
Exploratory assays (mL) 
 
 
 
 
 
 
 
60 
Gene expression profiling EDTA sample (mL) 
 
 
 
 
 
 
 
 
Gene expression profiling Li Heparin sample (mL) 
 
 
 
 
 
 
 
 
qPCR & gametocyte PCR (mL) 
 
 
 
 
 
 
 
2 
Visit Total Blood Volume (mL)^ 
- 
- 
- 
- 
- 
- 
- 
72 
Cumulative blood volume  (mL) 
119 
119 
119 
119 
119 
119 
119 
191 
Table 6c: Schedule of events for day of challenge – day 7 after challenge for volunteers in VAC069C-D (timeline/window are in relation to the day of 
challenge). C = challenge; (X) = If considered necessary. Visits continue according to schedule until diagnostic criteria are reached; when diagnostic criteria are 
met, venepuncture and procedures are followed according to Table 10c.*Physical observations include blood pressure, pulse and temperature. **Biochemistry 
comprises sodium, potassium, urea, creatinine, albumin, liver function tests. ^Blood volumes as given in the table are maximal volumes and smaller blood 
volumes may be taken at any visit, at the discretion of the investigator, with the exception of additional blood volume that may be required for any additional 
tests that may be performed if deemed clinically necessary. 
 
 

VAC069: A study of blood-stage controlled human Plasmodium vivax malaria infection. IRAS project ID: 252499 
VAC069 Clinical Trial Protocol, V8.0, 14th June 2022, University of Oxford.  Page 85 of 122 
Day 
C 
C+1 
C+2 
C+3 
C+4 
C+5 
C+6 
C+7 
Attendance number 
3 
- 
- 
- 
- 
4 
5 
6 
Timeline (days) (window) 
0 
1 
2 
3 
4 
5 
6 
7 
Physical observations* 
X 
 
 
 
 
X 
X 
X 
Medical history/examination 
(X) 
 
 
 
 
X 
X 
X 
Review contraindications 
X 
 
 
 
 
 
 
 
Daily phone call 
 
X 
X 
X 
X 
 
 
 
Review AEs & medications 
 
X 
X 
X 
X 
X 
X 
X 
Medic alert card provided 
X 
 
 
 
 
 
 
 
Diary card issued 
X 
 
 
 
 
 
 
 
Serum βhCG (mL) (females only) 
 
 
 
 
 
 
 
5 
Haematology (mL) 
 
 
 
 
 
 
 
2 
Biochemistry (mL)** 
 
 
 
 
 
 
 
3 
Exploratory assays (mL) 
 
 
 
 
 
 
 
22 
qPCR & gametocyte PCR (mL) 
 
 
 
 
 
2 
2 
2 
Visit Total Blood Volume (mL)^ 
- 
- 
- 
- 
- 
2 
2 
34 
Cumulative blood volume  (mL) 
119 
119 
119 
119 
119 
121 
123 
157 
Table 6d: Schedule of events for day of challenge – day 7 after challenge for volunteers in VAC069E (timeline/window are in relation to the day of 
challenge). C = challenge; (X) = If considered necessary. Visits continue according to schedule until diagnostic criteria are reached; when diagnostic criteria are 
met, venepuncture and procedures are followed according to Table 10d.*Physical observations include blood pressure, pulse and temperature. **Biochemistry 
comprises sodium, potassium, urea, creatinine, liver function tests. ^Blood volumes as given in the table are maximal volumes and smaller blood volumes may 
be taken at any visit, at the discretion of the investigator, with the exception of additional blood volume that may be required for any additional tests that may 
be performed if deemed clinically necessary. 
 
 
 
 

VAC069: A study of blood-stage controlled human Plasmodium vivax malaria infection. IRAS project ID: 252499 
VAC069 Clinical Trial Protocol, V8.0, 14th June 2022, University of Oxford.  Page 86 of 122 
 
 
Day 
C+8 C+8.5 
C+9 
C+9.5 C+10 C+10.5 C+11 C+11.5 
C+12 
C+12.5 C+13 C+13.5a C+14 C+14.5a 
Attendance number 
17 
18 
19 
20 
21 
22 
23 
24 
25 
26 
27 
28 
29 
30 
Timeline (days) (window) 
8 
8 
9 
9 
10 
10 
11 
11 
12 
12 
13 
13 
14 
14 
Physical observations* 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Medical history/examination 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
Review Aes & medications 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Serum βhCG (mL) (females only) 
 
 
 
 
 
 
 
 
 
 
 
 
5 
  
Haematology (mL) 
 
 
 
 
 
 
 
 
 
 
 
 
2 
  
Biochemistry (mL)** 
 
 
 
 
 
 
 
 
 
 
 
 
3 
  
Immunology (mL) 
 
 
 
 
 
 
 
 
 
 
 
  
30 
  
Immunology serum (mL) 
 
 
 
 
 
 
 
 
 
 
 
  
40 
  
Gene expression profiling (mL) 
15 
  
15 
  
15 
  
15 
  
15 
  
15 
  
15 
  
qPCR & gametocyte PCR (mL) 
3 
3 
3 
3 
3 
3 
3 
3 
3 
3 
3 
3 
3 
3 
Blood film(mL) 
1 
1 
1 
1 
1 
1 
1 
1 
1 
1 
1 
1 
1 
1 
Visit Total Blood Volume (mL) 
19 
4 
19 
4 
19 
4 
19 
4 
19 
4 
19 
4 
99 
4 
Cumulative blood volume  (mL) 
310 
314 
333 
337 
356 
360 
379 
383 
402 
406 
425 
429 
528 
532 
Table 7a: Schedule of events for days 8-14 after challenge for volunteers in VAC069A (timeline/window are in relation to the day of challenge). C = 
challenge; (X) = If considered necessary. Visits continue according to schedule until diagnostic criteria are reached; when diagnostic criteria are met, 
venepuncture and procedures are followed according to Table 10a.*Physical observations include blood pressure, pulse and temperature. **Biochemistry 
comprises sodium, potassium, urea, creatinine, albumin, liver function tests. aFrom C+13, visits will once daily only if qPCR <1000 genome copies/mL, i.e. 
C+13.5 and C+14.5 visits will be omitted if this threshold has not been reached. 
 
 
 
 
 

VAC069: A study of blood-stage controlled human Plasmodium vivax malaria infection. IRAS project ID: 252499 
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Day 
C+8 
C+9 
C+10 C+10.5 C+11 C+11.5 
C+12 
C+12.5 C+13 
C+13.5 
C+14 
C+14.5 
Attendance number 
6 
7 
8 
9 
10 
11 
12 
13 
14 
15 
16 
17 
Timeline (days) (window) 
8 
9 
10 
10 
11 
11 
12 
12 
13 
13 
14 
14 
Physical observations* 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Medical history/examination 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
Review AEs & medications 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Serum βhCG (mL) (Females only) 
 
 
 
 
 
 
 
 
 
 
5 
  
Haematology (mL) 
 
 
 
 
 
 
 
 
 
 
2 
  
Biochemistry (mL)** 
 
 
 
 
 
 
 
 
 
 
3 
  
Immunology plasma (mL) 
 
 
 
 
 
 
 
 
 
  
30 
  
Immunology serum (mL) 
 
 
 
 
 
 
 
 
 
  
30 
  
Gene expression profiling EDTA sample (mL) 
9 
3 
9 
 
3 
  
9 
  
3 
  
9 
  
Gene expression profiling Li Heparin sample (mL) 
3 
 
3 
 
 
 
3 
 
 
 
3 
 
DMFA (mL) 
 
 
 
 
 
 
 
 
 
 
10 
 
Direct feeding assay (mosquito feeding) 
 
 
 
 
 
 
 
 
 
 
X 
 
qPCR & gametocyte PCR (mL) 
3 
3 
3 
3 
3 
3 
3 
3 
3 
3 
3 
3 
Blood film(mL) 
1 
1 
1 
1 
1 
1 
1 
1 
1 
1 
1 
1 
Visit Total Blood Volume (mL) 
16 
7 
16 
4 
7 
4 
16 
4 
7 
4 
96 
4 
Cumulative blood volume  (mL) 
235 
242 
258 
262 
269 
273 
289 
293 
300 
304 
400 
404 
Table 7b: Schedule of events for days 8-14 after challenge for volunteers in VAC069B (timeline/window are in relation to the day of challenge). C = 
challenge; (X) = If considered necessary. Visits continue according to schedule until diagnostic criteria are reached; when diagnostic criteria are met, 
venepuncture and procedures are followed according to Table 10b.*Physical observations include blood pressure, pulse and temperature. **Biochemistry 
comprises sodium, potassium, urea, creatinine, albumin, liver function tests. 
 
 
 
 

VAC069: A study of blood-stage controlled human Plasmodium vivax malaria infection. IRAS project ID: 252499 
VAC069 Clinical Trial Protocol, V8.0, 14th June 2022, University of Oxford.  Page 88 of 122 
Day 
C+8 
C+9 
C+10 
C+10.5 
C+11 
C+11.5 
C+12 
C+12.5 
C+13 
C+13.5 
C+14 
C+14.5 
Attendance number 
5 
6 
7 
8 
9 
10 
11 
12 
13 
14 
15 
16 
Timeline (days) (window) 
8 
9 
10 
10 
11 
11 
12 
12 
13 
13 
14 
14 
Physical observations* 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Medical history/examination 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
Review AEs & medications 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Serum βhCG (mL) (females only) 
 
 
 
 
 
 
 
 
 
 
5 
  
Haematology (mL) 
 
 
 
 
 
 
 
 
 
 
2 
  
Biochemistry (mL)** 
 
 
 
 
 
 
 
 
 
 
3 
  
Exploratory Assays (mL) 
12 
 
12 
 
 
 
12 
 
 
 
82 
 
DMFA (mL) 
 
 
 
 
 
 
 
 
 
 
10 
 
Direct feeding assay (mosquito feeding) 
 
 
 
 
 
 
 
 
 
 
X 
 
qPCR & gametocyte PCR (mL) 
2 
2 
2 
2 
2 
2 
2 
2 
2 
2 
2 
2 
Visit Total Blood Volume (mL)^ 
14 
2 
14 
2 
2 
2 
14 
2 
2 
2 
94 
2 
Cumulative blood volume  (mL) 
205 
207 
221 
223 
225 
227 
241 
243 
245 
247 
341 
343 
Table 7c: Schedule of events for days 8-14 after challenge for volunteers in VAC069C (timeline/window are in relation to the day of challenge). C = challenge; 
(X) = If considered necessary. Visits continue according to schedule until diagnostic criteria are reached; when diagnostic criteria are met, venepuncture and 
procedures are followed according to Table 10c.*Physical observations include blood pressure, pulse and temperature. **Biochemistry comprises sodium, 
potassium, urea, creatinine, albumin, liver function tests. ^Blood volumes as given in the table are maximal volumes and smaller blood volumes may be taken 
at any visit, at the discretion of the investigator, with the exception of additional blood volume that may be required for any additional tests that may be 
performed if deemed clinically necessary. 
 
 

VAC069: A study of blood-stage controlled human Plasmodium vivax malaria infection. IRAS project ID: 252499 
VAC069 Clinical Trial Protocol, V8.0, 14th June 2022, University of Oxford.  Page 89 of 122 
Day 
C+8 
C+9 
C+10 
C+10.5$ 
C+11 
C+11.5$ 
C+12 
C+12.5$ 
C+13 
C+13.5$ 
C+14 
C+14.5$ 
Attendance number 
5 
6 
7 
8 
9 
10 
11 
12 
13 
14 
15 
16 
Timeline (days) (window) 
8 
9 
10 
10 
11 
11 
12 
12 
13 
13 
14 
14 
Physical observations* 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Medical history/examination 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
Review AEs & medications 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Serum βhCG (mL) (females only) 
 
 
 
 
 
 
 
 
 
 
5 
  
Haematology (mL) 
 
 
 
 
 
 
 
 
 
 
2 
  
Biochemistry (mL)** 
 
 
 
 
 
 
 
 
 
 
3 
  
Exploratory Assays (mL) 
 
 
 
 
 
 
12 
 
 
 
82 
 
DMFA (mL) 
 
 
 
 
 
 
 
 
 
 
 
 
Direct feeding assay (mosquito feeding) 
 
 
 
 
 
 
 
 
 
 
X 
 
qPCR & gametocyte PCR (mL) 
2 
2 
2 
2 
2 
2 
2 
2 
2 
2 
2 
2 
Visit Total Blood Volume (mL)^ 
2 
2 
2 
2 
2 
2 
14 
2 
2 
2 
94 
2 
Cumulative blood volume  (mL) 
193 
195 
197 
199 
201 
203 
217 
219 
221 
223 
317 
319 
Table 7d: Schedule of events for days 8-14 after challenge for volunteers in VAC069D (timeline/window are in relation to the day of challenge). C = 
challenge; (X) = If considered necessary. Visits continue according to schedule until diagnostic criteria are reached; when diagnostic criteria are met, 
venepuncture and procedures are followed according to Table 10d. $From day 10 post challenge onwards, visits will continue once a day in mornings, 
increasing to twice daily if the malaria qPCR count is >1000 genome copies/ml. *Physical observations include blood pressure, pulse and temperature. 
**Biochemistry comprises sodium, potassium, urea, creatinine, albumin, liver function tests. ^Blood volumes as given in the table are maximal volumes and 
smaller blood volumes may be taken at any visit, at the discretion of the investigator, with the exception of additional blood volume that may be required for 
any additional tests that may be performed if deemed clinically necessary. 
 
 

VAC069: A study of blood-stage controlled human Plasmodium vivax malaria infection. IRAS project ID: 252499 
VAC069 Clinical Trial Protocol, V8.0, 14th June 2022, University of Oxford.  Page 90 of 122 
Day 
C+7.5$ 
C+8 
C+8.5$ 
C+9 
C+9.5$ 
C+10 C+10.5$ C+11 C+11.5$ C+12 C+12.5$ C+13 C+13.5$ C+14 C+14.5$ 
Attendance number 
6 
7 
8 
9 
10 
11 
12 
13 
14 
15 
16 
17 
18 
19 
20 
Timeline (days) 
(window) 
7 
8 
8 
9 
9 
10 
10 
11 
11 
12 
12 
13 
13 
14 
14 
Physical 
observations* 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Medical 
history/examination 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
Review AEs & 
medications 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Serum βhCG (mL) 
(females only) 
 
 
 
 
 
 
 
 
 
 
 
 
 
5 
 
Haematology (mL) 
 
 
 
 
 
 
 
 
 
 
 
 
 
2 
 
Biochemistry (mL)** 
 
 
 
 
 
 
 
 
 
 
 
 
 
3 
 
Exploratory Assays 
(mL) 
 
 
 
12 
 
 
 
12 
 
 
 
12 
 
10 
 
qPCR & gametocyte 
PCR (mL) 
2 
2 
2 
2 
2 
2 
2 
2 
2 
2 
2 
2 
2 
2 
2 
Visit Total Blood 
Volume (mL)^ 
2 
2 
2 
14 
2 
2 
2 
14 
2 
2 
2 
14 
2 
22 
2 
Cumulative blood 
volume  (mL) 
159 
161 
163 
177 
179 
181 
183 
197 
199 
201 
203 
217 
219 
241 
243 
Table 7e: Schedule of events for days 8-14 after challenge for volunteers in VAC069E (timeline/window are in relation to the day of challenge). C = challenge; 
(X) = If considered necessary. Visits continue according to schedule until diagnostic criteria are reached; when diagnostic criteria are met, venepuncture and 
procedures are followed according to Table 10e. $From day 7 post challenge onwards, visits will continue once a day in mornings, increasing to twice daily if 
the malaria qPCR count is >1000 genome copies/ml. *Physical observations include blood pressure, pulse and temperature. **Biochemistry comprises sodium, 
potassium, urea, creatinine, liver function tests. ^Blood volumes as given in the table are maximal volumes and smaller blood volumes may be taken at any 
visit, at the discretion of the investigator, with the exception of additional blood volume that may be required for any additional tests that may be performed if 
deemed clinically necessary. 
 
 

VAC069: A study of blood-stage controlled human Plasmodium vivax malaria infection. IRAS project ID: 252499 
VAC069 Clinical Trial Protocol, V8.0, 14th June 2022, University of Oxford.  Page 91 of 122 
 
 
 
 
 
Day 
C+15 C+15.5a C+16 C+16.5a C+17 C+17.5a C+18 C+18.5a 
C+19 
C+19.5a C+20 C+20.5a 
Attendance number 
31 
32 
33 
34 
35 
36 
37 
38 
39 
40 
41 
42 
Timeline (days) (window) 
15 
15 
16 
16 
17 
17 
18 
18 
19 
19 
20 
20 
Physical observations 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Medical history/examination 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
Review Aes & medications 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Gene expression profiling (mL) 
15 
  
15 
  
15 
  
15 
  
15 
  
15 
  
qPCR & gametocyte PCR (mL) 
3 
3 
3 
3 
3 
3 
3 
3 
3 
3 
3 
3 
Blood film(mL) 
1 
1 
1 
1 
1 
1 
1 
1 
1 
1 
1 
1 
Visit Total Blood Volume (mL) 
19 
4 
19 
4 
19 
4 
19 
4 
19 
4 
19 
4 
Cumulative blood volume  (mL) 
551 
555 
574 
578 
597 
601 
620 
624 
643 
647 
666 
670 
 
Table 8a: Schedule of events for days 15-20 after challenge for volunteers in VAC069A (timeline/window are in relation to the day of challenge). C = 
challenge; (X) = If considered necessary. Visits continue according to schedule until diagnostic criteria are reached; when diagnostic criteria are met, 
venepuncture and procedures are followed according to Table 10a.*Physical observations include blood pressure, pulse and temperature. **Biochemistry 
comprises sodium, potassium, urea, creatinine, albumin, liver function tests. aFrom C+13, visits will once daily only if qPCR <1000 genome copies/mL, i.e. 
C+15.5, C+16.5, C+17.5, C+18.5, C+19.5 and C+20.5 visits will be omitted if this threshold has not been reached . 
 
 
 
 
 

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Day 
C+15 C+15.5 C+16 C+16.5 C+17 C+17.5 C+18 C+18.5 
C+19 
C+19.5 C+20 C+20.5 
Attendance number 
18 
19 
20 
21 
22 
23 
24 
25 
26 
27 
28 
29 
Timeline (days) (window) 
15 
15 
16 
16 
17 
17 
18 
18 
19 
19 
20 
20 
Physical observations 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Medical history/examination 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
Review AEs & medications 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Immunology plasma (mL) 
 
 
 
 
 
 
 
 
 
 
 
 
Immunology serum (mL) 
 
 
 
 
 
 
 
 
 
 
 
 
Gene expression profiling EDTA sample (mL) 
3 
  
9 
  
3 
  
9 
  
3 
  
9 
  
Gene expression profiling Li Heparin sample (mL) 
 
 
3 
 
 
 
3 
 
 
 
3 
 
qPCR & gametocyte PCR (mL) 
3 
3 
3 
3 
3 
3 
3 
3 
3 
3 
3 
3 
Blood film(mL) 
1 
1 
1 
1 
1 
1 
1 
1 
1 
1 
1 
1 
Visit Total Blood Volume (mL) 
7 
4 
16 
4 
7 
4 
16 
4 
7 
4 
16 
4 
Cumulative blood volume  (mL) 
411 
415 
431 
435 
442 
446 
462 
466 
473 
477 
493 
497 
 
Table 8b: Schedule of events for days 15-20 after challenge for volunteers in VAC069B (timeline/window are in relation to the day of challenge). C = 
challenge; (X) = If considered necessary. Visits continue according to schedule until diagnostic criteria are reached; when diagnostic criteria are met, 
venepuncture and procedures are followed according to Table 10b.*Physical observations include blood pressure, pulse and temperature. **Biochemistry 
comprises sodium, potassium, urea, creatinine, albumin, liver function tests. aFrom C+13, visits will once daily only if qPCR <1000 genome copies/mL, i.e. 
C+15.5, C+16.5, C+17.5, C+18.5, C+19.5 and C+20.5 visits will be omitted if this threshold has not been reached . 
 
 
 

VAC069: A study of blood-stage controlled human Plasmodium vivax malaria infection. IRAS project ID: 252499 
VAC069 Clinical Trial Protocol, V8.0, 14th June 2022, University of Oxford.  Page 93 of 122 
 
 
 
Day 
C+15 C+15.5 C+16 C+16.5 C+17 C+17.5 C+18 C+18.5 
C+19 
C+19.5 C+20 C+20.5 
Attendance number 
17 
18 
19 
20 
21 
22 
23 
24 
25 
26 
27 
28 
Timeline (days) (window) 
15 
15 
16 
16 
17 
17 
18 
18 
19 
19 
20 
20 
Physical observations 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Medical history/examination 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
Review AEs & medications 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Exploratory Assays (mL) 
 
 
12 
 
 
 
12 
 
 
 
12 
 
qPCR & gametocyte PCR (mL) 
2 
2 
2 
2 
2 
2 
2 
2 
2 
2 
2 
2 
Visit Total Blood Volume (mL)^ 
2 
2 
14 
2 
2 
2 
14 
2 
2 
2 
14 
2 
Cumulative blood volume  (mL) 
345 
347 
361 
363 
365 
367 
381 
383 
385 
387 
401 
403 
 
Table 8c: Schedule of events for days 15-20 after challenge for volunteers in VAC069C (timeline/window are in relation to the day of challenge). C = 
challenge; (X) = If considered necessary. Visits continue according to schedule until diagnostic criteria are reached; when diagnostic criteria are met, 
venepuncture and procedures are followed according to Table 10c.*Physical observations include blood pressure, pulse and temperature. **Biochemistry 
comprises sodium, potassium, urea, creatinine, albumin, liver function tests. ^Blood volumes as given in the table are maximal volumes and smaller blood 
volumes may be taken at any visit, at the discretion of the investigator, with the exception of additional blood volume that may be required for any additional 
tests that may be performed if deemed clinically necessary. 
 
 

VAC069: A study of blood-stage controlled human Plasmodium vivax malaria infection. IRAS project ID: 252499 
VAC069 Clinical Trial Protocol, V8.0, 14th June 2022, University of Oxford.  Page 94 of 122 
Day 
C+15 C+15.5$ C+16 C+16.5$ C+17 C+17.5$ C+18 C+18.5$ 
C+19 
C+19.5$ C+20 C+20.5$ 
Attendance number 
17 
18 
19 
20 
21 
22 
23 
24 
25 
26 
27 
28 
Timeline (days) (window) 
15 
15 
16 
16 
17 
17 
18 
18 
19 
19 
20 
20 
Physical observations 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Medical history/examination 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
Review AEs & medications 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Exploratory Assays (mL) 
 
 
12 
 
 
 
12 
 
 
 
12 
 
qPCR & gametocyte PCR (mL) 
2 
2 
2 
2 
2 
2 
2 
2 
2 
2 
2 
2 
Visit Total Blood Volume (mL)^ 
2 
2 
14 
2 
2 
2 
14 
2 
2 
2 
14 
2 
Cumulative blood volume  (mL) 
321 
323 
337 
339 
341 
343 
357 
359 
361 
363 
377 
379 
 
Table 8d: Schedule of events for days 15-20 after challenge for volunteers in VAC069D (timeline/window are in relation to the day of challenge). C = 
challenge; (X) = If considered necessary. Visits continue according to schedule until diagnostic criteria are reached; when diagnostic criteria are met, 
venepuncture and procedures are followed according to Table 10d. $From day 10 post challenge onwards, visits will continue once a day in mornings, 
increasing to twice daily if the malaria qPCR count is >1000 genome copies/ml. *Physical observations include blood pressure, pulse and temperature. 
**Biochemistry comprises sodium, potassium, urea, creatinine, albumin, liver function tests. ^Blood volumes as given in the table are maximal volumes and 
smaller blood volumes may be taken at any visit, at the discretion of the investigator, with the exception of additional blood volume that may be required for 
any additional tests that may be performed if deemed clinically necessary. 
 
 
 

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VAC069 Clinical Trial Protocol, V8.0, 14th June 2022, University of Oxford.  Page 95 of 122 
Day 
C+15 C+15.5$ C+16 C+16.5$ C+17 C+17.5$ C+18 C+18.5$ 
C+19 
C+19.5$ C+20 C+20.5$ 
Attendance number 
21 
22 
23 
24 
25 
26 
27 
28 
29 
30 
31 
32 
Timeline (days) (window) 
15 
15 
16 
16 
17 
17 
18 
18 
19 
19 
20 
20 
Physical observations 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Medical history/examination 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
Review AEs & medications 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Exploratory Assays (mL) 
12 
 
 
 
12 
 
 
 
12 
 
 
 
qPCR & gametocyte PCR (mL) 
2 
2 
2 
2 
2 
2 
2 
2 
2 
2 
2 
2 
Visit Total Blood Volume (mL)^ 
14 
2 
2 
2 
14 
2 
2 
2 
14 
2 
2 
2 
Cumulative blood volume  (mL) 
257 
259 
261 
263 
277 
279 
281 
283 
297 
299 
301 
303 
 
Table 8e: Schedule of events for days 15-20 after challenge for volunteers in VAC069E (timeline/window are in relation to the day of challenge). C = 
challenge; (X) = If considered necessary. Visits continue according to schedule until diagnostic criteria are reached; when diagnostic criteria are met, 
venepuncture and procedures are followed according to Table 10e. $From day 7 post challenge onwards, visits will continue once a day in mornings, 
increasing to twice daily if the malaria qPCR count is >1000 genome copies/ml. *Physical observations include blood pressure, pulse and temperature. 
**Biochemistry comprises sodium, potassium, urea, creatinine, liver function tests. ^Blood volumes as given in the table are maximal volumes and smaller 
blood volumes may be taken at any visit, at the discretion of the investigator, with the exception of additional blood volume that may be required for any 
additional tests that may be performed if deemed clinically necessary. 
 
 
 
 
 
 
 
 

VAC069: A study of blood-stage controlled human Plasmodium vivax malaria infection. IRAS project ID: 252499 
VAC069 Clinical Trial Protocol, V8.0, 14th June 2022, University of Oxford.  Page 96 of 122 
 
Table 9a: Schedule of events for days 21 - 27 after challenge for volunteers in VAC069B (timeline/window are in relation to the day of challenge). C = challenge; (X) = If 
considered necessary. Visits continue according to schedule until diagnostic criteria are reached; when diagnostic criteria are met, venepuncture and procedures are followed 
according to Table 10b.*Physical observations include blood pressure, pulse and temperature. **Biochemistry comprises sodium, potassium, urea, creatinine, albumin, liver 
function tests. a From C+21, visits may be reduced to once daily at the discretion of the Investigator (as agreed by the Chief Investigator) i.e. C+21.5, C+22.5, C+23.5, C+24.5, 
C+25.5, C+26.5 and C+27.5 visits may be omitted at the Investigator’s discretion  
Day 
C+21 
C+21.5 
C+22 
C+22.5 
C+23 
C+23.5 
C+24 
C+24.5 
C+25 
C+25.5 
C+26 
C+26.5 
C+27 
C+27.5 
Attendance number 
30 
31 
32 
33 
34 
35 
36 
37 
38 
39 
40 
41 
42 
43 
Timeline (days) (window) 
21 
21 
22 
22 
23 
23 
24 
24 
25 
25 
26 
26 
27 
27 
Physical observations 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Medical history/examination 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
Review AEs & medications 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Serum βhCG (mL) (females 
only) 
5 
 
 
 
 
 
 
 
 
 
 
 
 
 
Haematology (mL) 
2 
 
 
 
 
 
 
 
 
 
 
 
 
 
Biochemistry (mL)** 
3 
 
 
 
 
 
 
 
 
 
 
 
 
 
Immunology plasma (mL) 
30 
 
 
 
 
 
 
 
 
 
 
 
 
 
Immunology serum (mL) 
30 
 
 
 
 
 
 
 
 
 
 
 
 
 
Gene expression profiling 
EDTA sample (mL) 
3 
 
9 
 
3 
 
9 
 
3 
 
9 
 
3 
 
Gene expression profiling Li 
Heparin sample (mL) 
 
 
3 
 
 
 
3 
 
 
 
3 
 
 
 
qPCR & gametocyte PCR (mL) 
3 
3 
3 
3 
3 
3 
3 
3 
3 
3 
3 
3 
3 
3 
Blood film(mL) 
1 
1 
1 
1 
1 
1 
1 
1 
1 
1 
1 
1 
1 
1 
Visit Total Blood Volume (mL) 
77 
4 
16 
4 
7 
4 
16 
4 
7 
4 
16 
4 
7 
4 
Cumulative blood volume  
(mL) 
574 
578 
594 
598 
605 
609 
625 
629 
636 
640 
656 
660 
667 
671 

VAC069: A study of blood-stage controlled human Plasmodium vivax malaria infection. IRAS project ID: 252499 
VAC069 Clinical Trial Protocol, V8.0, 14th June 2022, University of Oxford.  Page 97 of 122 
 
 
Table 9b: Schedule of events for days 21 - 27 after challenge for volunteers in VAC069C (timeline/window are in relation to the day of challenge). C = 
challenge; (X) = If considered necessary. Visits continue according to schedule until diagnostic criteria are reached; when diagnostic criteria are met, 
venepuncture and procedures are followed according to Table 10c.*Physical observations include blood pressure, pulse and temperature. **Biochemistry 
comprises sodium, potassium, urea, creatinine, albumin, liver function tests. a From C+21, visits may be reduced to once daily at the discretion of the 
Investigator (as agreed by the Chief Investigator) i.e. C+21.5, C+22.5, C+23.5, C+24.5, C+25.5, C+26.5 and C+27.5 visits may be omitted at the Investigator’s 
discretion ^Blood volumes as given in the table are maximal volumes and smaller blood volumes may be taken at any visit, at the discretion of the 
investigator, with the exception of additional blood volume that may be required for any additional tests that may be performed if deemed clinically 
necessary.  
Day 
C+21 C+21.5a C+22 C+22.5a C+23 
C+23.5a 
C+24 
C+24.5a 
C+25 
C+25.5a 
C+26 
C+26.5a 
C+27 
C+27.5a 
Attendance number 
29 
30 
31 
32 
33 
34 
35 
36 
37 
38 
39 
40 
41 
42 
Timeline (days) (window) 
21 
21 
22 
22 
23 
23 
24 
24 
25 
25 
26 
26 
27 
27 
Physical observations 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Medical history/examination 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
Review AEs & medications 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Serum βhCG (mL) (females 
only) 
5 
 
 
 
 
 
 
 
 
 
 
 
 
 
Haematology (mL) 
2 
 
 
 
 
 
 
 
 
 
 
 
 
 
Biochemistry (mL)** 
3 
 
 
 
 
 
 
 
 
 
 
 
 
 
Exploratory Assays (mL) 
60 
 
12 
 
 
 
12 
 
 
 
12 
 
 
 
qPCR & gametocyte PCR (mL) 
2 
2 
2 
2 
2 
2 
2 
2 
2 
2 
2 
2 
2 
2 
Visit Total Blood Volume 
(mL)^ 
72 
2 
14 
2 
2 
2 
14 
2 
2 
2 
14 
2 
2 
2 
Cumulative blood volume  
(mL) 
475 
477 
491 
493 
495 
497 
511 
513 
515 
517 
531 
533 
535 
537 

VAC069: A study of blood-stage controlled human Plasmodium vivax malaria infection. IRAS project 
ID: 252499 
VAC069 Clinical Trial Protocol, V8.0, 14th June 2022, University of Oxford.  Page 98 of 122 
Day 
D/C+21 if 
undiagnoseda 
T+1 
T+2 
T+6 
C+28 
C+45 
C+90 
Attendance number 
43 
44 
45 
46 
47 
48 
49 
Timeline (days) 
(window) 
 
 
 
 
28(±4
) 
45(±7
) 
90(±7
) 
Physical 
observations* 
X 
X 
X 
X 
X 
X 
X 
Medical 
history/examination 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
Review Aes & 
medications 
X 
X 
X 
X 
X 
X 
X 
Review 
contraindications 
X 
 
 
 
 
 
 
Diary card collected 
 
 
 
 
X 
(X) 
(X) 
Antimalarials Initiated 
X 
 
 
 
 
 
 
Urine β-hCG (females 
only) 
X 
 
 
 
 
 
 
Serum βhCG (mL) 
(females only) 
5 
 
 
 
 
 
 
Haematology (mL) 
2 
2 
 
2 
2 
0 
2 
Biochemistry (mL)** 
3 
3 
 
3 
3 
0 
3 
Immunology plasma 
(mL) 
30b 
 
 
 
30 
 
30 
Immunology serum 
(mL) 
30b 
 
 
 
40 
4 
40 
Safety serum (mL)^ 
 
 
 
 
 
 
4 
Plasma for 
exploratory studies 
(mL) 
13 
 
 
 
 
 
 
Gene expression 
profiling (mL) 
35 
 
 
15 
1 
20 
 
qPCR & gametocyte 
PCR (mL) 
3 
3 
3 
3 
3 
3 
3 
Blood film(mL) 
1 
1 
1 
 
 
 
 
Visit Total Blood 
Volume (mL) 
122 
9 
4 
23 
79 
27 
82 
Cumulative blood 
volume  (mL) 
792 
801 
805 
828 
907 
934 
1016 
Table 10a: Schedule of events from diagnosis to 90 days after challenge for volunteers in 
VAC069A (timeline/window are in relation to the day of challenge). D = diagnosis; C = challenge; T = 
treatment; (X) = If considered necessary.*Physical observations include blood pressure, pulse and 
temperature. **Biochemistry comprises sodium, potassium, urea, creatinine, albumin, liver function 
tests. ^Serum will be sent for HBsAg, HCV antibodies, HIV antibodies, CMV antibodies and EBV 
antibodies. However, EBV and CMV serology will only be repeated post-challenge if the participant 
had negative serology results at screening.   aBloods to be taken immediately before drug treatment, 
if diagnostic criteria not met, treatment will be started at C+21 morning visit b If diagnosis is made at 
C+14.5 visit, immunology draw will not be taken, since a large volume bleed is taken for 
immunological analysis at morning C+14 visit

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VAC069 Clinical Trial Protocol, V8.0, 14th June 2022, University of Oxford.  Page 99 of 122 
Day 
D/C+28 if undiagnoseda 
T+1c 
T+2 c 
T+3 
T+6 
C+56 
C+96 
Attendance number 
44 
45 
46 
47 
48 
49 
50 
Timeline (days) (window) 
- 
- 
- 
- 
- 
56(±4) 
96(±7) 
Physical observations* 
X 
X 
X 
X 
X 
X 
X 
Medical history/examination 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
Review AEs & medications 
X 
X 
X 
X 
X 
X 
X 
Review contraindications 
X 
 
 
 
 
 
 
Diary card collected 
 
 
 
 
X 
(X) 
(X) 
Antimalarials Initiated 
X 
 
 
 
 
 
 
Urine β-hCG (females only) 
X 
 
 
 
 
 
 
Serum βhCG (mL) (females only) 
5 
 
 
 
 
 
 
Haematology (mL) 
2 
 
 
 
2 
 
2 
Biochemistry (mL)** 
3 
 
 
 
3 
 
3 
Diagnostic Serology^ 
 
 
 
 
 
 
4 
Immunology plasma (mL) 
30b 
 
 
 
 
30 
30 
Immunology serum (mL) 
40b 
 
 
 
 
40 
40 
Parasite cryopreservation 
20 
 
 
 
 
 
 
DMFA (mL) 
10 
 
 
 
 
 
 
Direct feeding assay (mosquito feeding) 
X 
 
 
 
 
 
 
Plasma for exploratory studies (mL) 
3 
 
 
 
 
 
 
Safety serum (mL)^ 
 
 
 
 
 
 
4 
Gene expression profiling EDTA sample 
(mL) 
9 
9 
 
9 
9 
17 
 
Gene expression profiling Li Heparin 
sample (mL) 
23 
3 
 
3 
3 
3 
 
qPCR & gametocyte PCR (mL) 
3 
3 
3 
 
 
 
 
Blood film(mL) 
1 
1 
1 
 
 
 
 
Visit Total Blood Volume (mL) 
149 
16 
4 
12 
17 
90 
83 
Cumulative blood volume  (mL) 
820 
836 
840 
852 
869 
959 
1042 
Table 10b: Schedule of events from diagnosis to 96 days after challenge for volunteers in VAC069B (timeline/window are in relation to the day of challenge). D = diagnosis; 
C = challenge; T = treatment; (X) = If considered necessary.*Physical observations include blood pressure, pulse and temperature. **Biochemistry comprises sodium, 
potassium, urea, creatinine, albumin, liver function tests. ^Diagnostic serology includes; HBsAg, HCV antibodies, HIV antibodies, CMV antibodies and EBV 

VAC069: A study of blood-stage controlled human Plasmodium vivax malaria infection. IRAS project ID: 252499 
VAC069 Clinical Trial Protocol, V8.0, 14th June 2022, University of Oxford.  Page 100 of 122 
antibodies. However, EBV and CMV serology will only be repeated post-challenge if the participant had negative serology results at screening.   aBloods to be 
taken immediately before drug treatment, if diagnostic criteria not met, treatment will be started at C+28 morning visit b If diagnosis is made at C+14.5 or C+21.5 visit, 
immunology draw will not be taken, since a large volume bleed is taken for immunological analysis at morning C+14 and C+21 visits. c Blood draws for microscopy and qPCR 
will continue until the volunteer has two consecutive qPCR readings with substantial reduction in genome copies per mL (and at least one negative thick film reading if 
treated on basis of thick film).  
 
 

VAC069: A study of blood-stage controlled human Plasmodium vivax malaria infection. IRAS project ID: 252499 
VAC069 Clinical Trial Protocol, V8.0, 14th June 2022, University of Oxford.  Page 101 of 122 
Day 
D/C+28 if undiagnoseda 
T+1c 
T+2 c 
T+3 
T+6 
T+9 
C+56 
C+96 
Attendance number 
43 
44 
45 
46 
47 
- 
48 
49 
Timeline (days) (window) 
- 
- 
- 
- 
- 
+2 
56(±14) 
96(±14) 
Physical observations* 
X 
X 
X 
X 
X 
 
X 
X 
Medical history/examination 
(X) 
(X) 
(X) 
(X) 
(X) 
 
(X) 
(X) 
Review AEs & medications 
X 
X 
X 
X 
X 
 
X 
X 
Review contraindications 
X 
 
 
 
 
 
 
 
Phone call 
 
 
 
 
 
X 
 
 
Diary card collected 
 
 
 
 
X 
 
(X) 
(X) 
Antimalarials Initiated 
X 
 
 
 
 
 
 
 
Urine β-hCG (females only) 
X 
 
 
 
 
 
 
 
Serum βhCG (mL) 
5 
 
 
 
 
 
 
 
Haematology (mL) 
2 
2 
 
2 
2 
 
2 
2 
Biochemistry (mL)** 
3 
3 
 
3 
3 
 
3 
3 
Safety serum^ 
 
 
 
 
 
 
 
4 
Exploratory Assays 
106 
12 
 
12 
12 
 
82 
70 
DMFA (mL) 
10 
 
 
 
 
 
 
 
Direct feeding assay (mosquito feeding)@ 
X 
 
 
 
 
 
 
 
qPCR & gametocyte PCR (mL)C 
2 
2 
2 
 
 
 
 
 
SARS-COV-2 PCR swab 
X 
 
 
 
 
 
 
 
Visit Total Blood Volume (mL) 
128 
19 
2 
17 
17 
0 
87 
79 
Cumulative blood volume  (mL) 
665 
684 
686 
703 
720 
720 
807 
886 
Table 10c: Schedule of events from diagnosis to 96 days after challenge for volunteers in VAC069C (timeline/window are in relation to the day of challenge 
or treatment). D = diagnosis; C = challenge; T = treatment; (X) = If considered necessary.*Physical observations include blood pressure, pulse and temperature. 
**Biochemistry comprises sodium, potassium, urea, creatinine, albumin, liver function tests. ^Diagnostic serology includes; HBsAg, HCV antibodies, HIV 
antibodies, CMV antibodies and EBV antibodies. However, EBV and CMV serology will only be repeated post-challenge if the participant had negative serology 
results at screening.  aBloods to be taken immediately before drug treatment, if diagnostic criteria not met, treatment will be started at C+28 morning visit b If 
diagnosis is made at C+14.5 or C+21.5 visit, immunology draw will not be taken, since a large volume bleed is taken for immunological analysis at morning 
C+14 and C+21 visits. c Blood draws for qPCR will continue daily until the volunteer has two consecutive qPCR readings with substantial reduction in genome 
copies per mL. @DFA is optional. Blood volumes as given in the table are maximal volumes and smaller blood volumes may be taken at any visit, at the 
discretion of the investigator, with the exception of additional blood volume that may be required for any additional tests that may be performed if deemed 
clinically necessary. 

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Day 
D/C+21 if undiagnoseda 
T+1c 
T+3c 
T+6 
T+9 
C+56 
C+96 
Attendance number 
29 
30 
31 
32 
33 
34 
35 
Timeline (days) (window) 
- 
- 
- 
±1 
±2 
56(±14) 
96(±14) 
Physical observations* 
X 
X 
X 
X 
 
X 
X 
Medical history/examination 
(X) 
(X) 
(X) 
(X) 
 
(X) 
(X) 
Review AEs & medications 
X 
X 
X 
X 
 
X 
X 
Review contraindications 
X 
 
 
 
 
 
 
Phone call 
 
 
 
 
X 
 
 
Diary card collected 
 
 
 
X 
 
(X) 
(X) 
Antimalarials Initiated 
X 
 
 
 
 
 
 
Urine β-hCG (females only) 
X 
 
 
 
 
 
 
Serum βhCG (mL) 
5 
 
 
 
 
 
 
Haematology (mL) 
2 
2 
2 
2 
 
2 
2 
Biochemistry (mL)** 
3 
3 
3 
3 
 
3 
3 
Serum storage 
 
 
 
 
 
 
4 
HIV, HBV, HCV, EBV, CMV serology (mL)^ 
 
 
 
 
 
 
5 
Exploratory Assays 
106b 
12 
12 
12 
 
82 
70 
DMFA (mL) 
10 
 
 
 
 
 
 
Direct feeding assay (mosquito feeding)@ 
X 
 
 
 
 
 
 
qPCR & gametocyte PCR (mL)C 
2 
2 
2 
 
 
 
 
SARS-COV-2 PCR swab 
X 
 
 
 
 
 
 
Visit Total Blood Volume (mL) 
128 
19 
19 
17 
0 
87 
84 
Cumulative blood volume  (mL) 
507 
526 
545 
562 
562 
649 
733 
Table 10d: Schedule of events from diagnosis to 96 days after challenge for volunteers in VAC069D (timeline/window are in relation to the day of challenge 
or treatment). D = diagnosis; C = challenge; T = treatment; (X) = If considered necessary.*Physical observations include blood pressure, pulse and temperature. 
**Biochemistry comprises sodium, potassium, urea, creatinine, albumin, liver function tests. ^EBV and CMV serology will only be repeated at the C+96 
timepoint if they were negative at screening.  aBloods to be taken immediately before drug treatment, if diagnostic criteria not met, treatment will be started 
at C+21 morning visit b If diagnosis is made at C+14.5 visit, 73ml of immunology bleed will not be taken, since a large volume bleed is taken for immunological 
analysis at morning C+14 visit. c Blood draws for qPCR will continue until the volunteer has two consecutive qPCR readings with substantial reduction in 
genome copies per mL. @DFA is optional. Blood volumes as given in the table are maximal volumes and smaller blood volumes may be taken at any visit, at the 
discretion of the investigator, with the exception of additional blood volume that may be required for any additional tests that may be performed if deemed 
clinically necessary. 

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Day 
D/C+21 if undiagnoseda 
T+1 
T+3 
T+6 
T+10 
C+56 
C+96 
Attendance number 
33 
34 
35 
36 
37 
38 
39 
Timeline (days) (window) 
- 
- 
(±0.5) 
(±1) 
(±2) 
56 (±14) 
96 (±14) 
Physical observations* 
X 
X 
X 
X 
X 
X 
X 
Medical history/examination 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
Review AEs & medications 
X 
X 
X 
X 
X 
X 
X 
Review contraindications 
X 
 
 
 
 
 
 
Diary card collected 
 
 
 
X 
 
 
 
Antimalarials Initiated 
X 
 
 
 
 
 
 
Urine β-hCG (females only) 
X 
 
 
 
 
 
 
Serum βhCG (mL) 
5b 
 
 
 
 
 
 
Haematology (mL) 
2b 
2 
2 
2 
2 
2 
6d 
Biochemistry (mL)** 
3b 
3 
3 
3 
3 
3 
3 
Serum storage 
 
 
 
 
 
 
4 
HIV, HBV, HCV, EBV, CMV serology (mL)^ 
 
 
 
 
 
 
5 
Exploratory Assays 
72 
12 
12 
12 
12 
72 
42 
qPCR & gametocyte PCR (mL)C 
2 
2 
2 
 
 
 
 
Visit Total Blood Volume (mL) 
84 
19 
19 
17 
17 
77 
60 
Cumulative blood volume  (mL) 
387 
406 
425 
442 
459 
519 
579 
Table 10e: Schedule of events for volunteers in VAC069E (timeline/window are in relation to the day of challenge or treatment). D = diagnosis; C = challenge; 
T = treatment; (X) = If considered necessary.*Physical observations include blood pressure, pulse and temperature. **Biochemistry comprises sodium, 
potassium, urea, creatinine, liver function tests. ^EBV and CMV serology will only be repeated at the C+96 timepoint if they were negative at screening.  
aBloods to be taken immediately before drug treatment, if diagnostic criteria not met, treatment will be started at C+21 morning visit.  b If diagnosis is made at 
C+14.5 visit, haematology and biochemistry blood tests will not be repeated as already taken at morning C+14 visit. c Blood draws for qPCR will continue until 
the volunteer has two consecutive qPCR readings with substantial reduction in genome copies per mL. d 4ml EDTA will be sent for alloantibody screening at 
C+96. Blood volumes as given in the table are maximal volumes and smaller blood volumes may be taken at any visit, at the discretion of the investigator, with 
the exception of additional blood volume that may be required for any additional tests that may be performed if deemed clinically necessary.

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10 ASSESSMENT OF SCIENTIFIC OBJECTIVES 
Statistical analysis will reference the following endpoints: 
10.1 
Primary endpoints 
Assessment of: 
 Safety and feasibility of primary P. vivax blood-stage CHMI, as measured by (S)AE 
occurrences and successful infection (development of detectable persistent parasitaemia 
by qPCR +/- clinical symptoms). 
 Choosing the optimal inoculation dose to take forward to future P. vivax CHMI studies will 
be decided based on the following algorithm: 
Ideal choice = the first group (2/2 volunteers) to reach diagnosis criteria (within 21 days). 
N.B. If both volunteers in Group 1 develop infection AND both volunteers in Group 2 (or 
3) reliably develop infection within 5 days of Group 1 (and within the 21-day window) 
then the lowest dose group should be chosen. 
 
10.2 
Secondary endpoints 
 Safety and feasibility of secondary and tertiary P. vivax controlled blood-stage CHMI, as 
measured by (S)AE occurrences and successful infection (development of detectable 
persistent parasitaemia by qPCR +/- clinical symptoms) 
 Immune response to primary, secondary and tertiary P. vivax and heterologous P. 
falciparum pre-treatment, as measured by antibody, B cell and T cell responses.  
 Gametocytaemia, as measured by qPCR in primary, secondary and tertiary P. vivax blood-
stage CHMI. 
 Transmissibility of gametocytes from the infected volunteer to Anopheline mosquito 
vector 

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11 ASSESSMENT OF SAFETY 
Safety of the CHMI will be assessed by analysing the frequency, incidence and nature of adverse 
events and serious adverse events arising during the study. 
11.1 
Definitions 
Adverse Event (AE): An AE is any untoward medical occurrence in a volunteer, including a dosing 
error, which may occur during or after administration of a study intervention (in this case, CHMI) 
and does not necessarily have a causal relationship with the intervention. An AE can therefore be 
any unfavourable 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.  
Adverse Reaction (AR): An AR is any untoward or unintended response to a study intervention. 
This means that a causal relationship between the intervention and an AE is at least a reasonable 
possibility, i.e. the relationship cannot be ruled out. All cases judged by either the reporting 
medical Investigator or the Sponsor as having a reasonable suspected causal relationship to the 
intervention (i.e. possibly, probably or definitely related to an intervention) will qualify as 
adverse reactions. 
Unexpected Adverse Reaction: An adverse reaction, the nature or severity of which is not 
consistent with CHMI, blood donation or antimalarial treatment (applicable product information) 
is considered as an unexpected adverse reaction. 
Serious Adverse Event (SAE): An SAE is an AE that results in any of the following outcomes, 
whether or not considered related to the study intervention. 
 Death (i.e. results in death from any cause at any time). 
 Life-threatening event (i.e. the volunteer was, in the view of the Investigator, at 
immediate risk of death from the event that occurred). This does not include an AE that, if 
it occurred in a more serious form, might have caused death. 
 Persistent or significant disability or incapacity (i.e. substantial disruption of one’s ability 
to carry out normal life functions). 
 Transfer of inpatient care to the NHS staff if the Investigators assess that a higher level of 
intervention and intense monitoring is required to manage symptoms following CHMI, 
blood donation, or drugs (over and above what can be provided by the Investigators in 
the research bay). Hospitalisation (including inpatient or outpatient hospitalisation for an 
elective procedure) for a pre-existing condition that has not worsened unexpectedly does 
not constitute a SAE. 
 An important medical event (that may not cause death, be life threatening, or require 
hospitalisation) that may, based upon appropriate medical judgment, jeopardise the 
volunteer and/or require medical or surgical intervention to prevent one of the outcomes 
listed above.  
 Congenital anomaly or birth defect. 
Serious Adverse Reaction (SAR): An adverse event (expected or unexpected) that is both serious 
and, in the opinion of the reporting Investigator or Sponsor, believed to be possibly, probably or 
definitely due to a study intervention or treatment, based on the information provided. 
Suspected Unexpected Serious Adverse Reactions (SUSARs): A SUSAR is a SAE that is 
unexpected and thought to be possibly, probably or definitely related to a study intervention. 

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11.2 
Causality assessment 
For every unsolicited AE, an assessment of the relationship of the AE to the study intervention(s) 
(CHMI) will be undertaken by the CI-delegated clinician. The relationship of the adverse event 
with the study procedures will be categorised as unrelated, unlikely to be related, possibly 
related, probably related or definitely related (Table 11). 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. Alternative causes of 
the AE, such as the natural history of pre-existing medical conditions, concomitant therapy, other 
risk factors and the temporal relationship of the event to CHMI will be considered and 
investigated. Causality assessment will take place during planned safety reviews, interim analyses 
and at the final safety analysis, except for SAEs, which should be assigned by the reporting 
Investigator. 
0 
No Relationship 
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 
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 
CHMI, blood donation or drug.  
2 
Possible 
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 CHMI, blood 
donation or similar drug. 
3 
Probable 
 
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 CHMI, blood 
donation or drug. 
4 
Definite 
 
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 CHMI, blood 
donation or drug.  
Table 11: Guidelines for assessing the relationship of study intervention to an AE 

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11.3 
Reporting procedures for all AEs (see SOP VC027) 
All AEs occurring post-CHMI, until 6 days after completion of antimalarial treatment (i.e. 9 days 
after DoD) will be recorded. We will NOT record bruising due to blood-taking or cannulation. 
Recording and reporting of all AEs will take place as detailed in SOP VC027. All AEs that result in a 
volunteer’s withdrawal from the study will be followed up until a satisfactory resolution occurs, 
the event is considered stable, or until a non-study related causality is assigned (if the volunteer 
consents to this). Serious adverse events (SAEs) will be collected throughout the entire trial 
period.  
Each adverse event will be graded by the participant according to the table for grading severity of 
adverse events (see Table 12). Severity gradings may be reviewed and discussed with the 
participants at the clinic visits. Volunteers will be instructed on how to self-assess the severity of 
these AEs. There will also be space on the diary card to self-document unsolicited AEs, and 
whether medication was taken to relieve the symptoms. 
The severity of clinical adverse events will be assessed by the Investigators according to the 
scales in Tables 12-13. 
 
GRADE 0 
None 
GRADE 1 
Mild: Transient or mild discomfort (< 48 hours); no medical 
intervention/therapy required 
GRADE 2 
Moderate: Mild to moderate limitation in activity – some assistance may be 
needed; no or minimal medical intervention/therapy required 
GRADE 3 
Severe: Marked limitation in activity, some assistance usually required; may 
require medical intervention/therapy  
Table 12: Severity grading criteria for AEs. 
 
Physical Observations 
Grade 1 
Grade 2 
Grade 3 
Tachycardia – beats per 
min* 
101-115 
116-130 
>130 
Hypotension (systolic) 
mm Hg 
85-89 
80-84 
<80 
Hypertension (systolic) 
mm Hg** 
141-159 
160-179 
>180 
Hypertension 
(diastolic) mm Hg** 
91-99 
100-109 
>110 
Fever °C 
37.6 – 38.0 
>38.0 
>39.0 
Table 13: Severity grading criteria for clinically significant abnormal physical observations. All 
observations should be measured at rest. *Only applies when resting heart rate is between 60 

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and 100 beats per minute. Use clinical judgement when characterising bradycardia in some 
healthy subject populations (e.g. conditioned athletes). **Systolic or diastolic hypertension may 
only be confirmed as clinically significant (and therefore an AE) if persistently present when 
observations are repeated (i.e. isolated measurements of hypertension are not clinically 
significant). 
11.3.1 Severity grading for laboratory AEs 
Severity grading for laboratory AEs are dependent on the OUH laboratory’s reference and will be 
graded according to the scales in the site-specific table that can be found in the Investigator file. 
These ranges will be based on FDA guidance relative to local laboratory reference ranges [82].  
11.3.2 Reporting procedures for serious AEs  
In order to comply with current regulations on serious adverse event reporting to regulatory 
authorities, the event will be documented accurately and notification deadlines respected. SAEs 
will be reported to an internal safety group (members of the study team) immediately (within 24 
hours) of the Investigators being aware of their occurrence, as described in the SOP OVC005.  
This safety group includes the Chief Investigator, who acts on behalf of the Sponsor for 
notification of SAEs. The Sponsor and Local Safety Monitor (LSM) will be notified of SAEs deemed 
possibly, probably or definitely related to study interventions; the LSM will be notified 
immediately (within 24 hours) of the Investigators’ being aware of their occurrence. SAEs will not 
normally be reported to the ethical committee(s) unless there is a clinically important increase in 
occurrence rate, an unexpected outcome, or a new event that is likely to affect safety of trial 
volunteers, at the discretion of the Chief Investigator and/or LSM.  
11.3.4 Reporting procedures for SUSARs  
Unexpected serious reactions would be reported to the Local Safety Monitor, ethical 
committees, Sponsor and other interested parties. For fatal and life-threatening SUSARS, this will 
be done within 7 calendar days after the Sponsor or delegate is first aware of the reaction. Any 
additional relevant information will be reported within 8 calendar days of the initial report. All 
other SUSARs will be reported within 15 calendar days. The Chief Investigator will also inform all 
investigators concerned of relevant information about SUSARs that could adversely affect the 
safety of participants.  
All SUSARs and deaths occurring during the study will be reported to the Sponsor. For all deaths, 
available autopsy reports and relevant medical reports will be made available for reporting to the 
relevant authorities. 
11.3.5 Procedures to be followed in the event of abnormal findings  
Abnormal clinical findings from medical history, examination or blood tests, will be assessed as to 
their clinical significance using the site-specific laboratory adverse event grading table kept in the 
trial master file. If a test is deemed clinically significant, it may be repeated, to ensure it is not a 
single occurrence. If a test remains clinically significant, the volunteer will be informed and 
appropriate medical care arranged as appropriate with the permission of the volunteer. 
Decisions to exclude the volunteer from enrolling in the trial or to withdraw a volunteer from the 
trial will be at the discretion of the Investigator. 
11.4 
Foreseeable medical occurrences  
The following medical occurrences are foreseeable: 

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 Clinical P. vivax or P. falciparum disease resulting in one or more of fever, tachycardia, 
hypotension, feverishness, chills, rigor, sweats, headache, anorexia, nausea, vomiting, 
myalgia, arthralgia, low back pain, fatigue, lymphopenia and thrombocytopenia; 
 Severe clinical P. vivax or P. falciparum disease resulting in inpatient transfer of care to 
the NHS;  
 Adverse reactions to Riamet®, Malarone, paracetamol or cyclizine, as detailed in the 
SmPCs for these medications. 
 
11.5 
Safety profile review of CHMI and antimalarial treatment 
The safety profile will be assessed on an on-going basis by the Investigators. 
 
11.6 
Local safety monitor 
An independent Local Safety monitor (LSM) will be appointed to provide real-time safety oversight. 
The LSM will review SAEs deemed possibly, probably or definitely related to study interventions. 
The LSM will be notified within 24 hours of the investigators’ being aware of their occurrence. The 
LSM has the power to terminate the study if deemed necessary following a study intervention-
related SAE. At the time of writing, the LSM will be Prof Brian Angus, a Clinical Tutor in Medicine, 
Honorary Consultant Physician and Director, Centre for Tropical Medicine at the University of 
Oxford. All correspondence between investigator and LSM will be conveyed by the investigator to 
the trial Sponsor. 
The LSM may be contacted for advice and independent review by the investigator or trial Sponsor 
in the following situations: 
• 
Following any SAE deemed to be possibly, probably, or definitely related to a study 
intervention. 
• 
Any other situation where the Investigator or trial sponsor feels independent advice or 
review is important. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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12 STATISTICS 
12.1  Sample size 
This is single site controlled human malaria infection study, aiming to assess the safety and 
feasibility of blood-stage controlled human P. vivax malaria infection using a newly produced 
cryopreserved inoculum source. 
In the first phase of the study, VAC069A, the inoculum will be injected at three different 
concentrations, to assess the safety and feasibility of each, with the view to use the most 
appropriate concentration in the subsequent challenges. The number of volunteers undergoing 
malaria challenge in the first phase will be 6, with 2 volunteers undergoing CHMI at each 
concentration. The primary objective of the first phase is to assess safety, therefore, this number 
limits the potential for harm whilst allowing establishment of safety.  
Subsequent phases of the study (VAC069B-D) will have larger numbers of volunteers (up to 8) 
per group, thus increasing the likelihood of detecting statistically significant differences in 
response to homologous rechallenge. 
This is the first study of its kind to be performed in Europe, and if successful, future challenge 
studies to assess efficacy of candidate vaccines will be powered appropriately.  
For VAC069E – 11 volunteers who underwent primary or secondary CHMI in the previous phase 
of the study (VAC069D) will be invited to continue in the study and undergo heterologous CHMI 
with P. falciparum. A minimum of 6 volunteers are likely to be required to detect significant 
differences in the clinical and immunological response to heterologous re-challenge. 
 

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13 QUALITY CONTROL AND QUALITY ASSURANCE PROCEDURES 
13.1  Investigator procedures 
Approved site-specific SOPs will be used at all clinical and laboratory sites. 
13.2 
Monitoring 
Monitoring will be performed externally by RGEA using established procedures, according to ICH 
Good Clinical Practice (GCP).  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 GCP. The Investigator sites will provide direct access to all trial 
related source data/documents and reports for the purpose of auditing by the Sponsor and 
inspection by local and regulatory authorities. 
13.3 
Modification to protocol 
No amendments to this protocol will be made without consultation with, and agreement of, the 
Sponsor. Any amendments to the trial that appear necessary during the course of the trial must 
be discussed by the Investigator and Sponsor concurrently. If agreement is reached concerning 
the need for an amendment, it will be produced in writing by the Chief Investigator and will be 
made a formal part of the protocol following ethical approval. 
An administrative change to the protocol is one that modifies administrative and logistical 
aspects of a protocol but does not affect the subjects’ safety, the objectives of the trial and its 
progress. An administrative change does not require UK ethical committee approval; however, 
the Research Ethics Committee (REC) will be notified in the event of any such change.  
The Investigator is responsible for ensuring that changes to an approved trial, during the period 
for which ethical committee(s) approval has already been given, are not initiated without ethical 
committee(s)’ review and approval except to eliminate apparent immediate hazards to the 
subject. 
13.4 
Protocol deviation  
Any deviations from the protocol will be documented in a protocol deviation form and filed in 
the site trial master file.  
13.5 
Audit & inspection  
The QA manager performs system based internal audits to check that  trials are being conducted, 
data recorded, analysed and accurately reported according to study protocols, departmental 
SOPs and in compliance with ICH GCP. The audit schedule includes laboratory activities. The 
internal audits will supplement the external monitoring process and will review processes not 
covered by the external monitor. 
The Sponsor, trial sites, ethical committee(s), and authorised individuals may carry out audit to 
ensure compliance with the protocol, GCP and appropriate regulations. GCP inspections may also 
be undertaken by the regulatory authority to ensure compliance with protocol and national 
regulations. The Sponsor will assist in any inspections. 
13.6 
Serious breaches 
A serious breach is defined as “A breach of GCP or the trial protocol which is likely to effect to a 
significant degree –  

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(a) the safety or physical or mental integrity of the subjects of the trial; or 
(b) the scientific value of the trial. 
In the event that a serious breach is suspected the Sponsor will be informed as soon as possible 
and in turn will notify the REC within 7 days.  
13.7 
Trial progress 
The progress of the trial will be overseen by the Chief Investigator. 
13.8 
Publication policy 
The 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 ICMJE guidelines and other contributors will be acknowledged. Data may also be used 
for a PhD or MD thesis.  
13.9 
Intellectual Property 
Ownership of intellectual property (IP) generated by employees of the University vests in the 
University.  The protection and exploitation of any new IP is managed by the University’s 
technology transfer office, Oxford University Innovations 

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14 ETHICS 
14.1 
Declaration of Helsinki 
The Investigator will ensure that this study is conducted according to the principles of the current 
revision of the Declaration of Helsinki 2008. 
14.2 
ICH guidelines for good clinical practice 
The Investigator will ensure that this study is conducted in full conformity to Medicine for Human 
use (Clinical Trials) Regulations 2004 (SI 2004 No. 1031) and its amendments and with the ICH 
guidelines for GCP (CPMP/ICH/135/95) July 1996.  
14.3 
Approvals 
A copy of the protocol, proposed informed consent form, other written volunteer information 
and the proposed advertising material will be submitted to an appropriate REC and the HRA for 
written approval. The Investigator will submit and, where necessary, obtain approval from the 
REC and the HRA for all subsequent amendments to the protocol and associated trial documents. 
A non-substantial amendment does not require REC approval; however, the REC will be notified 
in the event of any such change. The Investigator will notify deviations from the protocol or SAEs 
occurring at the site to the Sponsor and will notify the REC(s) of these if necessary in accordance 
with procedures. 
14.4 
Reporting  
The CI shall submit once a year throughout the study, or on request, an Annual Progress Report 
to the REC, HRA (where required), host organisation and Sponsor. In addition, an End of Study 
notification and final report will be submitted to the REC and Sponsor. 
14.5 
Volunteer confidentiality 
All data will be anonymised; volunteer data will be identified by a unique study number in the 
CRF and database. Separate confidential files containing identifiable information will be stored in 
secured locations. Only the Sponsor representative, Investigators, the clinical monitor, the 
ethical committee(s) and the regulatory authorities will have access to the records. Photographs 
taken (if required, with the volunteer’s written, informed consent) will not include the 
volunteer’s face and will be identified by the volunteer’s trial specific identification number only. 
Once developed, photographs will be stored as confidential records, as above. This material may 
be shown to other professional staff, used for educational purposes, or included in a scientific 
publication.  
If a participant is diagnosed with COVID-19 on a test carried out through this study, personal 
details of the participant (name, date of birth, contact details, ethnicity, NHS number) and date 
of diagnosis and symptom onset will be legally required to be passed onto UKHSA as COVID-19 is 
a notifiable disease. 
 

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15 DATA HANDLING AND RECORD KEEPING 
15.1 
Data handling 
The Chief Investigator will be the data manager with responsibility for delegating the receiving, 
entering, cleaning, querying, analysing and storing all data that accrues from the study. Data will 
be entered into the volunteers’ CRFs in a paper and/or electronic format (using the OpenClinica™ 
database). Electronic data will be stored on secure servers which are outsourced by 
OpenClinica™. OpenClinica™ meets FDA part 11B standards. This includes safety data, laboratory 
data and outcome data. Data are entered in a web browser on PCs and then transferred to the 
OpenClinica Database by encrypted (https) transfer. Safety data will also be collected through an 
electronic diary, which is stored on a secure server.  
15.2 
Record keeping 
The Investigators will maintain and retain appropriate medical and research records and 
essential documents for this trial in compliance with ICH E6 GCP and regulatory and institutional 
requirements for the protection of confidentiality of volunteers. The Chief Investigator, co-
investigators, clinical research nurses and authorised personnel will have access to records. The 
investigators will permit authorised representatives of the Sponsor, ethical committee(s), 
regulatory agencies, 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. 
15.3 
Source data and electronic case report forms (eCRFs) 
All protocol-required information will be collected in the electronic diaries and eCRFs designed 
by the Investigator. All source documents will be filed in the participants’ notes. Source 
documents are original documents, data, and records from which the volunteer’s eCRF data are 
obtained. For this study these will include, but are not limited to; volunteer consent form, blood 
results, GP response letters, laboratory records and correspondence. In the majority of cases, 
electronic diaries and eCRF entries will be considered source data as these are the site of the 
original recording (i.e. there is no other written or electronic record of data). In this study this will 
include, but is not limited to medical history, medication records, vital signs, physical 
examination records, urine assessments, blood results, adverse event data and details of study 
interventions. All source data and volunteer CRFs will be stored securely. 
15.4 
Data protection  
The study protocol, documentation, data and all other information generated will be held in 
strict confidence. No information concerning the study or the data will be released to any 
unauthorised third party, without prior written approval of the Sponsor. 
In the event of a participant testing positive for COVID-19 on a test carried out through this 
study, details of the participant (name, date of birth, contact details, ethnicity, NHS number) and 
date of diagnosis and symptom onset will be legally required to be passed onto UKHSA as COVID-
19 is a notifiable disease. 
 

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16 FINANCING AND INSURANCE 
16.1 
Financing 
The study is funded through a grant from The Wellcome Trust and MultiViVax, a European 
Commission Horizon 2020 funded project which intends to develop a highly effective multi-stage 
malaria vaccine to the point of proof-of-concept Phase II testing in Europe, prior to clinical trials 
in malaria-endemic areas. 
16.2 
Insurance 
The University has a specialist insurance policy in place which would operate in the event of any 
participant suffering harm as a result of their involvement in the research (Newline Underwriting 
Management Ltd, at Lloyd’s of London). NHS indemnity operates in respect of the clinical 
treatment that is provided. 
16.3 
Compensation 
Volunteers will be compensated for their time and for the inconvenience caused by procedures 
as below (see Table 17).   
- Screening visit  
 
 
 
 
 
 
£25  
Subsequent visits: 
- Travel expenses 
 
 
 
 
 
 
£15 per visit 
- Inconvenience of blood tests: 
 
 
 
 
£10 per blood donation 
- Time required for visit: 
 
 
 
 
 
£20 per hour 
- Admission (Illness) Compensation                                                  
£480 total 
- Direct mosquito feeding (optional)   
 
 
 
£80 per visit 
The backup volunteers who are not enrolled in the study will be compensated £200.  This is in 
addition to compensation for visits they may have attended. Volunteers partaking in direct 
mosquito bite feeding will also be compensated an additional £80 per visit.  
Time in Trial 
(approx.) 
Maximum 
No. of Visits 
Maximum Volume of 
Blood Taken (mL) 
Estimated 
Compensation 
Vols enrolled in VAC069A  
(3 Challenges)  
30 months 
148 
2971 
£6500 
Vols enrolled in VAC069B  
(3 Challenges)  
30 months  
148 
2868 
£6715 
Vols enrolled in VAC069C (3 
Challenges) 
18 months 
147 
2352 
£6375 
Vols enrolled in VAC069D (3 
Challenges) 
18 months 
105 
2199 
£6240 
Vols enrolled in VAC069E  
(1 Challenge) 
3 months 
39 
579 
£1970 
Table 14:  Estimated compensation amounts. Figures as given in table do not include compensation 
for direct mosquito bite feeding; any volunteer taking part in direct mosquito bite feeding will be 
compensated an additional £80 per visit. 

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VAC069 Clinical Trial Protocol, V8.0, 14th June 2022, University of Oxford.  Page 116 of 122 
17 APPENDICES 
Appendix A: laboratory values for exclusion 
Laboratory parameters for inclusion/exclusion in the trial will be considered on an individual 
basis, with Investigator discretion for interpretation of results and the need for repeated or 
further tests. In general, volunteers will be excluded if a result at screening constitutes what 
would qualify as a grade 1 (or higher) laboratory adverse event, according to the laboratory 
adverse event tables (filed in the TMF), on repeat of an abnormal test result. Urinalysis at 
screening will be assessed as per the table below: 
 
URINE ANALYSIS (using MULTISTIX) 
Protein* 
2+ or Protein creatinine ratio of ≥ 50 mg/mmol 
Blood£ 
2+ on two dipstick tests  
Glucose 
1+ 
Table 15: Urinalysis assessment.  
*In the event of the dipstick testing positive for protein with ≥1+ protein urine should be sent for 
a protein creatinine ratio. 
£ In the event of urine dipstick testing positive for ≥1+ blood with, or without, protein, a repeat 
dipstick test will be carried out to confirm haematuria. In female volunteers, a menstrual history 
will be taken to elicit whether the subject is currently menstruating and if they are, urine dipstick 
will be repeated after 1 - 2 weeks. If blood and/or proteinuria persist in any volunteer, an 
interpretation of the results will be undertaken by the Investigator on an individual basis to 
determine if they will be excluded from the trial, and the appropriate follow-up arranged. 
 
 
 
 
 
 
 
 

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VAC069 Clinical Trial Protocol, V8.0, 14th June 2022, University of Oxford.  Page 117 of 122 
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