Safety and Immunogenicity of a Protein Particle Malaria Vaccine Candidate, R21, Administered With and Without Matrix-M1 in Healthy UK Volunteers
Phase: PHASE1
Conditions: Malaria
Sponsor: University of Oxford

Clinical Trial Protocol: VAC053 Protocol v5.0 
 
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UNIVERSITY OF OXFORD 
 
 
 
 
 
 
 
Safety and immunogenicity of a protein particle malaria vaccine candidate, R21, 
administered with and without Matrix-M1 in healthy UK volunteers 
 
A Phase I Clinical Trial 
 
 
Study Reference: VAC 053 
Protocol Version Number: 5.0 
Date: 11th July 2016 
 
 
 
Chief Investigator: Professor A.V.S Hill 
Sponsor: University of Oxford 
Funder: European Commission Framework 7 
 
REC Number: 15/SC/0386 
EudraCT Number:  2015-002428-19 
 
 
 
 

Clinical Trial Protocol: VAC053 Protocol v5.0 
 
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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 Professor Adrian 
Hill. 
 
 
 
Statement of Compliance 
 
The trial will be conducted in compliance with the protocol, International Conference on 
Harmonisation Good Clinical Practice Guideline E6 (R1) (ICH-GCP) and the applicable 
regulatory requirements.   
 
 
 
Signatures 
 
“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.” 
 
 
Chief Investigator: 
 _______________________________ 
 
Date:  
 
 
 
 
Adrian Hill 
 
 
 
Professor of Human Genetics, University of Oxford 
 
 
 
 
 

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Chief Investigator 
Adrian V.S. Hill 
 
 
 
 
Centre for Clinical Vaccinology and Tropical Medicine  
Old Road, Headington, Oxford, OX3 7LE, UK 
Tel: 01865 617610  
Fax: 01865 857471 
Email: adrian.hill@ndm.ox.ac.uk 
 
Principal Investigator 
 
 
Professor David Lewis 
NIHR/Wellcome Trust Clinical Research Facility, 
Hammersmith Hospital,  
150 Du Cane Road,  
London, W12 0HS 
Email: d.lewis@imperial.ac.uk 
 
Trial Sites  
 
 
 
 
 
 
 
Project Manager 
 
 
 
 
 
Centre for Clinical Vaccinology and Tropical Medicine, Old Road, 
Headington, Oxford, OX3 7LE 
 
John Warin Ward, Oxford University Hospital NHS Trust, 
Churchill Hospital, Old Road, Headington, Oxford, OX3 7LE 
 
NIHR/Wellcome Trust Clinical Research Facility, Hammersmith 
Hospital, 150 Du Cane Road, London, W12 0HS 
 
Alison Lawrie 
Senior Project Manager 
Centre for Clinical Vaccinology and Tropical Medicine  
Old Road, Headington, Oxford, OX3 7LE, UK 
Tel: 01865 857382   
Fax: 01865 857471 
Email: alison.lawrie@ndm.ox.ac.uk 
 
Chair of Local Safety 
Committee 
Dr Brian Angus 
Centre for Clinical Vaccinology and Tropical Medicine  
Old Road, Headington, Oxford, OX3 7LE, UK 
Tel: 01865 553 20289 
Email: brian.angus@ndm.ox.ac.uk 
 
 
 

Clinical Trial Protocol: VAC053 Protocol v5.0 
 
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Sponsor: 
 
 
 
 
 
 
University of Oxford 
Named contact: Heather House, 
Clinical Trials and Research Governance 
Joint Research Office, Block 60, 
Churchill Hospital, 
Old Road, Headington, Oxford OX3 7LE  
Phone: 01865 572245 
Fax: 01865 572228 
Email: heather.house@admin.ox.ac.uk 
 
External Monitor  
Clinical Trials & Research Governance 
Joint Research Office, Block 60, 
Churchill Hospital, 
Old Road, Headington, Oxford OX3 7LE 
 
Modification history 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Version Date 
Modifications 
Author(s) 
2.0 
26/08/2015 Addition of the John Warin ward as 
an NHS site (pages 3 and 8). 
Correction of minor typographical 
errors (pages 3 and 4) 
Emma Bakpa 
2.1 
09/11/2015 Typographical error corrected in 
section 6.4. 
Emma Bakpa 
3.0 
24/11/2015 
 
 
Clarification of previous receipt of an 
investigational vaccine in exclusion 
criteria (page 43, Section 7.3.2) 
Clarification that safety data is 
collected and stored through an 
electronic diary on a secure server; 
this data is uploaded to the study 
database at the end of the trial. 
(Page 58, Section 9.6) 
Navin Venkatraman 
4.0 
08/06/2016 Addition of Group 4 – changes made 
throughout the document 
Navin Venkatraman 
5.0 
11/07/2016 New concentration of Matrix-M1 
added to be used for Group 4 
volunteers 
Navin Venkatraman 

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Table of Contents 
1SYNOPSIS ......................................................................................................................................... 8 
2 
ABBREVIATIONS .................................................................................................................. 11 
3  
BACKGROUND & RATIONALE .......................................................................................... 13 
3.1 
Impact of malaria and the need for a vaccine .................................................................. 13 
3.1.1 
Lifecycle of Plasmodium falciparum ............................................................................................ 13 
3.1.2 
Progress towards a P. falciparum vaccine ................................................................................. 14 
3.2 
Pre-erythrocytic stage as a vaccine target ........................................................................ 16 
3.3 
R21 vaccine development ....................................................................................................... 17 
3.4 
R21 - pre-clinical studies......................................................................................................... 18 
3.4.1 
Immunogenicity ................................................................................................................................. 18 
3.4.2 
Efficacy .................................................................................................................................................. 20 
3.5  
RTS,S/AS01 ................................................................................................................................... 22 
3.5.1 
Phase III evaluation ........................................................................................................................... 23 
3.6  
MATRIX-M1 .................................................................................................................................. 24 
3.6.1 
Pre-clinical studies ............................................................................................................................. 24 
3.6.2  
Clinical studies .................................................................................................................................... 25 
3.7  
Rationale ....................................................................................................................................... 34 
3.7.1 
Vaccine Development Strategy ..................................................................................................... 34 
4 
STUDY OVERVIEW ............................................................................................................... 35 
4.1.1 
First volunteers ................................................................................................................................... 36 
4.1.2 
Duration of study ............................................................................................................................... 36 
4.1.3 
Definition of Start and End of Trial .............................................................................................. 36 
4.2 
Potential Risks for volunteers ............................................................................................................. 36 
4.3 
Known Potential Benefits .................................................................................................................... 37 
5 
OBJECTIVES AND ENDPOINTS ......................................................................................... 38 
5.1 
Primary Objective ...................................................................................................................... 38 
5.1.1 
Primary Outcome Measures .......................................................................................................... 38 
5.2 
Secondary Objective ................................................................................................................. 38 
5.2.1 
Secondary Outcome Measures ..................................................................................................... 38 
6 
INVESTIGATIONAL PRODUCTS........................................................................................ 39 
6.1.1 
Description of R21c ........................................................................................................................... 39 
6.1.2 
R21 formulation and packaging .................................................................................................... 40 
6.1.3 
R21 Storage and Handling .............................................................................................................. 40 
6.2.1 
R21 mixed with Matrix-M1 ............................................................................................................. 40 
6.2.2 
Matrix-M1 formulation and packaging ....................................................................................... 41 
6.2.3 
Matrix-M1 Storage and Handling ................................................................................................. 41 
6.3 
Administration of Investigational Medicinal Products ................................................ 41 
6.4 
Vaccine Supply ............................................................................................................................ 41 
7 
RECRUITMENT AND WITHDRAWAL OF TRIAL VOLUNTEERS .............................. 41 
7.1 
Volunteers .................................................................................................................................... 41 
7.2 
Informed consent ....................................................................................................................... 42 

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7.3 
Inclusion and exclusion criteria ........................................................................................... 43 
7.3.1 
Inclusion Criteria ................................................................................................................................ 43 
7.3.2 
Exclusion Criteria ............................................................................................................................... 43 
7.3.3 
Effective contraception for female volunteers ........................................................................ 44 
7.3.4 
Prevention of ‘Over Volunteering’ ............................................................................................... 45 
7.3.5 
Re-vaccination exclusion criteria .................................................................................................. 45 
7.3.6 
Withdrawal of Volunteers............................................................................................................... 45 
7.4 
Compliance with Dosing Regime .......................................................................................... 46 
7.5 
Pregnancy ..................................................................................................................................... 46 
7.6 
Safety Stopping/ Holding Rules ............................................................................................ 46 
7.6.1 
Group holding rules .......................................................................................................................... 47 
7.6.2 
Individual stopping rules (will apply to all vaccinated individuals) ................................... 48 
8 
TREATMENT OF TRIAL VOLUNTEERS ........................................................................... 49 
8.1 
Study procedures ....................................................................................................................... 49 
8.2 
Observations ................................................................................................................................ 49 
8.3 
Blood Tests ................................................................................................................................... 49 
8.4 
Vaccinations ................................................................................................................................. 50 
8.5 
Study visits ................................................................................................................................... 51 
8.5.1 
Screening visit ..................................................................................................................................... 51 
8.5.2 
Day 0: Enrolment and vaccination visit ...................................................................................... 52 
8.5.3 
Sequence of Enrolment and Vaccination of Volunteers ....................................................... 52 
8.5.4 
Subsequent Visits: Day 1, 7, 14, 29, 35, 42, 57, 63, 70, 84, 238 ......................................... 52 
8.5.5 
First three volunteers (Groups 1-3) ............................................................................................. 53 
9 
ASSESSMENT OF SAFETY ................................................................................................... 56 
9.1 
Interim Safety Review .............................................................................................................. 56 
9.2 
Definitions .................................................................................................................................... 56 
9.2.1 
Adverse Event (AE) ............................................................................................................................ 56 
9.2.2 
Adverse Reaction (AR)...................................................................................................................... 56 
9.2.3 
Unexpected Adverse Reaction ...................................................................................................... 56 
9.2.4 
Serious Adverse Event (SAE) .......................................................................................................... 56 
9.2.5 
Serious Adverse Reaction (SAR) .................................................................................................... 57 
9.2.6 
Suspected Unexpected Serious Adverse Reaction (SUSAR)................................................. 57 
9.3 
Foreseeable Adverse Reactions: ........................................................................................... 57 
9.4 
Expected Serious Adverse Events ........................................................................................ 57 
9.5 
Causality Assessment ............................................................................................................... 57 
9.6 
Reporting Procedures for All Adverse Events ................................................................. 58 
9.6.1 
Reporting Procedures for Serious AEs (see SOP OVC005 Safety Reporting) .................. 58 
9.6.2 
Reporting Procedures for SUSARS ............................................................................................... 59 
9.6.3 
Development Safety Update Report ........................................................................................... 59 
9.7 
Assessment of severity ............................................................................................................. 59 
9.8 
Procedures to be followed in the event of abnormal findings ................................... 61 
9.9 
Local Safety Committee ............................................................................................................ 61 
9.9.1 
Safety Profile Review ........................................................................................................................ 61 
10 
 STATISTICS ........................................................................................................................ 62 

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11 
 QUALITY CONTROL AND QUALITY ASSURANCE PROCEDURES ...................... 63 
11.1 Investigator procedures .......................................................................................................... 63 
11.2 Monitoring .................................................................................................................................... 63 
11.3 Modification to protocol .......................................................................................................... 63 
11.4 Protocol deviation ..................................................................................................................... 63 
11.5 Audit & inspection ..................................................................................................................... 63 
11.6 Serious Breaches ........................................................................................................................ 64 
11.7 Trial Progress .............................................................................................................................. 64 
11.8 Publication Policy ...................................................................................................................... 64 
12 
ETHICS ................................................................................................................................. 65 
12.1 Declaration of Helsinki ............................................................................................................ 65 
12.2 ICH Guidelines for Good Clinical Practice ......................................................................... 65 
12.3 Informed Consent ...................................................................................................................... 65 
12.4 Research Ethics Committee (REC) ....................................................................................... 65 
12.5 Volunteer Confidentiality ....................................................................................................... 65 
13 
 DATA HANDLING AND RECORD KEEPING .............................................................. 66 
13.1 Data Handling .............................................................................................................................. 66 
13.2 Record Keeping........................................................................................................................... 66 
13.3 Source Data and Case Report Forms (CRFs) ..................................................................... 66 
13.4 Data Protection ........................................................................................................................... 66 
14 
 FINANCING AND INSURANCE ...................................................................................... 67 
14.1  Financing ...................................................................................................................................... 67 
14.2  Insurance ..................................................................................................................................... 67 
14.3  Compensation............................................................................................................................. 67 
15 
REFERENCES ...................................................................................................................... 68 
APPENDIX A  LABORATORY VALUES FOR EXCLUSION .................................................. 70 
 
 
 
 
 
 
 
 
 
 
 

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1 
SYNOPSIS 
 
Trial Title 
A Phase I study to assess the safety and immunogenicity of a protein 
particle malaria vaccine candidate, R21, administered with and without 
Matrix-M1 in healthy UK volunteers.  
 
Trial Identifier 
VAC053 
 
Clinical phase  
I  
 
Active ingredients of 
vaccines/products 
R21, a protein particle produced by using recombinant HBsAg particles 
expressing the central repeat and the C-terminus of the circumsporozoite 
protein (CSP), which will be administered either alone or adjuvanted with 
Matrix-M1.  
 
Finished products and 
doses 
1. R21 10g mixed with Matrix-M1 50g 
2. R21 50g without adjuvant  
3. R21 50g mixed with Matrix-M1 50g  
4. R21 2g mixed with Matrix-M1 50g 
 
Form 
Liquid (all finished products) 
 
Route 
Intramuscularly into the deltoid region of the arm 
 
Principal Investigator 
Adrian V. S. Hill  
 
Trial Centres 
Clinical Vaccinology & Tropical Medicine, University of Oxford, Churchill 
Hospital, Old Road, Headington, Oxford, OX3 7LE 
 
John Warin Ward, Oxford University Hospital NHS Trust, Churchill 
Hospital, Old Road, Headington, Oxford, OX3 7LE 
 
NIHR/Wellcome Trust Clinical Research Facility, Hammersmith Hospital, 
150 Du Cane Road, London, W12 0HS 
 
 
Planned Trial Period 
 
Q3 2015 to Q1 2017 
 
 
 

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Study 
Duration 
 
34 weeks per subject 
 
Primary 
Objective 
To assess the safety profile of the candidate vaccine R21 with and without the 
adjuvant Matrix-M1 in healthy adult volunteers  
 
Secondary 
Objective 
To assess the immunogenicity of the candidate vaccine R21 with and without 
the adjuvant Matrix-M1 in healthy adult volunteers  
Population Healthy UK adults aged 18 – 50 years 
 
Planned 
Sample 
34 volunteers 
 
Vaccination 
Schedule 
 
 
Follow-up 
duration 
26 weeks post final vaccine dose 
 
Primary 
Evaluation 
Criteria  
 
Actively and passively collected data on local and systemic adverse events 
The following parameters will be assessed for all study groups 
 Occurrence of solicited local reactogenicity signs and symptoms for 
7 days following the vaccination 
 Occurrence of solicited systemic reactogenicity signs and symptoms 
for 7 days following the vaccination 
 Occurrence of unsolicited adverse events for 28 days following the 
vaccination 
 Change from baseline for safety laboratory measures  
 Occurrence of serious adverse events during the whole study 
duration 
 
Volunteers will undergo clinical follow up for adverse events for a further 182 
days following completion of the vaccination regimen. 
 
 
Secondary 
Evaluation 
Measures of immunogenicity of R21 formulations with and without adjuvant 
may include: 

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Criteria  
 ELISA to quantify antibodies to CSP and NANP 
 ELISPOT to enumerate IFN- producing T cells 
 Ex vivo ELISPOT responses to NANP 
 Flow cytometry and intracellular cytokine staining to enumerate and 
functionally characterise immune cell populations such as; T cells (e.g. 
CD4+ and CD8+), B cells and dendritic cells 
 ELISPOT for enumeration of antibody-secreting cells (e.g. B cell ELISPOT 
responses to NANP) 
 Gene expression profiling including RNA analysis, DNA sequencing and 
other genotypic methods 
Study 
Design 
Open–labelled, non-randomised multicentre Phase I study 
 
 
 

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2 
ABBREVIATIONS 
 
AE 
 
   Adverse event 
AS01 
Adjuvant System 1 comprising the immunoenhancers QS-21   and 
MPL with proprietary liposomes. In the context of malaria vaccine 
development, AS01 has been tested as two formulations: AS01B and 
AS01E 
AR 
 
 
  Adverse reaction 
CBF  
 
 
  Clinical Bio-Manufacturing Facility 
CCVTM 
 
  Centre for Clinical Vaccinology and Tropical Medicine 
CHMI  
 
  Controlled human malaria infection 
CI 
 
 
  Confidence Interval 
CI 
 
 
  Chief Investigator 
CRF 
 
 
  Case report form or Clinical Research Facility 
CSP/CS  
 
  Circumsporozoite Protein  
CTL 
 
 
  Cytotoxic lymphocytes 
CTRG  
 
  Clinical Trials Research Governance 
Da 
 
 
  Dalton 
DSUR  
 
  Development Safety Update Report 
DTPwHepB/Hib   
 Diphtheria, 
tetanus, 
whole-cell 
pertussis, 
hepatitis 
B 
and 
Haemophilus Influenza type B 
ELISA  
 
 
Enzyme linked immunosorbent assay 
ELISPOT 
 
 
Enzyme linked immunospot assay 
EPI 
 
 
 
Expanded Programme of Immunisation 
GCP  
 
 
Good Clinical Practice 
GMP  
 
 
Good Manufacturing Practice 
HA 
 
 
 
Haemagglutinin 
HBsAg 
 
 
Hepatitis B surface antigen 
HCV  
 
 
Hepatitis C virus 
HI 
 
 
 
Haemagglutination inhibition 
HIV  
 
 
Human Immunodeficiency virus 
HLA  
 
 
Human leukocyte antigen 
ICH  
 
 
International Conference on Harmonisation 
IFN-


Interferon-gamma 
IM 
 
 
 
Intramuscular 
IMP  
 
 
Investigational Medicinal Product 
LPS  
 
 
Lipopolysaccharide 

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LSC  
 
 
Local Safety Committee 
LSM  
 
 
Local Safety Monitor 
MHRA 
 
 
Medicines and Healthcare products Regulatory Agency 
MM  
 
 
Matrix-M1 
MPL  
 
 
Monophosphoryl lipid A 
MRC  
 
 
Medical Research Council 
g 
 
 
 
microgram 
nm  
 
 
nanometer 
PIS 
 
 
 
Participant information sheet 
ppm  
 
 
parts per million  
QP 
 
 
 
Qualified Person 
QS 
 
 
 
Quillaja saponaria 
R21  
 
 
Protein particle malaria vaccine candidate 
RTS,S  
 
 
 A hybrid polypeptide consisting of a portion of the CS antigen of the 
malaria parasite P. falciparum strain NF54, fused to the amino 
terminal end of the Hepatitis B virus S protein 
SAE  
 
 
Serious Adverse Event 
SAR  
 
 
Serious Adverse Reaction 
SOP  
 
 
Standard Operating Procedure 
SUSAR 
 
 
Suspected Unexpected Serious Adverse Reaction 
TMF 
 
 
 Trial Master File 
UOXF  
 
 University of Oxford 
VE 
 
 
 Vaccine efficacy 
WHO  
 
 World Health Organisation 
 
 
 
 
 
 
 

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3  
BACKGROUND & RATIONALE 
3.1 
Impact of malaria and the need for a vaccine 
 
Falciparum malaria remains one of the leading infectious causes of morbidity and mortality 
worldwide, predominantly affecting children and pregnant women in sub-Saharan Africa.[1] 
There are over 430,000 deaths annually and a huge socioeconomic burden associated with 
this parasitic disease.[1, 2] In high transmission countries, a child presents with 1.6 - 5.4 
episodes of clinical malaria per year [1], with about 5% of malaria episodes turning to 
severe disease [3].  
 
The advent of artemisinin-combination therapy and increased uptake of insecticide-treated 
nets has resulted in significant recent reductions in mortality in many places. However, 
emergence of resistance to artemisinins and insecticides may hinder progress made 
towards the ultimate goal of eradication.[4] In this regard, the development of a vaccine 
would be an invaluable tool in the fight against malaria. Plasmodium falciparum is a 
complex pathogen, which is highly immunoevasive and the development of an efficacious 
vaccine has remained elusive for many years.  
 
3.1.1 Lifecycle of Plasmodium falciparum 
The lifecycle of P. falciparum is complex with stages in both human and mosquito hosts 
(Figure 1).  The bite of infected female Anopheles mosquitoes transmits malaria sporozoites 
to the human host where they travel via the bloodstream to the liver and invade 
hepatocytes.  Here, during the liver stage, they mature into merozoites for 6 to 7 days. 
Malaria parasites are not detectable in the blood stream during the liver stage. The 
hepatocytes then rupture, releasing a large number of merozoites into the bloodstream 
(blood stage of infection). Merozoites invade erythrocytes where they multiply and after 2 
days cause the erythrocyte to rupture, releasing progeny merozoites that in turn invade 
new erythrocytes. A small percentage of merozoites differentiate into gametocytes, which 
when ingested by a mosquito, unite with another gametocyte to create a zygote. The 
zygote matures and releases sporozoites which migrate to the mosquito’s salivary glands 
and are injected into the human when the mosquito feeds.  
 

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Figure 1. Lifecycle of Plasmodium falciparum 
 
3.1.2 Progress towards a P. falciparum vaccine 
Recently, there have been significant advances; the leading vaccine candidate RTS,S/AS01 
has been  tested in a Phase III study in African infants that completed very recently. RTS,S is 
based on the major malaria sporozoite surface protein the circumsporozoite protein, or CS 
protein.  
 
The pivotal Malaria-055 Phase III efficacy study is a large double-blinded, randomized, 
controlled multicentre trial, which includes 15,460 infants and young children in seven sub-
Saharan African countries (Burkina Faso, Gabon, Ghana, Kenya, Malawi, Mozambique, and 
Tanzania) with diverse malaria transmission settings. In children aged 5-17 months at first 
immunization, the estimated overall efficacy was a 55.8% (97.5% CI: 50.6, 60.4; p<0.001) 
reduction in the number of all malaria episodes during the first 12 months of follow-up, 
with 47.3% (95% CI: 22.4, 64.2; p<0.001) efficacy against severe, life-threatening malaria.[5] 
The vaccine did not perform as well in children vaccinated aged 6-14 weeks of age, in co-
administration with other EPI (expanded programme of immunisation) vaccines. Estimated 
overall efficacy in this age group over 12 months of follow-up was 33% for all malaria 
episodes, and 37% for severe, life-threatening malaria.[5]  
 
In July 2014, efficacy was reported for both age groups over 18 months of follow-up. In the 
5-17 month age group, the incidence of clinical malaria in the per-protocol population was 

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0.69/person-year in the RTS,S/AS01 group and 1.17/person-year in the control group, 
resulting in an overall VE of 46% (95% CI 42% to 50%). This waned over time, but it 
persisted throughout the 18-month period. Efficacy against severe malaria was 36% (95% CI 
15% to 51%) and reductions in both malaria hospitalizations (41%) and all-cause 
hospitalizations (19%) were noted over 18 months.[6] In the 6-14 week age group, an 
overall efficacy of 27% (95% CI 20% to 32%) was observed in the incidence of all episodes of 
clinical malaria.[6]  
 
The final results of this Phase 3 trial were published in April 2015.[7] 8922 children aged 5-
17 months were included in the modified intention-to-treat analysis and followed up for a 
median of 48 months (IQR 39-50).  9585 episodes of clinical malaria met the primary case 
definition in the control group who received a comparator vaccine at months 0, 1, 2 and 20. 
In comparison, there were 6616 episodes of clinical malaria in those that received 
RTS,S/AS01 at months 0, 1, 2 and 20, giving an overall vaccine efficacy of 36.3% (95% CI 
31.8-40.5). However, achieving this efficacy would require four immunisations at time 
points not currently in the standard schedule of infant immunisations and therefore be 
likely very expensive and logistically difficult to achieve. In those that received RTS,S/AS01 
at months 0, 1, 2 and a dose of comparator vaccine at month 20, overall vaccine efficacy 
was 28% (95% CI 23-33). Vaccine efficacy against severe malaria was 32% (95% CI 44-47) in 
those that received RTS,S/AS01 at months 0, 1, 2 and 20, but only 1% (95% CI -23-21) in 
those administered the vaccine at 0, 1 and 2 months.  
 
6537 young infants aged 6-12 weeks were included in the modified intention-to-treat 
analysis and followed up for a median of 38 months (IQR 34-41). These infants were 
administered the RTS,S/AS01 vaccine with other infant vaccinations at months 0, 1 and 2, 
and some received a fourth booster dose at a non-standard time point of 20 months.  
Overall vaccine efficacy against episodes of clinical malaria in those that received 
RTS,S/AS01 at months 0, 1, 2 and 20 was 26% (95% CI 20-32) and 18% (95% CI 12-24) in 
those receiving immunisations at 0, 1 and 2 months.  Efficacy against severe malaria was 
17% (95% CI -9-38) with four doses and 10% (95% CI -18-32) with three doses. There was no 
vaccine efficacy against malaria mortality but power was low.    
 
Though these results show significant low level efficacy against most endpoints, there 
remains an urgent need to improve efficacy to achieve World Health Organisation (WHO) 
goals - development of a suitable vaccine with at least 75% durable efficacy against clinical 
malaria by 2030. Potential explanations for the reduced immune response in 6-12 week old 
infants include: 1) the infant’s immature immune system; 2) the co-administration of RTS,S 
with other childhood vaccines (DTPw-HepB/Hib and oral poliovirus vaccines); 3) the 
absence of priming with hepatitis B vaccine or with P. falciparum infection; and 4) maternal 
antibodies. It is also possible that the excess of Hepatitis B surface antigen present in the 

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formulation interferes with the induction of a potent immune response to circumsporozoite 
protein.  
 
RTS,S targets the pre-erythrocytic circumsporozoite (CS) protein, which is the major 
functional protein that plays a key role in sporozoite development and hepatocyte 
invasion.[8] 80% of the molecules in each RTS,S particle are hepatitis B surface antigen, and 
only 20% are fusion proteins of the malaria circumsporozoite protein moiety fused to 
hepatitis B suface antigen. R21, to be tested in this trial, is a biosimilar protein particle that 
lacks the excess of HBsAg in RTS,S has been developed at the University of Oxford. Indeed 
R21 comprises only fusion protein moieties, i.e. as 100% of its molecules, in contrast to 
RTS,S which comprises 20% of these with the remaining 80% being HBsAg molecules (Figure 
2).  
 
3.2 
Pre-erythrocytic stage as a vaccine target 
 
The pre-erythrocytic stage of P. falciparum infection presents an attractive target for an 
efficacious human vaccine, as sufficient reduction in the number of viable merozoites 
reaching the blood from the liver will prevent parasitisation of red blood cells and initiation 
of the blood stage of infection.  Anti-CS antibodies can target sporozoites, facilitating 
destruction of sporozoites prior to hepatocyte invasion. As sporozoites travel from the skin 
to the liver within minutes, it may be difficult for a vaccine to achieve complete protection 
against P. falciparum based solely on antibodies to sporozoites. The liver stage of infection 
provides a longer window of opportunity for cell mediated immunity to recognize and 
destroy infected hepatocytes. Research suggests that, in isolation the RTS,S vaccine 
targeting the pre-erythrocytic stage antigen, the CS protein, and vaccines targeting ME-
TRAP, a liver-stage insert in Oxford’s viral vectored vaccines, do not delay the initial 
emergence of parasites in to the blood, nor the rate of parasite multiplication in the blood, 
but rather reduce the size of this initial inoculum.[9] A delay to patent blood stage infection 
in persons receiving these vaccines reflects a reduced liver-to-blood inoculum. The efficacy 
of these pre-erythrocytic vaccine strategies can be assessed experimentally by subjecting 
volunteers to inoculation with P. falciparum sporozoites by the bite of infected mosquitoes. 
Complete protection against blood-stage infection, or a delay in the time to patent blood 
stage infection in vaccinees compared to controls, reflect vaccine efficacy. 
 
There are a number of reasons that support the selection of circumsporozoite protein as a 
potential target for a malaria vaccine candidate. This protein is expressed on the sporozoite 
surface [10] and to a lesser degree on hepatic schizonts and plays a pivotal role in 
alignment and sporozoite invasion of hepatocytes.[8, 11] In vitro, antibodies directed 
against B cell epitopes derived from this protein can inhibit the infectivity of sporozoites to 
liver cells.[12] In murine models, passive transfer of antibodies to the immunodominant B 

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cell repeat epitope of the CS protein as well as active immunisation with constructs 
containing this epitope, confer protection against sporozoite challenge. [13, 14]  
Furthermore, it has been shown that adoptive transfer of CD8+ cytotoxic lymphocytes (CTL) 
or CD4+ T cell clones specific for epitopes on the CS protein can provide protection against 
a sporozoite challenge. [15, 16] Finally the leading malaria vaccine candidate, RTS,S, induces 
partial efficacy by inducing antibodies against the central repeat (NANP) of the 
circumsporozoite protein.  
 
3.3 
R21 vaccine development 
 
Manufacture of clinical grade R21 particle was undertaken at the University of Oxford 
Clinical Bio-Manufacturing Facility (CBF) (www.cbf.ox.ac.uk), with financial support from the 
UK Medical Research Council (MRC) and the EC FP7 programme.     
Figure 2:  Schematic diagram showing RTS,S and R21 fusion proteins. Both RTS,S and R21 
include the fusion protein of hepatitis B surface antigen to the C-terminus and central 
repeats of the circumsporozoite (CS) protein. These repeats comprise many copies of the 
four amino acid sequence NANP. R21 is a virus like-particle that results for spontaneous 
assembly of the R21 molecules. RTS,S, expressed in a different yeast types required 
expression of an four fold excess of the unfused hepatitis B surface antigen to allow it to 
form hybrid particles. Generation of virus-like particles by both RTS,S and R21 has been 
shown to be important for allowing induction of high level antibody responses. 
 
RTS,S/AS01 vaccine, induces very strong antibody responses to the conserved central 
repeat of circumsporozoite protein (CSP), of the order of 100 - 600 micrograms per ml, very 

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weak mainly IL-2 containing CD4+ T cells and no CD8+ T cells to CSP.[17]  The most 
reproducible correlate of protection in clinical studies is with antibody levels.[5, 17]   
 
We propose here to test clinically a biosimilar of the RTS,S vaccine called R21 adjuvanted 
with Matrix-M1 (MM).  As a biosimilar of the RTS,S vaccine, the R21 particle contains no P. 
falciparum sequences that are not present in RTS,S, which has been safely used in 
thousands of individuals.  It is a hybrid protein of the majority of the CS protein of P. 
falciparum fused to the hepatitis B surface antigen (Figure 2).  It spontaneously forms a 
particle just like RTS,S. However, we anticipate that R21 may be a more immunogenic 
particle than RTS,S in humans for two reasons.  It induces predominantly malaria rather 
than hepatitis antibodies in pre-clinical studies probably because it has a higher proportion 
of malaria to hepatitis antigen in its composition than RTS,S.  This is made possible by 
expressing R21 in the better expressing yeast Pichia pastoris, rather than in Saccharomyces 
cerevisiae. Secondly, in pre-clinical studies R21 has been found to be exceptionally 
immunogenic for induction of anti-NANP antibodies, likely the key protective immune 
mechanism of RTS,S, yielding titres of a mean of 800,000 ELISA (enzyme linked 
immunosorbent assay) units  (K Collins and A Hill, unpublished data). At the C-terminus of 
R21 a four amino acid sequence has been added, EPEA, which is required for efficient 
immunochromatographic purification of R21. This very short sequence is found many times 
in the proteome of malaria parasites and humans but has not, to our knowledge, been used 
previously as a vaccine component.    
3.4 
R21 - pre-clinical studies  
3.4.1 Immunogenicity 
 
Initial pre-clinical assessment of immunogenicity was undertaken in BALB/c mice that were 
immunised intramuscularly with 0.5g of R21 alone or in combination with an adjuvant 
(Alhydrogel, or “Abisco” which is almost identical to the Matrix M to be used in this trial 
and made by the same company). Immune responses including antibody levels to the 
central NANP repeat region and antigen–specific T cell responses were measured three 
weeks after a 3-dose immunisation schedule (Figures 3.1 & 3.3).  R21 + Abisco-100, a potent 
saponin-based adjuvant resulted in the greatest humoral immune response at each time 
point in the vaccination schedule.  

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Figure 
3.1:  Pre-clinical assessment of immunogenicity with 0.5g of R21 alone or in combination 
with an adjuvant (Alhydrogel, Abisco). 
 
The responses in all groups were boosted by a third immunisation and R21 + Abisco-100 
induced the highest titres of NANP specific IgG and the response for this group was 
significantly higher than both the R21 + Alhydrogel and R21 alone groups after the final 
immunisation (Figure 3.1). 
 
 
 
 
Figure 3.2:  Relative proportions of IgG to NANP and HBsAg after immunisation with R21 + 
Abisco-100 in BALB/c mice.  
 
 
NANP IgG ELISA
A - 1st immunisation
R21 alone
R21 + Alhydrogel 
R21 + Abisco
0
2
4
6
8
***
Endpoint titre (log 10)
B - 2nd immunisation
R21 alone
R21 + Alhydrogel 
R21 + Abisco
0
2
4
6
8
**
C - 3rd immunisation
R21 alone
R21 + Alhydrogel 
R21 + Abisco
0
2
4
6
8
*
*
1
2
3
4
5
6
7
1st immunisation
2nd immunisation
3rd immunisation
NANP IgG
HBsAg IgG
Endpoint titre (log 10)

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Antibodies to hepatitis B surface antigen were measured in the same study. As expected 
from the composition of R21 and RTS,S antibodies to hepatitis B surface antigen were much 
lower with R21 than RTS,S (Figure 3.2) probably reflecting a structure in R21 where the CS 
sequences are found on the exposed surface of the virus-like particle whereas the surface 
of RTS,S comprises both hepatitis B and malaria epitopes.  
CS-specific IFN-γ producing T cells measured after the third immunisation were only 
detected at a significant level in mice immunised with R21 + Abisco-100 (Figure 3.3). R21 
alone was ineffective at inducing CS-specific T cell responses on its own. Further 
comparison to other adjuvants including a squalene-based oil-in-water emulsion (Addavax) 
and a polyionic carbomer (Carbopol) showed that Abisco-100 was the best adjuvant to 
induce high levels of humoral and cell-mediated immunity.  
 
 
 
Figure 3.3:  CS-specific IFN-γ producing T cells measured after the third immunisation 
 
 
3.4.2 Efficacy 
 
Sporozoite challenge (1000 sporozoites per mouse injected intravenously) using transgenic 
P. berghei parasite were performed in BALB/c mice (Figure 3.4). R21 + adjuvant were given 
twice, eight weeks apart and mice were challenged three weeks after the second dose. Thin 
blood films looking microscopically for parasitaemia were performed daily from day 5 post-
challenge. Sterile protection was defined as remaining slide negative at day 14, and 
significant delay in development of 1% parasitaemia compared to non-immunised control 
mice was regarded as partial efficacy.  
 
R21 + Abisco-100 steriley protected 100% of the challenged mice (p=< 0.0001) and R21 + 
Matrix M steriley protected 87.5% (p=0.0002) and this was confirmed in a second 
p
R21 alone
R21 + Alhydrogel 
R21 + Abisco
0
200
400
600
800
1000
SFC/106 Splenocytes

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independent challenge. (p = < 0.0001). There was no significant difference between the two 
adjuvants.  
 
The durability of efficacy was assessed by undertaking sporozoite challenge in mice seven 
and fourteen weeks after immunisation. Efficacy was maintained well at seven weeks post 
immunisation with 75% of mice sterilely protected (6/8) and this was not significantly 
different when compared to efficacy at three weeks post immunisation (p=0.4468, by Log-
rank (Mantel-Cox) Test). At 14 weeks post immunisation however, sterile efficacy was 
reduced to 50% (2/4) and this was 37% lower than the efficacy at three weeks. This was not 
significantly lower probably due to the small number in the group (p=0.0636). 
 
Sterile efficacy 14 weeks after immunisation is 37% lower than efficacy three weeks after 
immunisation. This reduction in protective efficacy can however be boosted to 100% if mice 
are challenged once (three weeks post immunisation) within the 14 weeks. Therefore 
efficacy after vaccination and one sporozoite infection is very durable and 100% sterile 
efficacy is maintained for at least 14 weeks. 
 
A Adjuvant only
4
5
6
7
8
9
10 11 12 13 14 15
0
20
40
60
80
100
Adjuvant only
(Abisco/Matrix M)
Naive
Time to 1% parasitaemia
Percent survival
B R21+Abisco-100
4
5
6
7
8
9
10 11 12 13 14 15
0
20
40
60
80
100
R21 + Abisco
Controls
(Abisco + naives)
Time to 1% parasitaemia
Percent survival

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Figure 3.4 (A-F): Protective efficacy elicited by saponin based ISCOM adjuvants with R21 in a 
transgenic sporozoite model. BALB/c mice were immunised i.m. with 0.5µg R21 + adjuvant 
(Abisco-100 or Matrix M), twice eight weeks apart (n=8/group). Mice were challenged three 
weeks after the final vaccination by i.v. injection of 1000 sporozoites (P. berghei transgenic 
for P. falciparum CSP) along with eight naïve mice. Two groups of adjuvant control mice 
(n=5/group) were also challenged three weeks after receiving two shots of adjuvant (Abisco-
100 or Matrix M) i.m., eight weeks apart. Blood stage parasitemia was monitored from day 
5 after challenge by thin-film blood smear, and time to 1% parasitemia was calculated using 
linear regression. The results are presented in the Kaplan-Meier survival graphs and survival 
curves were compared by Log-rank (Mantel-Cox) Test. (A) Adjuvant control = no significant 
difference, (B) R21 + Abisco-100 p<0.0001, (C) R21 + Matrix M p=0.0002, (D) R21 + Matrix M 
repeat p=<0.0001, (E) R21 + Abisco vs R21 + Matrix M = no significant difference. Blood was 
taken three weeks after each vaccination (Day 21 and Day 77) for immunology and NANP 
specific IgG was assayed by ELISA (F), group mean responses shown and dotted line 
indicates the limit of detection. 
3.5  
RTS,S/AS01 
 
The RTS,S antigen consists of two proteins, RTS and S, that are expressed intracellularly in 
yeast and spontaneously assemble into mixed polymeric particulate structures. RTS,S is 
produced in Saccharomyces cerevisiae, genetically modified to contain the coding 
sequences of the RTS and S proteins. RTS is a hybrid polypeptide consisting of a portion of 
the CS antigen of the malaria parasite P. falciparum strain NF54, fused to the amino 
C R21+Matrix M
4
5
6
7
8
9
10 11 12 13 14 15
0
20
40
60
80
100
R21 + Matrix M
Controls
(Matrix M + naive)
Time to 1% parasitaemia
Percent survival
D Repeat challenge
4
5
6
7
8
9
10 11 12 13 14 15
0
20
40
60
80
100
R21 + Matrix M
Naive
Time to 1% parasitaemia
Percent survival
E R21+Abisco vs R21+Matrix M
4
5
6
7
8
9
10 11 12 13 14 15
0
20
40
60
80
100
R21+Abisco
R21+Matrix M
Time to 1% parasitaemia
Percent survival
F NANP IgG ELISA
0
1
2
3
4
5
6
7
8
R21 +Abisco
R21 + Matrix M
Day 21
Day 77
Endpoint titre (log 10)

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terminal end of the Hepatitis B virus S protein. S corresponds to the surface antigen of 
Hepatitis B virus (HBsAg) (Figure 2).  
 
The AS01 family of Adjuvant Systems contains 2 immunostimulants formulated with 
liposomes. The monophosphoryl lipid A (MPL) molecule consists of a chemically detoxified 
form of the parent lipopolysaccharide (LPS) from the Gram-negative bacterium Salmonella 
minnesota. QS21 is a natural saponin molecule purified from the bark of the South 
American tree, Quillaja saponaria. 
 
The RTS,S/AS malaria vaccines have been assessed in 14 clinical studies conducted in 
malaria-naïve adults and in five adult field studies conducted in Africa. It was initially 
assessed in malaria-naïve adults to establish proof-of-concept. These studies demonstrated 
efficacy, immunogenicity and an acceptable safety profile of the RTS,S/AS02 vaccine 
formulation.[18, 19] Subsequently, studies demonstrated protection against natural 
infection in semi-immune adult Gambian men.[20] The AS01 adjuvant system was preferred 
for further evaluation as it elicited superior cellular and humoral responses in comparison 
to RTS,S/AS02; improved efficacy was also demonstrated in Phase II studies.[21, 22]  
 
These results formed the basis to assess the RTS,S/AS malaria vaccine candidate in African 
children living in endemic regions. To date, eight Phase I/II clinical studies and one Phase III 
clinical study in subjects aged at least 5 months and three Phase I/II studies in infants aged 
less than 5 months of age have been completed. Efficacy data are presented above (section 
3.1.2).  
 
3.5.1 Phase III evaluation  
 
From March 2009 through January 2011, 15460 children were recruited to the first large-
scale randomised, controlled, double-blind trial evaluating the leading malaria vaccine 
candidate, RTS,S/AS01. There were two age categories: children 6 to 12 weeks of age and 
those 5 to 17 months of age at enrolment. Within each age category, there were three 
study groups: children who received all three doses of the immunisation schedule at 1-
month intervals and scheduled to receive a booster dose at 18 months, children who 
received the primary RTS,S/AS01 vaccination series without a booster and a control group 
who received a comparator vaccine.[5, 6]   
 
To date, four sets of results are available on the primary endpoint of the trial, vaccine 
efficacy.[5-7] (See Section 3.1.2 and Figure 4) Anti-CSP antibodies were measured in in the 
first 200 participants in each age category at each study site at enrolment and 1 month 
after the third dose of vaccine. The GMT was low at enrolment in all three study groups and 

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99.9% of children in the RTS,S/AS01 group were seropositive for anti-CSP Abs at 1 month 
after the third dose, with a GMT of 621 EU/ml (95% CI 592-652) in the older age 
category.[5] 
 
 
Figure 4. Cumulative Incidence of First or Only Episodes of Clinical Malaria (Primary Case 
Definition) in the Older Age Category. The cumulative incidence of the primary case 
definition in children 5 to 17 months of age at enrollment is shown during 12 months of 
follow-up after the administration of the third dose of vaccine in the per-protocol population 
(Panel A) and during 14 months of follow-up after the administration of the first dose of 
vaccine in the intention-to-treat population (Panel B).[5] 
 
3.6  
MATRIX-M1 
 
Matrix-M1 (MM) is a 40nm-sized complex containing the adjuvant-active saponin Quillaja 
saponaria, phospholipid and cholesterol. Quillaja saponins are triterpene glycoside 
substances derived from the tree Quillaja saponaria. The molecular weights of the different 
saponins range from 1800 - 2000 Da. In water, saponin in concentrations of 200-500 ppm 
exist as monomers; at higher concentrations they aggregate as micelles, with a molecular 
weight of approximately 100 000 Da. Saponins are surface-active compounds with a variety 
of applications including in agriculture, feed, food and beverage, mining, and veterinary 
vaccines, and are currently being investigated in human vaccine clinical trials. In aqueous 
solution, saponins are excellent adjuvants and are used in commercial veterinary vaccines, 
e.g., vaccines against foot-and-mouth disease, bovine mastitis, feline leukemia and equine 
influenza. An HPLC-purified fraction of the same saponin, called QS21, is a component of 
the AS01 adjuvant used in RTS,S/AS01. 
3.6.1 Pre-clinical studies 
In animal studies, MM has been shown to perform better than most other adjuvants, 
inducing a multifaceted response including antibody production, T cell responses and 

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recruitment of innate immune cells into draining lymph nodes.[25, 26] Mixed with a 
virosomal H9N2 avian influenza vaccine, Matrix-M1 induced enhanced antigen-specific 
humoral and CD8+ T cell responses.[27] Matrix-M1 administered with an intramuscular 
H5N1 virosomal influenza vaccine induced a strong immediate and long-term humoral and 
cellular immune response and showed a dose-sparing potential.[28] 
 
In pre-clinical studies, R21 adjuvanted with both Matrix-M1 and MF59 has demonstrated 
good antibody responses.[29] In addition, there was no interference with induction of 
antibodies or T cells when R21/MF59 was combined with viral vectors.[29] BALB/c mice 
immunised with 2 doses of R21/MM showed excellent efficacy (91.3% sterile protection) 
against transgenic malaria parasite challenge.[29] Combining protein and viral-vectored 
vaccines in murine malaria models has also previously been shown to have a synergistic 
effect resulting in much higher sterile efficacy (90%) than either vaccine individually.[30] 
 
3.6.2  Clinical studies 
Phase I clinical trials have provided evidence that MM appears safe and well tolerated in 
humans.  
 
Phase I human clinical trial of a Matrix M™-adjuvanted virosomal H5N1 vaccine[31] 
An open-label human phase I dose escalating clinical trial was conducted to evaluate the 
safety and tolerability of a pandemic influenza A/H5N1 virosomal vaccine formulated with 
Matrix M™ . Matrix M™ was constituted from Matrix A and Matrix C using a ratio of Matrix 
A: Matrix C of 91:9. 
In total 60 healthy adult volunteers (38 women and 22 males, 20-49 years old) were 
recruited. Volunteers were randomised into four groups of 15 subjects and vaccinated with 
two intramuscular (IM) injections into the deltoid muscle at an interval of 21 (±1) days. One 
group received 30 microgram of HA alone, the other groups received 50 microgram of 
Matrix M™ mixed with 1.5, 7.5 or 30 microgram HA (Table 1). Local and systemic adverse 
events (AE) were ascertained, and haematological, biochemical and immunological 
parameters were investigated. Local and systemic immunogenicity of the test items were 
assessed by ELISPOT (enzyme-linked immunospot assay) for antibody secreting cells and 
flow cytometry for detection of multifunctional CD4+ T cells. Serum samples were tested 
for detection of homologous and cross reactive HI antibodies using a modified 
haemagglutination inhibition assay. 
 
 
 
 

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Table 1. Details for the respective treatment groups included in the phase I clinical trial.  
Grou
p 
Treatment 
Number of 
Volunteers 
(female/male) 
Mean age 
Administration 
days 
1 
30 g HA 
15 (9/6) 
32 (20-41) 
d0, d21 
2 
1.5 g HA  
+ 50 g Matrix M-
2  
15 (10/5) 
29 (21-44) 
d0, d21 
3 
7.5 g HA  
+ 50 g Matrix M-
2  
15 (8/7) 
31 (22-42) 
d0, d21 
4 
30 g HA  
+ 50 g Matrix M-
2  
15 (11/4) 
31 (25-49) 
d0, d21 
  
There were no serious adverse events and no clinically relevant changes in haematological, 
biochemical, and immunological parameters. All four vaccine formulations were well 
tolerated with the majority of reported solicited adverse events described as mild to 
moderate in intensity usually resolving within 3 days of vaccination. The most common 
local reactions were pain or tenderness at the injection site, often lasting up to three days, 
and more frequent in volunteers receiving Matrix M™. Pain at the injection site was 
described as mild and transient, apart from in two volunteers who reported it as moderate 
(severe enough to affect daily activity). Systemic reactions were most commonly described 
as mild to moderate fatigue, headache and myalgia. Adverse events are detailed below in 
Fig 5. 
 
Antibody responses were enhanced when adjuvanted with virosomal H5N1 vaccine, 
allowing significant dose-sparing.[31]  
 
 

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Figure 5. Adverse events in volunteers receiving two doses of a virosomal influenza vaccine 
alone, or adjuvanted with Matrix M™. The vaccine dose and presence (+) or absence (−) of 

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Matrix M™ adjuvant is shown on the x-axis. * and ** indicates significantly more subjects 
reporting adverse events as compared to the 30 µg HA virosomal alone group, p < 0.05 and 
p < 0.01, respectively (Chi square test). Adopted from Cox et al.[31] 
 
Phase I clinical trial of a Matrix-M1-adjuvanted seasonal influenza vaccine (Vaxigrip) 
 
A human phase I clinical trial was conducted to evaluate the safety and tolerability of 
Matrix-M1 (Matrix A and C ratio, 85:15) formulated with a seasonal influenza vaccine 
(Vaxigrip, Sanofi-Pasteur), in an elderly population. The efficacy was also evaluated with 
respect to serological and cell mediated immunological parameters. The commercial 
influenza vaccine, Vaxigrip was a marketed, inactivated, trivalent, split virus seasonal 
influenza vaccine (season 2010/2011). The primary endpoint of the study was to assess 
safety of the investigational vaccine (Matrix M-1 formulated with Vaxigrip) in both young 
adults and elderly adults in comparison with the commercial vaccine without adjuvant. The 
secondary endpoint of the study was to assess a comprehensive battery of immunological 
parameters.  
The study started with vaccination of one cohort of 22 healthy adults aged 18-50 years, 
with one dose of the investigational vaccine to assess the adverse event profile. Vaccination 
of the elderly subjects started when the results from the first cohort were evaluated and 
found acceptable, no SAEs were detected. One cohort (age 65-75, n=44) received one dose 
of the commercial vaccine and another cohort (age 65-75, n=44) received one dose of the 
investigational vaccine (Table 2.1).  
All test items were administered intramuscularly once and blood samples were taken at 
days 0, 7, 28, 90 and 150 post vaccination. Haematological parameters, blood biochemical 
and immunological parameters were investigated day 0 and 7.  
 
 
Table 2.1. Details for the respective treatment groups included in the phase I clinical trial 
with Matrix-M1/Vaxigrip.  
 
The Matrix M-1 adjuvanted investigational vaccine was well tolerated. An expected 
increase of side-effects compared to the unadjuvanted comparator vaccine was recorded. 
No severe side effects were found. The only local side effect with an increased incidence for 
Cohort
Age group
Subjects (n)
Vaccine
HA (µg)
Matrix M (µg)
1
Adults
18-50 y
22
Vaxigrip +
Matrix-M1
3x15
50
2
Elderly
65-75 y
44
Vaxigrip
3x15
0
3
Elderly
65-75 y
44
Vaxigrip +
Matrix-M1
3x15
50

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the investigational vaccine was pain. There were an overall increase of systemic side events, 
but all were predominantly mild and transient (Table 2.2).  
 
Table 2.2. Local and systemic side events for the elderly cohorts in the phase I clinical trial 
with Matrix-M1/Vaxigrip. 
 
In summary, this was the first clinical trial in which the Matrix M-1 formulation of Matrix 
M™ was administered to humans. Matrix M-1 appeared safe and well tolerated in 66 adult 
clinical trial volunteers, at a dose of 50µg, formulated with a commercial influenza vaccine. 
There was a tendency to more adverse events in the Matrix M-1- adjuvanted versus non 
adjuvanted vaccinations, but the adverse event profile was still excellent. 
 
Phase I clinical trial of ChAd63 ME-TRAP / MVA ME-TRAP heterologous prime boost malaria 
vaccination adjuvanted with Matrix M™ 
 
VAC048 is an open-label Phase I study that has been undertaken at the University of Oxford 
evaluating the safety and immunogenicity of MM administered in combination with 
heterologous prime-boost vaccination with ChAd63 ME-TRAP and MVA ME-TRAP. The study 
groups are shown below.  
 
Group Name 
Vaccination Regimen 
Number of 
Volunteers 
Vaccination 1 (Day 0) 
Vaccination 2 (Day 56) 
Control 
ChAd63 ME-TRAP 5 x 1010 vp  
MVA ME-TRAP 2 x 108 pfu  
6 
Low dose 
Matrix M™ 
ChAd63 ME-TRAP 5 x 1010 vp  
mixed with Matrix-M1 25µg  
MVA ME-TRAP 2 x 108 pfu  
mixed with Matrix-M1 25µg  
8 
Standard 
Dose Matrix 
M™  
ChAd63 ME-TRAP 5 x 1010 vp  
mixed with Matrix-M1 50µg  
MVA ME-TRAP 2 x 108 pfu  
mixed with Matrix-M1 50µg  
8 
Table 3. Study groups in VAC048. 
Vaxigrip + Matrix-
M1 (n=44)
Vaxigrip
(n=44)
Local
side
effects
Pain
27%
9%
Erythema
14%
14%
Induration
2%
2%
Systemic 
side 
effects
Malaise
2%
0%
Fever
0%
0%
Myalgia
7%
0%
Headache
7%
5 %

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Overall, vaccines were safe and generally well tolerated. Most adverse events were mild in 
nature and resolved within 48 hours of vaccination. The local and systemic adverse event 
profile for the low dose (25g) and standard dose (50g) MM groups are shown below.  
  
-  
ChAd63 ME-TRAP 5 x 1010 vp IM only 
 
 
 
n=6 
25 +  
ChAd63 ME-TRAP 5 x 1010 vp mixed with Matrix M-1 25µg IM 
 
n=9 
50 + 
ChAd63 ME-TRAP 5 x 1010 vp mixed with Matrix M-1 50µg IM 
 
n=8 
 
‘Other’ (Control group) = bruising at vaccine site and sensation of sensitive skin 
‘Other’ (25 +) = bruising at vaccine site 
‘Other’ (50+) = shoulder pain 
Figure 6.1: VAC048 Reactogenicity results. The figure shows the percentage of subjects that 
developed local adverse effects after vaccination with ChAd63 ME-TRAP mixed with Matrix 
M-1 (25µg or 50g) IM in comparison to subjects who received ChAd63 ME-TRAP 5 x 1010 
vp IM only. Only AEs deemed definitely, possibly or probably related to vaccination are 
reported. The highest intensity of each AE per subject is listed.  
0
10
20
30
40
50
60
70
80
90
100
- 25
+
50
+
- 25
+
50
+
- 25
+
50
+
- 25
+
50
+
- 25
+
50
+
- 25
+
50
+
Pain
Redness
Swelling
Warmth
Itch
Other
% of volunteers 
Local adverse events related to 
ChAd63 ME-TRAP +/- Matrix M 
Severe
Moderate
Mild

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-  
ChAd63 ME-TRAP 5 x 1010 vp IM  
 
 
 
 
             n=6 
25 +  
ChAd63 ME-TRAP 5 x 1010 vp mixed with Matrix M-1 25µg IM 
 
n=9 
50 + 
ChAd63 ME-TRAP 5 x 1010 vp mixed with Matrix M-1 50µg IM 
 
n=8 
 
‘Other’ (control group) = back pain and postural dizziness 
‘Other’ (25 +) = dizziness, rigors and lightheadedness 
‘Other’ (50 +) = lower backache, cough, sore throat, runny nose, rigors and difficulty 
sleeping 
Figure 6.2: VAC048: Reactogenicity results. The figure shows the percentage of subjects 
that developed systemic adverse effects after vaccination with ChAd63 ME-TRAP mixed 
with Matrix M-1 (25µg or 50g) IM in comparison to subjects who received ChAd63 ME-
TRAP 5 x 1010 vp IM only. Only AEs deemed definitely, possibly or probably related to 
vaccination are reported. The highest intensity of each AE per subject is listed. 

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- 
MVA ME-TRAP 2 x 108 pfu IM  
  
 
 
 
 
n=6 
25 +  
MVA ME-TRAP 1.45 x 108 pfu or 2 x 108 mixed with Matrix M-1 25µg IM n=8 
50 + 
MVA ME-TRAP 2 x 108 pfu mixed with Matrix M-1 50µg IM  
             n=8 
 
‘Other’ (Control) severe burning sensation in arm 
‘Other’ (50 +) two volunteeers reported mild bruising at the vaccine site  
Figure 6.3: VAC048: Reactogenicity results. The figure shows the percentage of subjects 
that developed local adverse effects after vaccination with MVA ME-TRAP mixed with 
Matrix M-1 (25µg or 50g) IM in comparison to subjects who received MVA ME-TRAP 2 x 
108 pfu IM only. The first two volunteers in the low dose group only received 1.45 x 108 pfu 
instead of 2 x 108 pfu. Only AEs deemed definitely, possibly or probably related to 
vaccination are reported. The highest intensity of each AE per subject is listed. 
0
10
20
30
40
50
60
70
80
90
100
- 25
+
50
+
- 25
+
50
+
- 25
+
50
+
- 25
+
50
+
- 25
+
50
+
- 25
+
50
+
Pain
Redness Swelling Warmth
Itch
Other
% of volunteers 
Local adverse events related to MVA 
ME-TRAP +/- Matrix M 
Severe
Moderate
Mild

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- 
MVA ME-TRAP 2 x 108 pfu IM  
  
 
 
 
 
n=6 
25 +  
MVA ME-TRAP 1.45 x 108 pfu or 2 x 108 mixed with Matrix M-1 25µg IM n=8 
50 + 
MVA ME-TRAP 2 x 108 pfu mixed with Matrix M-1 50µg IM  
 
n=8 
 
‘Other’ (Control) – moderate backache and mild abdominal cramps 
‘Other’ (25+) – mild palpitations and feeling shaky; severe rigors 
‘Other’ (50+) – moderate dizziness, feeling shaky, abdominal pain, chills and rigors; mild 
lower back ache 
Figure 6.4: VAC048: Reactogenicity results. The figure shows the percentage of subjects 
that developed systemic adverse effects after vaccination with MVA ME-TRAP mixed with 
Matrix M-1 (25µg or 50g) IM in comparison to subjects who received MVA ME-TRAP 2 x 
108 pfu IM only. The first two volunteers in the low dose group only received 1.45 x 108 pfu 
instead of 2 x 108 pfu. Only AEs deemed definitely, possibly or probably related to 
vaccination are reported. The highest intensity of each AE per subject is listed. 
 
 
 
 
 
 

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3.7  
Rationale 
 
3.7.1 Vaccine Development Strategy 
 
R21 has been developed at the Jenner Institute, University of Oxford. It is produced by 
using recombinant HBsAg particles expressing the central repeat and the C-terminus of the 
circumsporozoite protein (CSP) and has been GMP manufactured in Pichia pastoris. This is a 
biosimilar protein particle to RTS,S which also targets the pre-erythrocytic circumsporozoite 
protein, the major functional protein in sporozoite development and hepatocyte invasion. 
R21 lacks the excess of HBsAg in RTS,S (See Figure 2) and has been shown to be highly 
immunogenic (> 105 ELISA units after two immunisations) and have at least comparable 
immunogenicity and similar high level efficacy to RTS,S in animal studies. (K Collins and A 
Hill, unpublished data). To date, safety and immunogenicity observed in VAC053 is very 
promising and antibody levels observed are comparable to previous studies done in Oxford 
with the 
leading 
malaria 
vaccine 
candidate, 
RTS,S. 
Furthermore, 
comparable 
immunogenicity is observed at both the tested doses of R21 adjuvanted with Matrix M. 
(See Figure 7). Therefore, we are proposing to add an additional group to the study to test 
an even lower dose of R21 of 2g as this could lead to significant dose-sparing if it is shown 
to be equally immunogenic.  
 
Figure 7. Mean IgG antibody responses observed in Groups 1 and 3 of the VAC053 trial in 
comparison to historical data with RTS,S.  
 
Matrix-M1 is an attractive adjuvant, as it, and other matrix formulations of Quillaja 
saponins, show good safety profiles, and the ability to enhance both cellular and humoral 
immune responses to a range of vaccines. Preclinical data presented in Section 3.4 
demonstrate the potential for Matrix-M1 to enhance the immunogenicity of R21. 

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In order to progress the development of a potentially more efficacious anti-sporozoite 
malaria vaccine, the proposed clinical trial, VAC053, has two objectives. The first is to assess 
the safety of R21 administered as a mixture with the Matrix-M1 adjuvant. The second is to 
determine the effects of Matrix-M1 on the immunogenicity of the vaccine. 
 
If these studies show,  
 Satisfactory safety of R21 adjuvanted with Matrix-M1, and 
 Matrix-M1 generates sufficient enhancement of the humoral immunogenicity of 
R21 with acceptable safety,  
this will support future clinical evaluation of the efficacy of the vaccine using controlled 
human malaria infection (CHMI) where sporozoites are delivered by mosquito bite.  
4 
STUDY OVERVIEW 
 
This is an open label Phase I study of a protein particle malaria vaccine candidate in healthy 
volunteers. R21 will be administered intramuscularly alone or with the adjuvant Matrix-M1.  
 
There will be 4 study groups, with Groups 1, 3 and 4 containing 10 volunteers and Group 2 
containing 4 volunteers as shown in Table 4. 
  
Volunteers will be first recruited to Group 1 and subsequently to Groups 2-4 with interim 
clinical safety reviews (See Section 8.5.3). There will also be staggered enrolment for the 
first three volunteers within Groups 1-3. Volunteers will be allocated to study group by 
selecting eligible volunteers for enrolment in the order in which they were deemed eligible 
following screening. 
 
4.1 
Study Groups 
 
Table 4: Study Groups 
 

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4.1.1 First volunteers 
The first volunteer in Group 1 will be vaccinated alone and then reviewed 72 hours 
following vaccination. If there are no safety concerns, another two Group 1 volunteers may 
be immunised, at least one hour apart, and reviewed in a further 72 hours. Providing there 
are no safety concerns as assessed by the CI and LSM, the remaining volunteers in Group 1 
and the first volunteer in Group 3 may be vaccinated.  
The first volunteer in Group 3 will be vaccinated alone and then reviewed 72 hours 
following vaccination. If there are no safety concerns, another two volunteers in Group 3 
may be immunised, at least one hour apart, and reviewed in a further 72 hours. Providing 
there are no safety concerns as assessed by the CI and LSM, the remaining volunteers in 
Group 3 may be vaccinated.  
In parallel, following review of the first Group 3 volunteer at 72 hours post-vaccination, the 
first volunteer in Group 2 will be vaccinated alone and then reviewed 72 hours following 
vaccination. If there are no safety concerns, another two Group 2 volunteers may be 
immunised, at least one hour apart, and reviewed in a further 72 hours. Providing there are 
no safety concerns as assessed by the CI and LSM, the remaining volunteer in Group 2 may 
be vaccinated.  
Group 4 has been added to the trial to assess whether a further reduction in dose would 
still elicit comparable immunogenicity to Groups 1 and 3. There is no requirement for 
staggered enrolment of the first three volunteers in Group 4 as R21 has already been 
administered to humans in much higher doses (10g in Group1 and 50g in Group 3). 
 
All volunteers will be issued with the telephone number of the investigators and 
encouraged to contact the investigators if there are any problems. An investigator will be 
available 24 hours a day. 
 
4.1.2 Duration of study  
The total duration of the study will be 34 weeks from the day of enrolment for all 
volunteers.  
 
4.1.3 Definition of Start and End of Trial 
The start of the trial is defined as the date of the first vaccination of the first volunteer. The 
end of the trial is the date of the last visit of the last volunteer. 
 
4.2 
Potential Risks for volunteers 
The potential risk to participants is considered as low. The potential risks are those 
associated with phlebotomy and vaccination. 
 

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Phlebotomy:  
The maximum volume of blood drawn over the study period (approximately 620ml) 
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.  
 
Vaccination:  
R21 has not been used in humans before, and therefore will be initially administered at the 
lower dose of 10g before progressing to the higher dose of 50g in Groups 2 and 3. 
Potential expected risks from vaccination include local effects such as pain, redness, warmth 
and swelling and systemic effects including a mild self-limiting flu-like illness.  
 
There has been previous experience with saponin-based adjuvants administered to humans 
with a range of vaccines. Matrix M™ (constituted of Matrix A : Matrix C at a ratio of 91:9) 
has been administered at a dose of 50 µg, mixed with a virosomal influenza vaccine, to 45 
adult volunteers in the Phase I clinical trial presented above[31]. Vaccination was well 
tolerated, with adverse events related to vaccination generally being mild and resolving 
within three days. The formulation of Matrix M™ to be used in this study, Matrix-M1, is 
constituted of Matrix A : Matrix C at a ratio of 85:15. This formulation has been used in the 
VAC048 trial and the results presented above demonstrate an acceptable safety profile, 
despite the mixed administration with liver-stage viral vectors in that trial, with most 
symptoms being mild in nature.  
 
As with any vaccine, Guillain-Barré syndrome or immune-mediated reactions that can lead 
to organ damage including serious allergic reactions may occur but this should be extremely 
rare. Serious allergic reactions including anaphylaxis could also occur and for this reason 
volunteers will be vaccinated in a clinical area where Advanced Life Support trained 
physicians, equipment and drugs are immediately available for the management of any 
serious adverse reactions (AR). 
 
4.3 
Known Potential Benefits 
Volunteers will not benefit directly from participation in this study. However, it is hoped 
that the information gained from this study will contribute to the development of a safe 
and effective malaria vaccine regime. The only benefits for participants would be 
information about their general health status. 

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5 
OBJECTIVES AND ENDPOINTS 
The number of volunteers has been chosen to generate adequate safety and 
immunogenicity data to meet these objectives, whilst minimising the number of volunteers 
exposed to a new vaccination regimen.  
 
5.1 
Primary Objective 
To assess the safety and tolerability of R21 with and without adjuvant Matrix-M1 in healthy 
volunteers.  
 
5.1.1 Primary Outcome Measures 
The specific endpoints for safety and reactogenicity will be actively and passively collected 
data on adverse events.  
 
 
The following parameters will be assessed for all study groups 
 Occurrence of solicited local reactogenicity signs and symptoms for 7 days 
following the vaccination 
 Occurrence of solicited systemic reactogenicity signs and symptoms for 7 days 
following the vaccination 
 
Occurrence of unsolicited adverse events for 28 days following the vaccination 
 Change from baseline for safety laboratory measures  
 Occurrence of serious adverse events during the whole study duration 
 
Volunteers will undergo clinical follow up for adverse events for a further 182 days 
following completion of the vaccination regimen. The duration of follow up reflects the 
desire to obtain longer term safety data with the first use of R21 in humans. 
5.2 
Secondary Objective 
To assess the cellular and humoral immunogenicity of R21 in humans with and without 
adjuvant Matrix-M1 in healthy volunteers. 
 
5.2.1 Secondary Outcome Measures 
Comparison of immunogenicity of the Matrix-M1 – adjuvanted vaccination regimens, 
versus R21 alone.  
 
Measures of immunogenicity may include: 
 
 ELISA to quantify antibodies to CSP and NANP 

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 ELISPOT to enumerate IFN-γ producing T cells 
 Ex vivo ELISPOT responses to NANP 
 Flow cytometry and intracellular cytokine staining to enumerate and functionally 
characterise immune cell populations such as; T cells (e.g. CD4+ and CD8+), B 
cells and dendritic cells 
 ELISPOT for enumeration of antibody-secreting cells (e.g. B cell ELISPOT 
responses to NANP) 
 Gene expression profiling including RNA analysis, DNA sequencing and other 
genotypic methods 
 
The immunoassay of most interest is the antibody response to NANP because this 
correlates with vaccine efficacy after RTS,S/AS01 administration, and induction of antibody 
levels comparable to or greater than RTS,S/AS01 would suggest likely vaccine efficacy.  
 
Other exploratory immunology may be carried out in collaboration with other specialist 
laboratories, including laboratories outside of Europe. This would involve transfer of 
serum/plasma and/or peripheral blood mononuclear cells (PBMC), but samples would be 
anonymised. Volunteers will be consented for this. 
6 
INVESTIGATIONAL PRODUCTS 
 
The following vaccinations will be given in this study:  
 
1. R21 10g mixed with Matrix-M1 50g 
2. R21 50g given alone without adjuvant  
3. R21 50g mixed with Matrix-M1 50g 
4. R21 2g mixed with Matrix-M1 50g 
 
6.1 Formulation, Dose, Storage and Accountability of Investigational Medicinal Products 
 
6.1.1 Description of R21c 
R21c has been developed and produced at the Jenner Institute, University of Oxford. The 
R21 vaccine consists of recombinant HBsAg particles expressing the central repeat and the 
C-terminus of the circumsporozoite protein from Plasmodium falciparum strain NF54. R21 
is a biosimilar to RTS,S expressed in the yeast Pichia Pastoris. It is 14 amino acids smaller 
than the RTS fusion protein at the C-terminus of the CSP sequence, has an additional four 
amino acids (EPEA, called C-tag) at the C-terminus, and lacks the excess of HBsAg in RTS,S 
(See Figure 2).  
 

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To increase the efficiency of biomanufacture and likely reduce the final cost of goods of this 
potential vaccine a C-tag has been encoded at the C-terminus of R21. The C-tag is a four 
amino acid tag (E-P-E-A) located at the C-terminus of the protein to allow immunoaffinity 
purification of the R21. To permit affinity purification of proteins carrying this C-tag, a 
chromatography resin utilising a cross-linked camelid nanobody developed by the company 
BAC BV (Naarden, The Netherlands, now part of Thermo Fisher) specifically binds the tag. 
This affinity chromatography resin was used for the purification of R21.  
 
R21 will be used at a dose of 10g in Group 1, 50g in Groups 2 and 3 and 2g in Group 4. 
  
6.1.2 R21 formulation and packaging 
R21 is formulated in formulation buffer at a target concentration to allow the extraction of 
25µg from each vial. The drug product is filled into 2mL glass vials with a 13 mm grey 
bromobutyl rubber freeze-dry stopper (CE Marked, supplied by Adelphi Tubes) and a 13 
mm complete tear, clear lacquered aluminium seal. The nitrogen filled vials are supplied 
sterile.  The containers and closures are tested for compliance with defined specifications. 
The vials are made from Ph Eur Type 1 glass. 
 
6.1.3 R21 Storage and Handling  
Long term, R21 vaccine is stored frozen at a nominal temperature of -80°C. 
 
6.2.1 R21 mixed with Matrix-M1 
 
Matrix-M1 to be used in Group 1and 3 volunteers was manufactured in compliance with 
cGMP by Apoteket Produktion & Laboratorier AB (APL) Formvägen 5B, SE-903 03 Umeå, 
Sweden. It is supplied as a sterile 1mg/ml solution in 2ml glass vials. Matrix-M1 (85 parts 
Matrix A and 15 parts of Matrix C) is obtained by simply mixing Matrix A and C, followed by 
dilution in PBS to 1 mg/ml, filtration though filter 0.22 μm and filling into vials in a volume 
of 2 ml. Matrix M-1 is a slightly-opalescent non-viscous liquid. 
 
Matrix-M1 to be used in Group 4 volunteers was manufactured in compliance with cGMP 
by Apotek Produktion & Laboratorier AB (APL) Formvägen 5B, SE-903 03 Umeå, Sweden. It 
is supplied as a sterile 0.75 mg/ml solution in 3ml glass vials. Matrix-M1 (85 parts Matrix A 
and 15 parts of Matrix C) is obtained by simply mixing Matrix A and C, followed by dilution 
in PBS to 0.75 mg/ml, filtration though filter 0.22 μm and filling into vials in a volume of 2 
ml. Matrix M-1 is a colourless slightly-opalescent non-viscous liquid. 
 
 

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A mixture of R21 with Matrix M-1 50µg will be administered to volunteers in Groups 1, 3 
and 4. Matrix-M1 and R21 will be mixed at the bedside immediately prior to administration 
and will be administered intramuscularly within one hour of thawing of R21. 
 
6.2.2 Matrix-M1 formulation and packaging 
Matrix-M1 to be used in Group 1and 3 volunteers is formulated at a concentration of 1 
mg/mL in PBS. The drug product is filled into sterile brown glass vials.  
 
Matrix-M1 to be used in Group 4 volunteers is formulated at a concentration of 0.75 mg/mL 
in PBS. The drug product is filled into sterile brown glass vials.  
 
6.2.3 Matrix-M1 Storage and Handling  
Matrix-M1 is stored refrigerated at 2 to 8°C and protected from light. 
 
All movements of the vaccines and adjuvants will be documented.  Accountability, storage, 
shipment and handling of R21 and Matrix-M1 will be in accordance with relevant local SOPs 
and forms.  
6.3 
Administration of Investigational Medicinal Products 
On vaccination day, R21 will be allowed to thaw to room temperature. It will be administered 
within 1 hour of removal from the freezer, either alone, or mixed with Matrix-M1. The 
investigational products will be administered intramuscularly into the deltoid of the non-
dominant arm. All volunteers will be observed in the unit for 1 hour (±10 minutes) after 
vaccination.  During administration of the investigational products, Advanced Life Support 
drugs and resuscitation equipment will be immediately available for the management of 
anaphylaxis.  Vaccination will be performed and the IMPs handled according to the relevant 
SOPs. 
6.4 
Vaccine Supply 
R21 will be formulated and vialed under Good Manufacturing Practice conditions at the 
CBF, University of Oxford. At the CBF the vaccines will be certified and labelled for trial 
VAC053 by a Qualified Person (QP) before transfer to the clinical site.  
7 
RECRUITMENT AND WITHDRAWAL OF TRIAL VOLUNTEERS 
7.1 
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: 

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 In public places, including buses and trains, with the agreement of the 
owner/proprietor. 
 In newspapers or other literature for circulation. 
 On radio via announcements. 
 On a website or social media site operated by our group or with the agreement of 
the owner or operator (including on-line recruitment through our web-site).  
 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 or the Hammersmith 
NIHR/Wellcome Trust Imperial Clinical Research Facility (CRF), London. 
 On stalls or stands at exhibitions or fairs. 
 Via presentations (e.g. presentations at lectures or invited seminars). 
 Direct mail-out: This will involve obtaining names and addresses of adults via the 
most recent Electoral Roll. The contact details of individuals who have indicated that 
they do not wish to receive postal mail-shots would be removed prior to the 
investigators being given this information. The company providing this service is 
registered under the Data Protection Act 1998. Investigators would not be given 
dates of birth or ages of individuals but the list supplied would only contain names 
of those aged between 18-50 years (as per the inclusion criteria). 
 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. 
 Hammersmith NIHR Wellcome Trust Imperial CRF Database of Healthy Volunteers: 
We may contact individuals from this database who have previously expressed an 
interest in receiving information about future studies for which they may be eligible.  
7.2 
Informed consent 
All volunteers will sign and date the informed consent form before any study specific 
procedures are performed. 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 general principles 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 

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 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 
 The study involves research of an investigational vaccine 
 There is no direct benefit from participating 
 The volunteer’s GP will be contacted to corroborate their medical history 
 The volunteer’s blood samples taken as part of the study will be stored indefinitely 
and samples may be sent outside of the UK and Europe to laboratories in 
collaboration with the University of Oxford. These will be anonymised. 
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, they will sign and date two 
copies of the consent form, one for them to take away and keep, and one to be stored in 
the case report form (CRF) – this is a paper or electronic document used to collect data 
relating to a particular volunteer. These forms will also be signed and dated by the 
Investigator. 
7.3 
Inclusion and exclusion criteria 
This study will be conducted in healthy adults, who meet the following inclusion and 
exclusion criteria: 
 
7.3.1 Inclusion Criteria 
The volunteer must satisfy all the following criteria to be eligible for the study: 
 Healthy adults aged 18 to 50 years  
 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 
 For females only, willingness to practice continuous effective contraception (see 
below) during the study and a negative pregnancy test on the day(s) of screening 
and vaccination 
 Agreement to refrain from blood donation during the course of the study 
 Provide written informed consent 
 
7.3.2 Exclusion Criteria 
The volunteer may not enter the study if any of the following apply: 
 Participation in another research study involving receipt of an investigational 
product in the 30 days preceding enrolment, or planned use during the study period  

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 Prior receipt of an investigational vaccine likely to impact on interpretation of the 
trial data. 
 Administration of immunoglobulins and/or any blood products within the three 
months preceding the planned administration of the vaccine candidate 
 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 any component of 
the vaccine 
 Any history of anaphylaxis in relation to vaccination 
 Pregnancy, lactation or willingness/intention to become pregnant during the study 
 History of cancer (except basal cell carcinoma of the skin and cervical carcinoma in 
situ) 
 History of serious psychiatric condition likely to affect participation in the study 
 Any other serious chronic illness requiring hospital specialist supervision 
 Suspected or known current alcohol abuse as defined by an alcohol intake of greater 
than 42 units every week 
 Suspected or known injecting drug abuse in the 5 years preceding enrolment 
 Seropositive for hepatitis B surface antigen (HBsAg) 
 Seropositive for hepatitis C virus (antibodies to HCV)  
 History of clinical malaria (any species) 
 Travel to a malaria endemic region during the study period or within the previous six 
months 
 Any clinically significant abnormal finding on screening biochemistry or haematology 
blood tests or urinalysis 
 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 
 
7.3.3 Effective contraception for female volunteers 
Female volunteers are required to use an effective form of contraception during the course 
of the study. As this is a Phase I, first-in-human, study there is no information about the 
effect of these vaccines on a foetus. 

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Acceptable forms of contraception for female volunteers include: 
 Established use of oral, injected or implanted hormonal methods of contraception. 
 Placement of an intrauterine device (IUD) or intrauterine system (IUS). 
 Total abdominal hysterectomy 
 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). 
7.3.4 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). 
 
7.3.5 Re-vaccination exclusion criteria 
The following adverse events associated with vaccine immunisation constitute absolute 
contraindications to further administration of vaccine. If any of these events occur during 
the study, the subject must be withdrawn and followed until resolution of the event, as 
with any adverse event: 
 Anaphylactic reaction following administration of vaccine 
 Pregnancy 
The following adverse events constitute contraindications to administration of vaccine at 
that point in time; if any one of these adverse events occurs at the time scheduled for 
vaccination, the subject may be vaccinated at a later date, or withdrawn at the discretion of 
the Investigator. The subject must be followed until resolution of the event as with any 
adverse event: 
 Acute disease at the time of vaccination. (Acute disease is defined as the presence 
of a moderate or severe illness with or without fever). All vaccines can be 
administered to persons with a minor illness such as diarrhoea, mild upper 
respiratory infection with or without low-grade febrile illness, i.e. temperature of 
≤37.5°C/99.5°F. 
 Temperature of >37.5°C (99.5°F) at the time of vaccination. 
7.3.6 Withdrawal of Volunteers 
In accordance with the principles of the current revision of the Declaration of Helsinki and 
any other applicable regulations, a volunteer has the right to withdraw from the study at 

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any time and for any reason, and is not obliged to give his or her reasons for doing so. The 
Investigator may withdraw the volunteer at any time in the interests of the volunteer’s 
health and well-being. In addition the volunteer may withdraw/be withdrawn 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. 
 An AE, which requires discontinuation of the study involvement or results in inability 
to continue to comply with study procedures. 
The reason for withdrawal will be recorded in the CRF. If withdrawal is due to an AE, 
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 is withdrawn from the study may be replaced, if that is 
possible within the specified time frame. The Local Safety Monitor (LSM) may recommend 
withdrawal of volunteers.  
Any volunteer who fails to attend for two or more follow-up visits during the study will be 
deemed to have withdrawn from the study. 
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.  
In all cases of subject withdrawal, excepting those of complete consent withdrawal, long-
term safety data collection, including some procedures such as safety bloods, will continue 
as appropriate if subjects have received one or more vaccine doses.  
7.4 
Compliance with Dosing Regime 
All doses in this vaccine study will be administered by the Investigator and recorded in the 
CRF. The study medication will be at no time in the possession of the volunteer and 
compliance will not, therefore, be an issue. 
7.5 
Pregnancy 
Should a volunteer become pregnant during the trial, she will be followed up as other 
volunteers and in addition will be followed until pregnancy outcome. We will not routinely 
perform venepuncture in a pregnant volunteer. 
7.6 
Safety Stopping/ Holding Rules 
Safety holding rules have been developed considering the fact that this is a first-in-human 
dose escalation study.  
 

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‘Solicited adverse events’ are those listed as foreseeable adverse events in section 9.3 of 
the protocol, occurring within the first 7 days after vaccination (day of vaccination and six 
subsequent days). ‘Unsolicited adverse events’ are adverse events other than the 
foreseeable AEs occurring within the first 7 days, or any AEs occurring after the first 7 days 
after vaccination. 
 
7.6.1 Group holding rules 
For safety reasons the first volunteer to receive a new vaccine dose in Groups 1-3 will be 
vaccinated alone and we will wait 72 hours before vaccinating subsequent volunteers. Two 
further volunteers may be vaccinated 72 hours after the first, and then at least another 72 
hours gap will be left before vaccinating the rest of the volunteers receiving that dose of 
vaccine. 
 
Solicited local adverse events: 
 
If more than 25% of doses of a vaccine are followed by Grade 3 solicited local 
adverse event beginning within 2 days after vaccination (day of vaccination and 
one subsequent day) and persisting at Grade 3 for >48 hrs. 
 
Solicited systemic adverse events:  
 
If more than 25% of doses of a vaccine are followed by Grade 3 solicited systemic 
adverse event beginning within 2 days after vaccination (day of vaccination and 
one subsequent day) and persisting at Grade 3 for >48hrs.  
 
Unsolicited adverse events: 
 
If more than 25% of volunteers develop a Grade 3 unsolicited adverse event 
(including the same laboratory adverse event) that is considered possibly, probably 
or definitely related to vaccination and persists at Grade 3 for > 48hrs.  
 A serious adverse event considered possibly, probably or definitely related to 
vaccination occurs 
 Death occurs 
 A life-threatening reaction occurs 
If a holding rule has been met and following an internal safety review it is deemed 
appropriate to restart dosing, a request to restart dosing with pertinent data must be 
submitted to the regulatory authority as a request for a substantial amendment. The 
internal safety review will consider:  
 The relationship of the AE or SAE to the vaccine. 
 The relationship of the AE or SAE to the vaccine dose, or other possible causes of 
the event. 

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 If appropriate, additional screening or laboratory testing for other volunteers to 
identify those who may develop similar symptoms and alterations to the current 
Participant Information Sheet (PIS) are discussed. 
 New, relevant safety information from ongoing research programs on the 
various components of the vaccine. 
The local ethics committee and vaccine manufacturers will also be notified if a holding rule 
is activated or released.  
All vaccinated volunteers will be followed for safety until resolution or stabilisation (if 
determined to be chronic sequelae) of their AEs. 
 
7.6.2 Individual stopping rules (will apply to all vaccinated individuals) 
In addition to the above stated group holding rules, stopping rules for individual volunteers 
will apply (i.e., indications to withdraw individuals from further vaccinations). If any of the 
events listed below occur and are considered possibly, probably or definitely related to 
vaccination the volunteer will be withdrawn from further vaccination.  
 
Local reactions: Injection site ulceration, abscess or necrosis 
 
Laboratory AEs: 
 
the volunteer develops a Grade 3 laboratory adverse event considered possibly, 
probably or definitely related within 7 days after vaccination and persisting 
continuously at Grade 3 for > 72hrs. 
 
Systemic solicited adverse events:  
 
the volunteer develops a Grade 3 systemic solicited adverse event considered 
possibly, probably or definitely related within 2 days after vaccination (day of 
vaccination and one subsequent day) and persisting continuously at Grade 3 for > 
72hrs. 
 
Unsolicited adverse events: 
 
the volunteer has a Grade 3 adverse event, persisting continuously at Grade 3 for 
>72hrs. 
 
the volunteer has a serious adverse event. 
 
the volunteer has an acute allergic reaction or anaphylactic shock following the 
administration of vaccine investigational product. 
If a volunteer has an acute illness (moderate or severe illness with or without fever) or a 
fever (oral temperature greater than 37.5°C) at the scheduled time of administration of 
investigational product, the volunteer will not receive the vaccine at that time. The vaccine 
may be administered to that volunteer at a later date within the time window specified in 
the protocol (see Table 6) or they may be withdrawn from the study at the discretion of the 
Investigator.  

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All vaccinated volunteers will be followed for safety until the end of their planned 
participation in the study or until resolution or stabilisation (if determined to be chronic 
sequelae) of their AEs, providing they consent to this.  
In addition to these pre-defined criteria, the study can be put on hold upon advice of the 
Local Safety Monitor, Chief Investigator, Study Sponsor, regulatory authority, Ethical 
Committee(s) or Local Safety Committee, for any single event or combination of multiple 
events which, in their professional opinion, jeopardise the safety of the volunteers or the 
reliability of the data. 
8 
TREATMENT OF TRIAL VOLUNTEERS 
This section describes the clinical procedures for evaluating study participants and follow-
up after administration of study vaccine.  
8.1 
Study procedures 
Procedures will be performed on the visit time points indicated in the schedules of 
attendance (Table 6). All volunteers will have the same schedule of clinic attendances and 
procedures, except the first three volunteers in Groups 1-3 will have an extra visit at three 
days post Vaccination 1 for additional safety assessment. All subjects will receive three 
vaccinations, four weeks apart, and undergo follow-up for a total of 34 weeks (26 weeks 
following the final vaccination). The total volume of blood donated during the study will be 
620ml. Additional visits or procedures may be performed at the discretion of the 
investigators, e.g., further medical history and physical examination, or urine microscopy in 
the event of positive urinalysis. 
8.2 
Observations 
Pulse, blood pressure and temperature will be measured at the time-points indicated in the 
schedule of procedures and may also be measured as part of a physical examination if 
indicated at other time-points. 
8.3 
Blood Tests 
Blood will be drawn for the following laboratory tests and processed: 
1. At Oxford University Hospitals’ NHS Trust, or Hammersmith Hospital using NHS 
standard procedures: 
 Haematology; Full Blood Count  
 Biochemistry; Sodium, Potassium, Urea, Creatinine, Albumin, Liver Function 
Tests  
 Diagnostic serology; HBsAg, HCV antibodies, HIV antibodies (specific consent 
will be gained prior to testing blood for these blood-borne viruses) 
 Immunology; Human Leukocyte Antigen (HLA) typing 
2. At University of Oxford research laboratories: 

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 Exploratory Immunology; Immunogenicity will be assessed by a variety of 
immunological assays. This includes antibodies to CSP and NANP, ex vivo ELISpot 
assays for interferon gamma and flow cytometry assays, functional antibody 
assays and B cell analyses. Other exploratory immunological assays including 
cytokine analysis, other antibody assays, DNA analysis of genetic polymorphisms 
potentially relevant to vaccine immunogenicity and gene expression studies 
amongst others may be performed at the discretion of the Investigators. 
3. Urinalysis; Urine will be tested for protein, blood and glucose at screening. For 
female volunteers only, urine will be tested for beta-human chorionic 
gonadotrophin (β-HCG) at screening and immediately prior to each vaccination. 
4. Collaboration with other specialist laboratories in the UK, Europe and outside of 
Europe for further exploratory immunological tests may occur. This would involve 
the transfer of serum or plasma and/or PBMC to these laboratories, but these would 
remain anonymised. Informed consent for this will be gained from volunteers. 
Immunological assays will be conducted according to the procedures established in the test 
laboratories. With the volunteers’ informed consent, any leftover cells and serum/plasma 
will be frozen indefinitely for future immunological analysis of malaria-specific or vaccine-
related responses. This may include human DNA and RNA analysis to search for correlates 
of vaccine immunogenicity and efficacy.  
8.4 
Vaccinations  
Before each vaccination, the on-going eligibility of the volunteer will be reviewed. All 
vaccines will be administered intramuscularly according to SOP VC002 Vaccination as 
described in Section 6.3. The injection site will be covered with a sterile dressing and the 
volunteer will stay in the CCVTM for observation, in case of immediate adverse events. 
Observations will be taken 30 minutes after vaccination (+/- 5 minutes) and the sterile 
dressing removed and injection site inspected. Observations will also be taken at 60 
minutes (+/- 10 minutes) before the volunteer leaves. An oral thermometer, tape measure 
and diary card (paper or electronic) will be given to each volunteer, with instructions on 
use, along with the emergency 24 hour telephone number to contact the on-call study 
physician if needed. 
 
Diary cards will collect information on the timing and severity of the following solicited AEs: 
Local solicited AEs 
Systemic solicited AEs 
Pain 
Fever 
Redness 
Feverishness/Chills 
Warmth 
Joint pains 
 Itch 
Muscle pains 
 
Fatigue 

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Headache 
 
Malaise 
Table 5: Solicited AEs as collected on post vaccination diary cards 
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. 
8.5 
Study visits 
The study visits and procedures will be undertaken by one of the clinical trials team. The 
procedures to be included in each visit are documented in the schedule of attendances. 
Each visit is assigned a time-point and a window period, within which the visit will be 
conducted. 
 
8.5.1 Screening visit 
All potential volunteers will have a screening visit, which may take place up to 90 days prior 
to vaccination. Informed consent will be taken before screening, as described in section 7.2. 
If consent is obtained, the screening procedures indicated in the schedule of attendances 
will be undertaken. To avoid unnecessary additional venepuncture, if the appropriate blood 
test results for screening are available for the same volunteer from a screening visit for 
another Jenner Institute Clinical Trials group vaccine study, these results may be used for 
assessing eligibility (provided the results date is within the 3 months preceding enrolment 
in VAC053). 
The subject’s general practitioner will be contacted with the written permission of the 
subject after satisfactory screening as notification that the subject has volunteered for the 
study and to ascertain any significant medical history. 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 
(www.tops.org.uk). 
Potential subjects will be informed that there may be leftover samples of their blood (after 
all testing for this study is completed), and that such samples may be stored indefinitely for 
possible future research (exploratory immunology), including genotypic testing of genetic 
polymorphisms potentially relevant to vaccine immunogenicity. Subjects will be able to 
decide if they will permit such future use of any leftover samples. 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. 
Abnormal clinical findings from the medical history, physical examination, urinalysis or 
blood tests at screening will be assessed as detailed in Appendix A. Abnormal blood tests 
following enrolment will be assessed according to site-specific laboratory adverse event 

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grading tables which are filed in the trial master file (TMF). 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. 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. 
 
8.5.2 Day 0: Enrolment and vaccination visit 
The eligibility of the volunteer will be reviewed at the end of the screening visit and again 
when all results from the screening visit have been considered. If eligible, a day 0 visit will 
be scheduled for the volunteer to receive the vaccine. Volunteers will not be considered 
enrolled in the study until they have received a vaccine. The vaccine will be administered as 
described above in sections 6.3 and 8.4. 
 
8.5.3 Sequence of Enrolment and Vaccination of Volunteers 
The first Group 1 volunteer to receive Vaccination 1 will be vaccinated alone. If there are no 
safety concerns following review of this volunteer at 3 days post-vaccination, a further two 
Group 1 volunteers may receive Vaccination 1.  If there are no safety concerns following 
review of these volunteers at 3 days post-vaccination as assessed by the CI and LSM, the 
remaining seven Group 1 volunteers and the first volunteer in Group 3 may receive 
Vaccination 1.  
 
The first Group 3 volunteer to receive Vaccination 1 will be vaccinated alone. If there are no 
safety concerns following review of this volunteer at 3 days post-vaccination, a further two 
Group 3 volunteers may receive Vaccination 1.  If there are no safety concerns following 
review of these volunteers at 3 days post-vaccination as assessed by the CI and LSM, the 
remaining seven Group 3 volunteers will receive Vaccination 1. In parallel, following review of 
the first Group 3 volunteer at 3 days post-vaccination, the first Group 2 volunteer to receive 
Vaccination 1 will be vaccinated alone. If there are no safety concerns following review of this 
volunteer at 3 days post-vaccination, a further two Group 2 volunteers will receive Vaccination 
1.  If there are no safety concerns following review of these volunteers at 3 days post-
vaccination as assessed by the CI and LSM, the remaining Group 2 volunteer will receive 
Vaccination 1. There will be no staggered enrolment of volunteers in Group 4 as R21 has 
already been previously administered to humans in much higher doses (10g in Group1 and 
50g in Group 3). 
 
8.5.4 Subsequent Visits: Day 1, 7, 14, 29, 35, 42, 57, 63, 70, 84, 238 
On subsequent visits, the volunteers will be assessed for local and systemic adverse events, 
using diary cards (paper or electronic), interim history, physical examination and blood tests 

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at the time-points indicated in the schedule of attendances (Table 6). Blood will also be 
taken for exploratory immunology analysis. 
 
8.5.5 First three volunteers (Groups 1-3) 
The first three volunteers in Groups 1-3 will be required to attend an additional visit at Day 
3 post-vaccination 1 to assess for local and systemic adverse events. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Table 6. Schedule of attendances for Groups 1-4. S = screening visit; (X) = if considered 
necessary ^ = Vital signs includes pulse, blood pressure and temperature; $ = Biochemistry 
will include Sodium, Potassium, Urea, Creatinine, Albumin and Liver function tests. £ = 
Attendance 
Number 
1S 
2 
3 3a*
4 
5 
6 
7 
8 
9 
10 
11 
12 
13 
14 
15 
Timeline** 
(days) 
≤ 
90
0 
1 
3 
7 
14 
28 
29 
35 
42 
56 
57 
63 
70 
84 
238 
(weeks) 
 
0 
 
 
1 
2 
4 
 
5 
6 
8 
 
9 
10 
12 
34 
Time window 
(days) 
 
 
+1 +1 
±2 
±3 
±2 
+1 
±2 
±3 
±2 
+1 
±2 
±3 
±2 
±14 
Informed 
Consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Review 
contraindications,
inclusion and 
exclusion criteria 
X 
X 
 
 
 
 
X 
 
 
 
X 
 
 
 
 
 
Vaccination 
 
X 
 
 
 
 
X 
 
 
 
X 
 
 
 
 
 
Vital signs^ 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
(X) 
Ascertainment of 
adverse events 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
(X) 
Diary cards 
provided 
 
X 
 
 
 
 
X 
 
 
 
X 
 
 
 
 
 
Diary cards 
collected 
 
 
 
 
 
 
X 
 
 
 
X 
 
 
 
X 
 
Medical History, 
Physical 
Examination 
X 
(X) (X) (X) 
(X) 
(X) 
(X) 
 
(X) 
 
(X) 
 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
Biochemistry$, 
Haematology (ml) 5 
5 
 
 
5 
 
5 
 
5 
 
5 
 
5 
 
5 
 
Exploratory 
immunology£ (ml)
 
50 
 
 
50 
50 
50 
 
50 
50 
50 
 
50 
50 
50 
50 
Gene expression 
profiling (ml)  
 
3 
3 
 
3 
 
 
3 
3 
 
 
3 
3 
 
 
 
Urinalysis  
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Urinary HCG 
(women only) 
X 
X 
 
 
 
 
X 
 
 
 
X 
 
 
 
 
 
HLA typing (ml) 
 
4 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
HBsAg, HCV Ab, 
HIV serology (ml) 5 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Blood volume per 
visit 
10
62 
3 
0 
58 
50 
55 
3 
58 
50 
55 
3 
58 
50 
55 
50 
Cumulative blood 
volume 
10
72 
75 75 133 183 238 241 299 349 404 407 465 515 570 620% 

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Exploratory immunology includes antibodies to CSP and NANP, B cell ELISPOT to NANP, ex 
vivo ELISPOT responses to interferon- 
*The first three volunteers in Groups 1-3 will be reviewed at 3 days post-vaccination 1. 
**  Timeline is approximate only. Exact timings of visits relate to the day on enrolment, ie, 
each visit must occur at indicated number of days after enrollment  time window. 
% Cumulative blood volume for Oxford volunteers if blood taken as per schedule, and 
excluding any repeat safety blood test that may be necessary. Volunteers at other sites may 
have a slightly higher cumulative volume. 

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9 
ASSESSMENT OF SAFETY 
Safety will be assessed by the frequency, incidence and nature of adverse events and 
serious adverse events arising during the study. 
9.1 
Interim Safety Review 
Prior to dose escalation of each vaccine the local safety monitor will be consulted to 
provide a review of safety data and adverse events in volunteers before proceeding to the 
next vaccine dose. Interim safety data may also be made available to manufacturers (in 
coded format) as specified in the contract with the manufacturer(s). 
9.2 
Definitions 
9.2.1 Adverse Event (AE) 
An AE is any untoward medical occurrence in a volunteer, which may occur during or after 
administration of an Investigational Medicinal Product (IMP) 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. 
9.2.2 Adverse Reaction (AR) 
An AR is any untoward or unintended response to an IMP. This means that a causal 
relationship between the IMP and an AE is at least a reasonable possibility, i.e., the 
relationship cannot be ruled out. All cases judged by the reporting medical Investigator as 
having a reasonable suspected causal relationship to an IMP (i.e. possibly, probably or 
definitely related to an IMP) will qualify as adverse reactions. 
9.2.3 Unexpected Adverse Reaction  
An adverse reaction, the nature or severity of which is not consistent with the applicable 
product information (e.g., IB for an unapproved IMP). 
9.2.4 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  
 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 severe 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). 
 Hospitalisation, regardless of length of stay, even if it is a precautionary measure for 
continued 
observation. 
Hospitalisation 
(including 
inpatient 
or 
outpatient 

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hospitalisation for an elective procedure) for a pre-existing condition that has not 
worsened unexpectedly does not constitute a serious AE. 
 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. Examples of such medical events include allergic 
reaction requiring intensive treatment in an emergency room or clinic, blood 
dyscrasias, or convulsions that do not result in inpatient hospitalisation. 
 Congenital anomaly or birth defect. 
9.2.5 Serious Adverse Reaction (SAR) 
An adverse event (expected or unexpected) that is both serious and, in the opinion of the 
reporting Investigator or Sponsors, believed to be possibly, probably or definitely due to an 
IMP or any other study treatments, based on the information provided. 
9.2.6 Suspected Unexpected Serious Adverse Reaction (SUSAR) 
A serious adverse reaction, the nature and severity of which is not consistent with the 
information about the medicinal product in question set out in the IB or Summary of 
Product Characteristics (SmPC). 
9.3 
Foreseeable Adverse Reactions:  
The foreseeable ARs following vaccination with R21 and Matrix-M1 include injection site 
pain, erythema, warmth, swelling, pruritus, myalgia, arthralgia, headache, fatigue, fever, 
feverishness, malaise and nausea.   
9.4 
Expected Serious Adverse Events 
No serious adverse events are expected in this study. 
9.5 
Causality Assessment 
For every unsolicited AE, an assessment of the relationship of the event to the 
administration of the vaccine will be undertaken. An intervention-related AE refers to an AE 
for which there is a probable or definite relationship to administration of a vaccine. An 
interpretation of the causal relationship of the intervention to the AE in question will be 
made, based on the type of event; the relationship of the event to the time of vaccine 
administration; and the known biology of the vaccine therapy (Table 7). 
 
 
 
 
 

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0 
No 
Relationship 
No temporal relationship to study product and 
Alternate aetiology (clinical state, environmental or other 
interventions); and 
Does not follow known pattern of response to study product 
1 
Unlikely 
Unlikely temporal relationship to study product and 
Alternate aetiology likely (clinical state, environmental or 
other interventions) and 
Does not follow known typical or plausible pattern of 
response to study product. 
2 
Possible 
 
Reasonable temporal relationship to study product; or 
Event not readily produced by clinical state, environmental 
or other interventions; or 
Similar pattern of response to that seen with other vaccines 
3 
Probable 
 
Reasonable temporal relationship to study product; and 
Event not readily produced by clinical state, environment, or 
other interventions or  
Known pattern of response seen with other vaccines 
4 
Definite 
 
Reasonable temporal relationship to study product; and 
Event not readily produced by clinical state, environment, or 
other interventions; and  
Known pattern of response seen with other vaccines 
Table 7. Guidelines for assessing the relationship of vaccine administration to an AE. 
 
9.6 
Reporting Procedures for All Adverse Events (see SOP VC027) 
All AEs occurring in the 28 days following each vaccination observed by the Investigator or 
reported by the volunteer, whether or not attributed to study medication, will be recorded. 
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, 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.  
 
9.6.1 Reporting Procedures for Serious AEs (see SOP OVC005 Safety Reporting) 
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 on the SAE forms to members of the study team 
immediately the Investigators become aware of their occurrence, as described in SOP 

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OVC005. Copies of all reports will be forwarded for review to the Chief Investigator (as the 
Sponsor’s representative) within 24 hours of the Investigator being aware of the suspected 
SAE. The local safety committee (LSC) will be notified of SAEs that are deemed possibly, 
probably or definitely related to study interventions; the LSC will be notified immediately 
(within 24 hours) of the Investigators’ being aware of their occurrence. SAEs will not 
normally be reported immediately 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 LSC. In 
addition to the expedited reporting above, the Investigator shall include all SAEs in the 
annual Development Safety Update Report (DSUR) report. 
9.6.2 Reporting Procedures for SUSARS 
The Chief Investigator will report all SUSARs to the MHRA and ethical committee(s) within 
required timelines (15 days for all SUSARs, unless life threatening in which case 7 days, with 
a final report within a further 8 days (total 15). 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. 
9.6.3 Development Safety Update Report 
A Development Safety Update Report (DSUR) will be submitted by the Sponsor to the 
competent authority and ethical committee on the anniversary of the first approval date 
from the regulatory authority for each IMP. 
9.7 
Assessment of severity 
The severity of clinical and laboratory adverse events will be assessed according to the 
scales in Tables 8-10. 
 
Adverse Event  
Grade  
Intensity  
Pain at injection site 
1 
Pain that is easily tolerated 
2 
Pain that interferes with daily activity 
3 
Pain that prevents daily activity 
Erythema 
at 
injection 
site* 
1 
>3 - ≤50 mm 
2 
>50 - ≤100 mm 
3 
>100 mm 
Swelling at injection site 
1 
>1 - ≤20 mm 

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Table 8. Severity grading criteria for local adverse events. 
 *erythema ≤3mm is an expected consequence of skin puncture and will therefore 
not be considered an adverse event. 
 
Grade 1  
(mild) 
Grade 2  
(moderate) 
Grade 3  
(severe) 
Fever (oral) 
37.6°C - 38.0°C 
38.1°C – 39.0°C 
>39.0°C 
Tachycardia (bpm)* 
101 - 115 
116 – 130 
>130 
Bradycardia 
(bpm)** 
50 – 54 
40 – 49 
<40 
Systolic 
hypertension 
(mmHg) 
141 - 159 
160 – 179 
≥180 
Diastolic 
hypertension 
(mmHg) 
91 - 99 
100 – 109 
≥110 
Systolic 
hypotension 
(mmHg)*** 
85 - 89 
80 – 84 
<80 
Table 9. Severity grading criteria for physical observations 
*Taken after ≥10 minutes at rest 
**When resting heart rate is between 60 – 100 beats per minute. Use clinical judgement 
when characterising bradycardia among some healthy subject populations, for example, 
conditioned athletes. 
***Only if symptomatic (e.g. dizzy/ light-headed) 
 
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; 
medical intervention/therapy required, hospitalisation possible 
Table 10. Severity grading criteria for local and systemic AEs. 
2 
>20 - ≤50 mm 
3 
>50 mm 

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9.8 
Procedures to be followed in the event of abnormal findings 
Eligibility for enrolment in the trial in terms of laboratory findings will be assessed as 
detailed in Appendix A. Abnormal clinical findings from medical history, examination or 
blood tests will be assessed as to their clinical significance throughout the trial. Laboratory 
adverse events will be assessed using the site-specific tables in the TMF. 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 and 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. 
9.9 
Local Safety Committee 
A Local Safety Committee (LSC) will be appointed to provide real-time safety oversight. The 
LSC will review SAEs deemed possibly, probably or definitely related to study interventions. 
The LSC will be notified within 24 hours of the Investigators’ being aware of their 
occurrence. The LSC has the power to place the study on hold if deemed necessary 
following a study intervention-related SAE. At the time of writing the LSC will be chaired by 
Dr Brian Angus, a Clinical Tutor in Medicine, Honorary Consultant Physician and Director, 
Centre for Tropical Medicine at the University of Oxford. There will be a minimum of two 
other appropriately qualified committee members. All correspondence between 
Investigator and LSC will be conveyed by the Investigator to the trial Sponsor. 
The chair of the LSC 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. 
 
9.9.1 Safety Profile Review 
The safety profile will be assessed on an on-going basis by the Investigators. The LSM will 
perform independent external safety reviews prior to dose escalations. The Chief 
investigator, Principal Investigator, and relevant Investigators (as per the trial delegation 
log) will also review safety issues and SAEs as they arise.   

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10 
 
STATISTICS 
This is an observational and descriptive safety study, where volunteers will be vaccinated 
with R21 alone or in combination with adjuvant Matrx-M1. 34 volunteers will be vaccinated 
in total - 10 with 10g R21 adjuvanted with 50g Matrix-M1, 4 with 50g R21 alone, 10 
with 50g R21 adjuvanted with 50g Matrix-M1 and a further 10 with 2g R21 adjuvanted 
with 50g Matrix-M1. This sample size should allow an initial estimation to be made of the 
frequency and magnitude of outcome measures, rather than aiming to obtain statistical 
significance for differences between groups.  
 
Sample Size Selection 
This is a descriptive phase I first in human trial that will balance the safety of volunteers 
with the aims to assess the vaccine’s safety profile and immunogenicity after selected doses 
of the vaccines. The primary dose comparison will be between Groups 1, 3 and 4, which will 
each have 10 subjects. Group 2 is a smaller group because we anticipate that the 
immunogenicity of the R21 without adjuvant is very likely indeed to be considerably lower 
than R21 with adjuvant, based on both pre-clinical data and general performance of non-
adjuvanted protein-based vaccines. This should be demonstrable with just four vaccinees 
being administered vaccine without adjuvant. CSP-specific immunogenicity will be the key 
immunological readout assessed by a variety of immunological assays.  
 
 
 
 
 
 
 
 
 
 
 

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11  
QUALITY CONTROL AND QUALITY ASSURANCE PROCEDURES 
 
11.1 
Investigator procedures 
Approved site-specific standard operating procedures (SOPs) will be used at all clinical and 
laboratory sites. 
11.2 
Monitoring 
Monitoring will be performed according to ICH GCP by Clinical Trials Research Governance 
(CTRG). Following written SOPs, the monitors will verify that the clinical trial is conducted 
and data are generated, documented and reported in compliance with the protocol, GCP 
and the applicable regulatory requirements. The Investigator sites will provide direct access 
to all trial related source data/documents and reports for the purpose of monitoring and 
auditing by the Sponsor and inspection by local and regulatory authorities. 
11.3 
Modification to protocol 
No substantial amendments to this protocol will be made without consultation with, and 
agreement of, the Sponsor. Any substantial 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 and regulatory approval. 
The Investigator is responsible for ensuring that changes to an approved trial, during the 
period for which regulatory and ethical committee(s) approval has already been given, are 
not initiated without regulatory and ethical committee(s)’ review and approval except to 
eliminate apparent immediate hazards to the subject. 
11.4 
Protocol deviation  
Any deviations from the protocol will be documented in a protocol deviation form and filed 
in the trial master file.  
11.5 
Audit & inspection  
The QA manager will perform internal audits to check that the trial is being conducted; data 
recorded, analysed and accurately reported according to the protocol, Sponsor’s SOPs and 
in compliance with ICH GCP. The audits will also include laboratory activities according to an 
agreed audit schedule. The internal audits will supplement the external monitoring process 
and will review processes not covered by the external monitor. 
 
The Sponsor, trial sites, and ethical committee(s) may carry out audit to ensure compliance 
with the protocol, GCP and appropriate regulations. GCP inspections may also be 

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undertaken by the MHRA to ensure compliance with protocol and the Medicines for Human 
Use (Clinical Trials) Regulations 2004. The Sponsor will assist in any inspections and will 
formally respond to the MHRA as part of the inspection procedure. 
 
11.6 
Serious Breaches 
The Medicines for Human Use (Clinical Trials) Regulations contain a requirement for the 
notification of "serious breaches" to the MHRA within 7 days of the Sponsor becoming 
aware of the breach.  
A serious breach is defined as “A breach of GCP or the trial protocol which is likely to effect 
to a significant degree  
(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 within one 
working day. 
11.7 
Trial Progress 
The progress of the trial will be overseen by the Chief Investigator.  
11.8 
Publication Policy 
The Investigators will be involved in reviewing drafts of the manuscripts, abstracts, press 
releases and any other publications arising from the study. Data from the study may also be 
used as part of a thesis for a PhD or MD. 

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12 
ETHICS 
12.1 
Declaration of Helsinki 
The Investigators will ensure that this study is conducted according to the principles of the 
current revision of the Declaration of Helsinki. 
12.2 
ICH Guidelines for Good Clinical Practice 
The Investigators will ensure that this study is conducted in full conformity with the ICH 
Good Clinical Practice (GCP), the requirements of the Medicines for Human Use (Clinical 
Trial) Regulations 2004, and local regulatory requirements. 
12.3 
Informed Consent 
Written, informed consent will be obtained, as described in section 6.2 
12.4 
Research Ethics Committee (REC) 
A copy of the protocol, proposed informed consent form, other written volunteer 
information and the proposed advertising material will be submitted to a REC for written 
approval. The Chief Investigator will submit and, where necessary, obtain approval from the 
REC for all subsequent substantial amendments to the protocol and informed consent 
document.  
12.5 
Volunteer Confidentiality 
All data will be anonymised: volunteer data will be identified by a unique study number in 
the CRF and database. A separate confidential file containing identifiable information will 
be stored in a secured location in accordance with the Data Protection Act 1998. Only the 
Sponsor representative, Investigators, the clinical monitor, the REC and the MHRA will have 
access to the records. Photographs taken of vaccination sites (if required, with the 
volunteer’s written, informed consent) will not include the volunteer’s face and will be 
identified by the date, trial code and subject’s unique identifier. 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. 

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13  
DATA HANDLING AND RECORD KEEPING 
13.1 
Data Handling 
The Chief Investigator will be the data manager with responsibility for receiving, entering, 
cleaning, querying, analysing and storing all data that accrues from the study. The data will 
be entered into the volunteers’ CRFs in a paper and/or electronic format (using 
OpenClinica™ database). Electronic data will be stored on secure servers which are 
outsourced by OpenClinica™. Data will be entered in a web browser on PCs in the CCVTM 
building and then transferred to the OpenClinica Database by encrypted (Https) transfer. 
OpenClinica™ meets FDA part 11B standards. This includes safety data, laboratory data 
(both clinical and immunological) and outcome data. 
13.2 
Record Keeping 
The Investigators will maintain appropriate medical and research records 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 and 
clinical research nurses will have access to records. The Investigators will permit authorised 
representatives of the Sponsor(s), as well as ethical and regulatory agencies 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. 
13.3 
Source Data and Case Report Forms (CRFs) 
All protocol-required information will be collected in CRFs designed by the Investigator. All 
source documents will be filed in the CRF. Source documents are original documents, data, 
and records from which the volunteer’s CRF data are obtained. For this study, these will 
include, but are not limited to, volunteer consent form, blood results, GP response letters, 
laboratory records, diaries, and correspondence. In the majority of cases, CRF entries will 
be considered source data as the CRF is 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 vaccinations. All source data 
and volunteer CRFs will be stored securely.  
13.4 
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. 

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14 
 
FINANCING AND INSURANCE 
14.1  Financing 
The study is funded through MultiMalVax, an EC FP7 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. 
14.2  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). 
14.3  Compensation 
Volunteers will be compensated for their time and for the inconvenience caused by 
procedures. They will be compensated £25 for attending the screening visit. For all other 
trial visits as outlined in Table 6, compensation will be calculated according to the following: 
 Travel expenses: 
o £10 per visit. Where travel expenses are greater than £10 per visit because 
the volunteer lives outside the city of the trial site, the volunteer will be 
given further reimbursement to meet the cost of travel necessary for study 
visits. 
 Inconvenience of blood tests: 
o £10 per blood donation 
 Time required for visit:  
o £20 per hour 
 
 
 
 
 
 
 
 
 
 

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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 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 site-
specific laboratory adverse event tables (stored in TMF). 
 
Urinalysis at screening will be assessed as per the table below: 
URINE ANALYSIS (using MULTISTIX) 
Protein* 
2+ or Protein creatinine ratio of ≥50mg/mmol 
Blood£ 
2+ on two dipstick tests  
Glucose 
1+ 
*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 in 
volunteers 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, they will be excluded from the trial, and the 
appropriate follow-up arranged.