A Phase Ia Study to Assess Safety and Immunogenicity of the Plasmodium Falciparum Malaria Vaccine Candidate Pfs48/45 in Matrix-M Adjuvant in Healthy Adults Living in the UK
Phase: EARLY_PHASE1
Conditions: Malaria, Plasmodium Falciparum
Sponsor: University of Oxford

VAC085 Clinical Trial Protocol, V2.0 11 January 2023, University of Oxford 
Page 1 of 66 
 
 
 
 
 
 
 
 
 
 
A Phase Ia study to assess safety and immunogenicity of the Plasmodium falciparum malaria 
vaccine candidate Pfs48/45 in Matrix-M adjuvant in healthy adults living in the UK 
 
Study Reference: VAC085 
Protocol Version: 2.0 
Date: 11 January 2023 
 
 
IRAS Project ID: 1004817 
REC reference: 22/SC/0291 
EudraCT ID: 2021-006763-26 
NCT05400746 
 
 
Chief Investigator: Angela M Minassian 
Sponsor: University of Oxford 
 
 
 
 
 
 

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MODIFICATION HISTORY 
Version Date 
Author(s) 
Modifications 
1.0 
22 March 2022 
Cyndi Goh 
David Mekhaiel 
Angela Minassian 
Sumi Biswas 
Created 
1.1 
11 May 2002 
Cyndi Goh 
Wording of group holding rules 
amended as per MHRA request 
2.0 
11 January 2023 Angela Minassian 
Rewording of 14.5 Storage and use of 
data 
Rewording of 8.2.4 Vaccination and re-
vaccination exclusion criteria 
Rewording of 9.2.5 Management of 
post-vaccination fever wording 
Bleed table pg 45 – D70 plasma and 
serum tube numbers. Total blood draw 
unchanged.  
Update to sponsor email address  
Update PHE to UK HSA 
 
 
 
 
 
 

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A Phase Ia study to assess safety and immunogenicity of the Plasmodium falciparum malaria 
vaccine candidate Pfs48/45 in Matrix-M adjuvant in healthy adults living in the UK 
Study Code:  VAC085 
Chief Investigator 
 
 
 
 
 
 
Investigator 
 
 
 
 
 
 
Trial Site 
Dr Angela M Minassian 
Centre for Clinical Vaccinology and Tropical Medicine, 
Jenner Institute, University of Oxford,  
Churchill Hospital, Old Road, Headington, 
Oxford, OX3 7LE 
Email: angela.minassian@ndm.ox.ac.uk 
 
Prof Sumi Biswas  
Jenner Institute, University of Oxford,  
Old Road Campus Research Building, 
Roosevelt Drive, Headington,  
Oxford, OX3 7DQ 
Email: sumi.biswas@ndm.ox.ac.uk 
 
Centre for Clinical Vaccinology & Tropical Medicine 
Jenner Institute, University of Oxford,  
Churchill Hospital, Old Road, Headington,  
Oxford, OX3 7LE 
PI at Site: Dr Angela M Minassian 
 
Sponsoring Institution 
 
 
 
 
University of Oxford 
Research Governance, Ethics and Assurance Team, 
Research Services, University of Oxford 
Joint Research Office, Boundary Brook House 
Churchill Drive, Headington, 
Oxford, OX3 7GB 
Tel: 01865 616480  
Email: rgea.sponsor@admin.ox.ac.uk  
 
 
 
 
 
 

VAC085 Clinical Trial Protocol, V2.0 11 January 2023, University of Oxford 
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External Monitor  
 
 
 
 
Appledown Clinical Research Ltd. 
Orchard End, 
Greenlands Lane, 
Prestwood, 
Bucks, HP16 9QX 
Tel: 01494 867676/07941 271635 
Email: ceri.mckenna@appledown.net 
 
Local Safety Monitor  
 
 
 
 
 
 
Funder 
Prof Brian Angus 
Centre for Clinical Vaccinology and Tropical Medicine, 
Churchill Hospital, Old Road, Headington, 
Oxford, OX3 7LE 
Tel: 01865 220289 
Email: brian.angus@ndm.ox.ac.uk 
 
European Commission (Horizon 2020) 
Rue de la Loi 200 / Wetstraat 200 
1040 
PO Box 1049 
Brussels 
Belgium 
 
The University of Oxford Internal Funds 
Nuffield Department of Medicine (NDM) 
Henry Wellcome Building for Molecular Physiology 
Old Road Campus, Headington 
Oxford, OX3 7BN 
 
 
 
 
 
 
 

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Confidentiality Statement 
This document contains confidential information that must not be disclosed to anyone other 
than the Sponsor, the Investigator Team, and members of the Independent Ethics Committee.  
This information cannot be used for any purpose other than the evaluation or conduct of the 
clinical investigation without the prior written consent of Dr Angela M Minassian. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Investigator Agreement 
“I have read this protocol and agree to abide by all provisions set forth therein. 
I agree to comply with the principles of the International Conference on Harmonisation Tripartite 
Guideline on Good Clinical Practice.”   
Dr Angela Minassian 
 
[redacted for public release] 
 
13 Jan 2023 
Chief Investigator 
Dr Angela M Minassian 
 
 
Investigator Signature 
 
Date  
 
Conflict of Interest 
1. “According to the Declaration of Helsinki, 2008, I have read this protocol, and declare 
no/the following (delete as appropriate) conflict of interest” 
Dr Angela Minassian 
 
[redacted for public release] 
 
 
 
13 Jan 2023 
Chief Investigator 
Dr Angela M Minassian 
 
 
Investigator Signature 
 
Date  
 
Details:____ n/a ________ _________________________________________________________ 
 
2.  “According to the Declaration of Helsinki, 2008, I have read this protocol, and declare no 
conflict of interest” 
 
Prof Sumi Biswas 
 
[redacted for public release] 
 
 
13 Jan 2023 
Investigator 
Prof Sumi Biswas 
 
Investigator Signature 
 
 
Date  
 
Details:_____n/a________________________________________________________________ 
 
 
 
 
 

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Table of Contents 
Modification History ................................................................................................................ 2 
1 Synopsis .............................................................................................................................. 10 
2 Abbreviations ...................................................................................................................... 12 
3 Background & Rationale ...................................................................................................... 15 
3.1 The need for a vaccine against Plasmodium falciparum malaria .................................................15 
3.2 Lifecycle of the P. falciparum malaria parasite ...........................................................................16 
3.3 Progress in vaccines targeting malaria .......................................................................................17 
3.4 Rationale for a transmission-blocking vaccine against P. falciparum malaria...............................18 
3.5 Challenges and directions in P. falciparum transmission-blocking vaccine development .............19 
3.6 Pfs48/45 as an antigen ..............................................................................................................20 
3.7 Clinical trials of Pfs48/45-based vaccines ...................................................................................22 
4 Pfs48/45 in Matrix-M Vaccine development ......................................................................... 23 
4.1 Characteristics of Pfs48/45 in Matrix-M .....................................................................................23 
4.2 Vaccine development strategy ..................................................................................................23 
4.3 Pfs48/45 immunogenicity and ex vivo efficacy in pre-clinical studies ..........................................24 
4.4 Clinical experience with Pfs48/45 in Matrix-M ...........................................................................26 
5 Investigational Products ...................................................................................................... 27 
5.1 Pfs48/45 ...................................................................................................................................27 
5.1.1 Vaccine supply .............................................................................................................................................27 
5.1.2 Vaccine formulation and packaging .............................................................................................................27 
5.1.3 Storage of Vaccines ......................................................................................................................................27 
5.2 Matrix-M ..................................................................................................................................27 
5.2.1 Matrix-M adjuvant supply ............................................................................................................................27 
5.2.2 Matrix-M adjuvant formulation and packaging ...........................................................................................27 
5.2.3 Matrix-M adjuvant storage and Handling ....................................................................................................27 
5.3 Administration of Pfs48/45 in Matrix-M adjuvant ......................................................................27 
6 Objectives and endpoints ..................................................................................................... 29 
6.1 Primary Objectives ....................................................................................................................29 
6.1.1 Primary Outcome Measures ........................................................................................................................29 
6.2 Secondary Objectives ................................................................................................................29 
6.2.1 
Secondary Immunological Outcome Measures ......................................................................................29 
7 Description and justification of study design ........................................................................ 30 
7.1 
Study rationale ...................................................................................................................30 
7.2  
Rationale for selected doses ...............................................................................................30 
7.3  
Study overview ..................................................................................................................30 
7.4 
Study groups ......................................................................................................................31 
7.4.1 First volunteers ............................................................................................................................................31 
7.4.2 Dose escalation ............................................................................................................................................31 
7.4.3 Group 1 ........................................................................................................................................................31 
7.4.4 Group 2 ........................................................................................................................................................32 

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7.4.5 Group 3 ........................................................................................................................................................32 
7.5 
Duration of study ...............................................................................................................32 
7.5.1 Definition of the start and end of the trial ...................................................................................................32 
7.5.2 Duration of volunteer participation .............................................................................................................32 
7.6 
Potential risks for volunteers ..............................................................................................32 
7.6.1 
Phlebotomy ............................................................................................................................................32 
7.6.2 Vaccinations .................................................................................................................................................32 
7.7 
Potential benefits for volunteers ........................................................................................33 
8 Recruitment and withdrawal for trial volunteers .................................................................. 34 
8.1 
Informed consent ...............................................................................................................34 
8.2  
Inclusion and exclusion criteria ...........................................................................................35 
8.2.1 
Inclusion criteria .....................................................................................................................................35 
8.2.2 
Exclusion criteria .....................................................................................................................................35 
8.2.3 
Prevention of ‘Over Volunteering’ ..........................................................................................................36 
8.2.4 Vaccination and re-vaccination exclusion criteria .......................................................................................36 
8.2.5 Concomitant medications ............................................................................................................................37 
8.3 
Withdrawal of volunteers ...................................................................................................37 
8.4 
Pregnancy ..........................................................................................................................38 
9. Treatment of trial volunteers .............................................................................................. 39 
9.1 
Trial sites ...........................................................................................................................39 
9.2 
Study procedures ...............................................................................................................39 
9.2.1 
Observations ...........................................................................................................................................39 
9.2.2 
Blood tests ..............................................................................................................................................39 
9.2.3 
 Urinalysis ................................................................................................................................................40 
9.2.4 Vaccinations .................................................................................................................................................40 
9.2.5 Management of post-vaccination fevers .....................................................................................................40 
9.2.6 E-diary ..........................................................................................................................................................41 
9.2.7 Photographs .................................................................................................................................................41 
9.3 
Study visits .........................................................................................................................41 
9.3.1 
Screening visits .......................................................................................................................................41 
9.3.2 Enrolment and first vaccination (V1) with Pfs48/45 in Matrix-M adjuvant (all groups) ..............................42 
9.3.3 Reviews post-vaccination on days 1, 7 and 14 (all groups) ..........................................................................42 
9.3.4 Second vaccination (V2) with Pfs48/45 in Matrix-M adjuvant on day 28 (all groups) .................................42 
9.3.5 Reviews post-vaccination on days 29, 35 and 42 (all groups) ......................................................................43 
9.3.6 Third vaccination (V3) with Pfs48/45 in Matrix-M adjuvant on day 56 (all groups) ....................................43 
9.3.7 Reviews post-vaccination on days 57, 63, 70 and 84 ...................................................................................43 
9.3.8 Long-term follow up post-vaccination on days 140 and 240 .......................................................................44 
10 Assessment of Safety ......................................................................................................... 46 
10.1 Definitions ..............................................................................................................................46 
10.1.1 Adverse Event (AE) .....................................................................................................................................46 
10.1.2 Adverse Reaction (AR)................................................................................................................................46 
10.1.3 Serious Adverse Event (SAE) ......................................................................................................................46 
10.1.4 Serious Adverse Reaction (SAR) .................................................................................................................47 
10.1.5 Suspected Unexpected Serious Adverse Reaction (SUSAR) .......................................................................47 
10.1.6 Foreseeable Adverse Reactions .................................................................................................................47 
10.2 Causality Assessment ..............................................................................................................47 
10.3 Reporting Procedures for all AEs ..............................................................................................48 
10.4 Reporting Procedures for SAEs .....................................................................................................................50 

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10.4.1 Reporting Procedures for SUSARS .........................................................................................50 
10.5 Development Safety Update Report ........................................................................................50 
10.6 AEs of Special Interest .............................................................................................................51 
10.7 Procedures to be followed in the event of abnormal findings ...................................................51 
10.8 Local Safety Monitor ...............................................................................................................51 
10.9 Safety Stopping/Holding Rules ................................................................................................51 
10.9.1 Group holding rules (applies to Groups 1 and 2/3) ...................................................................................51 
10.9.2 Individual stopping rules ............................................................................................................................52 
10.10 Contraception and pregnancy ................................................................................................53 
11 Statistics ............................................................................................................................ 54 
11.1  
Sample size ........................................................................................................................54 
12 Quality control and quality assurance procedures .............................................................. 55 
12.1  
Investigator procedures ......................................................................................................55 
12.2 
Monitoring .........................................................................................................................55 
12.3 
Modification to protocol .....................................................................................................55 
12.4 
Protocol deviation ..............................................................................................................55 
12.5 
Audit & inspection ..............................................................................................................55 
12.6 
Serious breaches ................................................................................................................56 
12.7 
Publication policy ...............................................................................................................56 
12.8 
Intellectual Property ...........................................................................................................56 
13 Ethics ................................................................................................................................. 57 
13.1 
Declaration of Helsinki .......................................................................................................57 
13.2 
GCP guidelines ...................................................................................................................57 
13.3 
Approvals ...........................................................................................................................57 
13.5 
Volunteer confidentiality ....................................................................................................57 
14 Data handling and record keeping ..................................................................................... 58 
14.1 
Data handling .....................................................................................................................58 
14.2 
Record keeping ..................................................................................................................58 
14.3 
Source data and electronic case report forms (eCRFs) ..........................................................58 
14.4 
Data protection ..................................................................................................................58 
14.5 
Storage and use of data ......................................................................................................58 
15 Financing and insurance .................................................................................................... 60 
15.1 
Financing ...........................................................................................................................60 
15.2 
Insurance ...........................................................................................................................60 
15.3 
Compensation ....................................................................................................................60 
16 Appendices ........................................................................................................................ 61 
17 References ......................................................................................................................... 62 

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1 SYNOPSIS 
Title 
A Phase Ia study to assess safety and immunogenicity of the Plasmodium 
falciparum malaria vaccine candidate Pfs48/45 in Matrix-M adjuvant in 
healthy adults living in the UK 
Trial Centre 
Clinical Centre for Vaccinology and Tropical Medicine, Churchill Hospital, 
Old Road, Headington, Oxford, OX3 7LE, UK  
Trial Identifier 
VAC085   
Clinical Phase  
Phase Ia 
Design 
Open label, single-site, first-in-human, dose-escalation Phase Ia study 
Population 
Healthy adults aged 18 – 45 years 
Sample Size 
Total: 24-30 volunteers 
Group 1: 8-10 volunteers receiving three doses of 10 µg Pfs48/45 in 50 µg 
Matrix-M on days 0, 28 and 56 
Group 2: 8-10 volunteers receiving three doses of 50 µg Pfs48/45 in 50 µg 
Matrix-M on days 0, 28 and 56 
Group 3: 8-10 volunteers receiving two doses of 50 µg Pfs48/45 in 50 µg 
Matrix-M on days 0 and 28, followed by one dose of 10 µg Pfs48/45 in 50 µg 
Matrix-M on day 56 
Follow-up 
duration 
Participants will be followed for approximately 8 months 
Planned trial 
period  
Approximately 12 months from first vaccination of the first volunteer 
Primary Objective To assess safety and tolerability of the Pfs48/45 in Matrix-M vaccine in 
healthy adult volunteers 
Secondary 
Objectives 
 
 
To assess the humoral and cellular immunogenicity of the Pfs48/45 in 
Matrix-M vaccine, when administered to healthy adult volunteers in the 
various regimes 
To assess ex vivo efficacy of the Pfs48/45 in Matrix-M vaccine using 
membrane feeding assays, when administered to healthy adult volunteers 

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Investigational 
Products 
1. Pfs48/45 – recombinant protein transmission-blocking malaria 
vaccine candidate 
2. Matrix-M – saponin based vaccine adjuvant  
Form 
1. Pfs48/45: liquid, stored at -80 oC (nominal) 
2. Matrix-M: liquid, stored at +2oC to +8oC 
Dose 
Group 1:  
a. Pfs48/45 - 10 µg, administered in a three-dose schedule on days 
0, 28 and 56 
b. Matrix-M – 50 µg, administered with each Pfs48/45 dose on 
days 0, 28 and 56 
Group 2: 
a. Pfs48/45 – 50 µg, administered in a three-dose schedule on 
days 0, 28 and 56 
b. Matrix-M – 50 µg, administered with each Pfs48/45 dose on 
days 0, 28 and 56 
Group 3:  
a. Pfs48/45 – 50 µg, administered in a two-dose schedule on days 
0 and 28 followed by 10 µg administered in a one-dose schedule 
on day 56 
b. Matrix-M – 50 µg, administered with each Pfs48/45 dose on 
days 0, 28 and 56 
Route 
Intramuscular injection (IM) in the deltoid region of the arm 
 
 

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2 ABBREVIATIONS 
ACT 
 
 
Artemisinin-combination therapies 
AE 
 
 
Adverse event 
ALS 
 
 
Advanced life support 
AR 
 
 
Adverse reaction 
ß-hCG  
 
Beta human chorionic gonadotropin 
CCVTM 
 
Centre for Clinical Vaccinology and Tropical Medicine 
CD1 
 
 
Cluster of differentiation 1 
ChAd63 
 
Chimpanzee adenovirus 63 
CI 
 
 
Chief investigator 
COVID-19 
 
Coronavirus disease 2019 
CRF 
 
 
Case report form  
DMFA  
 
Direct membrane feeding assay 
DNA 
 
 
Deoxyribonucleic acid 
DSUR  
 
Development safety update report 
ELISA  
 
Enzyme-linked immunosorbent assay 
ELISPOT 
 
Enzyme-linked immunospot assay 
EM 
 
 
Electron microscopy 
FDA 
 
 
United States Food and Drug Administration 
GCP 
 
 
Good clinical practice 
GLURP-R0 
 
Glutamate rich protein R0 
GMP  
 
Good manufacturing practice 
GP 
 
 
General practitioner 
HBsAg  
 
Hepatitis B surface antigen 
HCV 
 
 
Hepatitis C virus 
HIV 
 
 
Human immunodeficiency virus 
HLA 
 
 
Human leukocyte antigen 
ICH 
 
 
International Conference on Harmonisation 
IgG 
 
 
Immunoglobulin G 
IM 
 
 
Intramuscular 
IMP 
 
 
Investigational medicinal product 
kDa 
 
 
Kilodaltons  
LSM 
 
 
Local safety monitor 
mAb 
 
 
Monoclonal antibody 
MFA 
 
 
Membrane feeding assay 

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MHRA  
 
Medicines and Healthcare products Regulatory Agency 
MPC 
 
 
Multi-protein complexes 
MVA  
 
Modified vaccinia Ankara 
NDM  
 
Nuffield department of medicine 
NK 
 
 
Natural killer 
P. falciparum  
Plasmodium falciparum 
P. vivax 
 
Plasmodium viax 
PCR 
 
 
Polymerase chain reaction 
PHE 
 
 
Public Health England 
PIS 
 
 
Participant information sheet 
PPE 
 
 
Personal protective equipment 
PPM 
 
 
Parasite plasma membrane 
PV 
 
 
Parasitophorous vacuole  
PVM 
 
 
Parasitophorous vacuole membrane 
QP 
 
 
Qualified person 
RBM 
 
 
Roll back malaria 
REC 
 
 
Research Ethics Committee 
RGEA  
 
Research Governance, Ethics and Assurance Team 
RNA 
 
 
Ribonucleic acid 
S2 
 
 
Schneider 2 
SAE 
 
 
Serious adverse event 
SAR 
 
 
Serious adverse reaction 
SDEA  
 
Safety Data Exchange Agreement 
SMFA  
 
Standard membrane feeding assay 
SOP 
 
 
Standard operating procedure 
STOP-TRANS  
Safety and efficacy of R0.6C vacccine 
SUSAR  
 
Suspected unexpected serious adverse reaction 
TBI 
 
 
Transmission blocking immunity 
TBV 
 
 
Transmission blocking vaccine 
TOPS  
 
The overvolunteering prevention system 
TMF 
 
 
Trial master file 
TRA 
 
 
Transmission reducing activity 
UOXF  
 
University of Oxford 
VIMT  
 
Vaccines to interrupt malaria transmission 
VLP 
 
 
Virus-like particle 

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WHO  
 
World Health Organisation 

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3 BACKGROUND & RATIONALE 
3.1 The need for a vaccine against Plasmodium falciparum malaria  
Malaria is a potentially life-threatening parasitic infection and a major public health concern. 
Globally, 3.4 billion people are thought to be at risk of infection [1, 2], and in 2019 a reported 229 
million cases and 409 thousand deaths occurred from this disease [3]. Children under five years of 
age are the most severely affected, with 67% of worldwide malaria-related deaths occurring in this 
age group in 2019 [3].  
Of the five main species known to infect humans, Plasmodium falciparum (P. falciparum) is the 
most prevalent and the most important cause of morbidity and mortality. Plasmodium vivax also 
adds significantly to worldwide morbidity, with Plasmodium ovale, Plasmodium malariae and the 
zoonotic species Plasmodium knowlesi contributing to a lesser extent [4]. Geographically, P. 
falciparum is the predominant species in four of the five malaria endemic continents, and is 
overwhelmingly the primary malaria parasite in the WHO African Region, where it accounted for 
99.7% of all malaria cases in 2018; the same region accounted for 94% of all malaria cases and 
deaths worldwide [5]. 
Since 2000, following concerted worldwide efforts, there have been huge gains in the global fight 
against all-cause malaria, leading to a reduction in overall mortality from 736,000 in 2000 to 
409,000 in 2019 [3]. In the WHO African Region, malaria control interventions accounted for 70% 
of the 943 million fewer malaria cases occurring between 2001 and 2015, averting an estimated 
663 million malaria cases [6, 7]. This has largely been achieved through increased coverage of 
insecticide treated nets, estimated to account for 68% of the averted cases, as well as increased 
accessibility to artemisinin-combination therapies (ACTs) and increased indoor residual spraying, 
estimated to have contributed 19% and 13% respectively to prevented cases [8]. Other 
interventions, such as increased use of rapid diagnostic tests and intermittent prophylactic therapy 
for pregnant women and seasonal prophylaxis for children also play an important role [9-15]. In 
addition, in low-transmission areas and regions where artemisinin-resistant strains are emerging, 
single, low-dose primaquine is now recommended, in combination with ACT, as a gametocytocidal 
agent for non-pregnant adults and children above one year of age with parasitological evidence of 
P. falciparum infection [16]. However, limited direct evidence for safety for use in populations with 
glucose-6-phosphate dehydrogenase deficiency (G6PDD) and lack of access to diagnostics for pre-
treatment identification of G6PDD has resulted in ongoing safety concerns and poor levels of 
implementation. The high rates of gametocyte carriage in asymptomatic individuals in high-
transmission settings are also considered too large for adjunctive primaquine therapy to have any 
impact, limiting its use for malaria control across many African settings [17]. 
Despite nearly 20 years of advances, the WHO reports that the rate of progress in both cases and 
deaths has slowed worldwide since 2014 [18]. In some cases, including the ten highest burden 
African countries, the rate of progress has reversed [19]. Furthermore, current strategies are 
complicated by the rise in insecticide-resistant Anopheles mosquitoes and antimalarial resistant 
parasite strains in South East Asia and China, underlining the need for new tools for malaria control 
[20].  
The Roll Back Malaria (RBM) Partnership was launched in 1998 by the WHO, the United Nations 
Children’s Fund, the United Nations Development Programme and the World Bank. A major goal 
of The RBM Partnership is to support the development of vaccines against malaria as a key future 
strategy for reducing mortality from malaria. The WHO Global Vaccine Research Forum has set out 
a strategic framework for malaria vaccine development in the Malaria Vaccine Technology 
Roadmap, defining the goals for global malaria vaccine development community [21]. The 

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Roadmap calls for the development of vaccines against both P. falciparum and P. vivax by 2030 to 
achieve two key objectives: protective efficacy of at least 75 percent against clinical malaria and 
reduction of parasite transmission to substantially reduce incidence of human malaria infection, 
enabling elimination in multiple settings.  
3.2 Lifecycle of the P. falciparum malaria parasite 
In the pre-erythrocytic stage of human infection, the bite of an infected female Anopheles 
mosquito transmits malaria sporozoites to the human host where they travel via the bloodstream 
to the liver and invade hepatocytes (liver-stage). Here, they mature into merozoites for 6 to 7 days, 
after which the hepatocytes rupture releasing thousands of merozoites into the bloodstream. 
Merozoites then invade erythrocytes where they multiply and after 2 days cause the erythrocyte 
to rupture, releasing progeny merozoites that in turn invade new erythrocytes (blood-stage). It is 
the blood-stage of infection that is associated with clinical disease and potentially severe or fatal 
complications. The liver stage is asymptomatic.  
During the blood-stage infection, a small percentage of parasites undergo an alternative 
development pathway, differentiating into sexual male and female gametocyte forms. Over 5-12 
days, the intra-erythrocytic gametocytes progress through four developmental forms (stages I–IV) 
before reaching maturity, as stage V gametocytes [22-24]. The earlier stages of sexual 
development are largely absent from peripheral blood and are thought to sequester 
extravascularly in the bone marrow, before returning to the circulation as mature Stage V 
gametocytes [25]. The mature gametocytes are infectious to the mosquito vector, permitting 
transmission from human to Anopheline host.  
Following ingestion by the mosquito at the blood meal, environmental shifts, including changes in 
temperature and pH, as well as exposure to xanthurenic acid, trigger gametogenesis [26]. The 
female intra-erythrocytic sexual forms emerge as macrogametes, whilst male gametocytes 
undergo three rounds of DNA replication before exflagellation to release eight motile 
microgametes [24].  Extracellular macro and microgametes will unite within the mosquito’s midgut 
to undergo fertilisation, forming zygotes, which go on to become motile ookinetes that traverse 
the midgut epithelium. At the basal lamina, following meiotic division, the ookinete further 
develops into the oocyst. Multiple further rounds of division follow, eventually culminating in 
rupture of the oocyst and release of sporozoites. The sporozoites subsequently migrate to the 
mosquito’s salivary glands, where they will be injected into the human host at the next blood meal 
[22-24]. This lifecycle is summarised in Figure 1. 
 

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Figure 1: Lifecycle of P. falciparum malaria. Figure reproduced from the Centers for Disease 
Control and Prevention website [27]. 
3.3 Progress in vaccines targeting malaria 
Malaria vaccines can be subdivided into three categories based on the stages of the parasite life 
cycle targeted: pre-erythrocytic (liver-stage) vaccines, blood-stage vaccines and transmission-
blocking vaccines (Figure 2). Of the three stages, there has been most progress made with pre-
erythrocytic vaccines; the malaria vaccine RTS,S/ASO1E [28, 29] received approval from the 
European Medicines Agency in 2015 and pilot implementation programmes amongst African 
children were launched by the WHO in 2019.  
Since 2000, the rate of new malaria vaccine trials registered at ClinicalTrials.gov has remained 
steady at approximately 10 trials per year [30]. Initially, there was a strong focus on the subunit 
vaccine RTS,S. Over time, trials investigating whole-sporozoite pre-erythrocytic stage vaccines and 
transmission-blocking vaccines have increased in frequency.  
There has been increasing interest in the use of vaccines for malaria elimination. A proposed 
vaccine to interrupt malaria transmission (VIMT) could include antigens expressed during the three 
stages of the parasite lifecycle in order to reduce or halt the spread of parasites in the community 
[31]. 
 
 

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Figure 2: Lifecycle stages of Plasmodium and vaccine candidates that target each stage. Figure 
reproduced from a review by Duffy and Gorres, 2020 [32]. 
3.4 Rationale for a transmission-blocking vaccine against P. falciparum malaria 
Transmission-blocking vaccines (TBVs) aim to induce antibodies against sexual-stage antigens, 
which are taken up in the blood meal and act to prevent sexual development in the mosquito host, 
thus blocking onward malaria transmission. Uniquely, TBVs do not aim to provide protection 
against infection or disease in the vaccinated individual but instead to reduce risk of infection at 
the population level, through interruption of the transmission cycle. 
The first observations of transmission-blocking immunity (TBI) were reported in 1958, when lower 
infectivity to mosquitoes of the avian Plasmodium species, P. fallax and P. gallinaceum, were 
described in turkeys and chickens which had been immunised with formalin-killed parasitised 
erythrocytes [33]. However, it was not until almost 20 years later, in 1976, that the phenomenon 
was reproduced and the underlying mechanism delineated as antibody-mediated inhibition of 

VAC085 Clinical Trial Protocol, V2.0 11 January 2023, University of Oxford 
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sexual parasite development in the mosquito midgut [34]. In 1983, following developments in 
methods for in vitro culture of the sexual stages of P. falciparum, BALB/c mice were immunised 
with P. falciparum gametes leading to the generation of the first transmission-blocking monoclonal 
antibodies (mAbs), which could arrest parasite development in the mosquito midgut [35, 36]. 
Through surface radio-isotope labelling of gametes followed by precipitation with these 
transmission-blocking mAbs, the first list of transmission-blocking targets were identified [36].  
The gold standard ex vivo model for establishing transmission-reducing (conferring decreased 
oocyst intensity) or transmission-blocking (reduction in mosquito infection prevalence) effects, is 
the standard membrane feeding assay (SMFA), wherein mosquitoes are fed cultured gametocytes 
in the presence of whole serum or purified IgG, from either a test or control source. Mosquitoes 
are then dissected approximately a week later and oocyst development in the mosquito is 
quantified by microscopic visualisation using mercury-bromide staining, and compared between 
intervention and control groups. For determination of TBI against varied parasitic strains, direct-
membrane feeding assays (DMFAs), using gametocytes derived from field isolates, may also be 
employed. Vaccine-induced antibodies generated against the TBV candidate antigens first 
described in 1983, have reproducibly demonstrated capacity to reduce P. falciparum, since their 
identification [36-38]. 
3.5 Challenges and directions in P. falciparum transmission-blocking vaccine development 
The TBV candidates first identified in 1983, Pfs25, Pfs48/45 and Pfs230, named due to their 
molecular mass, remain the most well-studied and developed vaccine candidates to date [36-38]. 
The Pfs25 paralogue, Pfs28, was later identified as a further target mediator of TBI. Pfs28, 
structurally and genetically similar to Pfs25, has demonstrated a synergistic effect alongside Pfs25 
pre-clinically [39], although this was not reproduced in a similar study investigating the P. vivax 
equivalent, Pvs28 [40]. Pfs48/45 is highly important in male fertility, with knockout studies 
resulting in male gametes that are incapable of adhering to and penetrating female gametes, with 
consequent significant reductions in oocyst development [41]. Pfs230 has an important role in the 
interaction of exflagellated microgametes with adjacent red blood cells for formation of 
exflagellation centres and subsequent oocyst development. However, knockout studies for both 
pfs48 and pfs230 genes suggest that their function is non-essential, since disrupted strains display 
highly reduced oocyst formation, but not complete lack of development [41, 42]. 
More recently, PfHAP2, essential for fusion of the male and female gametocyte during fertilization, 
has also been demonstrated to induce transmission-blocking in the murine model [43]. In addition, 
in a novel approach, mosquito-derived antigens are under investigation to target immune 
responses to prevent parasite penetration of the mosquito midgut [44, 45]. However, these 
candidates, alongside other potential new transmission-blocking targets of interest, remain at 
early pre-clinical stages of investigation.   
Classical transmission-blocking candidates can be considered to fall into two broad categories: 
those that target antigens which are expressed before fertilisation and those that are only 
expressed in the post-fertilisation stages within the mosquito. Pfs48/45 and Pfs230 are the 
principal pre-fertilisation targets, whilst Pfs25 is the leading post-fertilisation candidate, along with 
Pfs28 [37]. Naturally-acquired immunity to pre-fertilisation antigens, which are present in the 
human host, have been identified in sero-epidemiological studies in The Gambia, Papua New 
Guinea and Cameroon, including anti-Pfs230 and anti-Pfs48/45 antibody responses, which 
correlated with transmission-reducing activity (TRA) [46-54]. This suggests that vaccine-induced 
responses against these targets may benefit from boosting in natural infection. Conversely, 
repression of translation prior to development in the mosquito vector means that post-fertilisation 

VAC085 Clinical Trial Protocol, V2.0 11 January 2023, University of Oxford 
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antigens do not come under immune pressure, resulting in very limited polymorphism. 
Polymorphisms in the pre-fertilisation antigens are more frequent, although are still less common 
than in blood-stage antigens [55].  
The induction of sustained, high-titre antigen-specific antibody responses, maintained over a 
minimum of a transmission season, will be critical for an efficacious TBV. Data from an inter-species 
comparative assessment of Pfs25-specific IgG antibody from mouse, rabbit, monkey and human 
sera also suggest that higher human antibody titres are required to produce transmission-blocking 
effects relative to animal models used in pre-clinical studies, with human IgG producing a 50% 
reduction in oocyst intensity by SMFA (IC50) at an estimated concentration of 85.6 g/mL 
compared to 15.9 g/mL, 4.2 g/mL, 41.2 g/mL for mice, rabbit and monkey sera, respectively 
[56]. The requirement for high human antibody titres for effecting TRA was reproduced in a clinical 
trial of the Pfs25-EPA/Alhydrogel vaccine candidate, in which the IC50 was estimated as 57.2 μg/ml 
(95% CI 44.7, 76.8) in sera from vaccinated subjects [57].  
Further to the development of vaccines developed specifically targeting transmission stages, 
vaccines candidates targeting the liver or blood-stages of malaria infection, if effective, may also 
have a role in reducing transmission, by preventing blood-stage infection and therefore 
gametocyte development entirely, or efficient blood-stage parasite clearance. The modest and 
short-lived efficacy demonstrated by the most advanced malaria vaccine candidate, RTS,S, may 
limit the impact on malaria transmission of this pre-erythrocytic vaccine alone [28, 29]. However, 
the combination of multiple antigens, targeting different stages has the potential to provide 
synergistic effects to enhance vaccine efficacy. Pre-clinical studies evaluating combinations of TBVs 
with vaccines targeting other life-cycle stages, or TBI as mediated by monoclonal antibodies (mAb), 
in combination with mAbs against the sporozoite stage circumsporozoite protein have 
demonstrated promising results [58, 59]. Additional investigation of this multi-stage strategy will 
likely be an important investigational approach in the development of an effective malaria vaccine 
for malaria control and reduced parasite transmission. 
3.6 Pfs48/45 as an antigen 
Pfs48/45 is a cysteine motif-rich protein expressed during P. falciparum gametocyte maturation. 
These gametocytes display multi-protein complexes (MPCs) on the plasma membrane, comprised 
of the PfCCp protein family of six secreted proteins. The PfCCp-based MPCs are linked to the 
gametocyte plasma membrane via interactions with Pfs230, a binding-partner of the 
glycophosphatidylinositol (GPI)-anchored Pfs48/45 (Figure 2). This MPC assembly step is a crucial 
step for sexual reproduction of the parasite [60].  

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Figure 3: Schematic representation of the Plasmodium falciparum multi-protein complex (MPC) 
composition and assembly during gametogenesis. Processing of Pfs230 during gametogenesis is 
represented by the release of a protein fragment. EM = erythrocyte membrane; PPM = parasite 
plasma membrane; PV = parasitophorous vacuole; PVM = parasitophorous vacuole membrane. 
Figure reproduced from Simon et al, 2015 [60]. 
A major bottleneck to Pfs48/45 vaccine development is the challenge of producing the properly 
folded recombinant protein in sufficiently high yields. As Pfs48/45 is cysteine-rich, correct 
formation of disulfide bridges is essential. This refolding into the native structure is required for 
the induction of transmission-blocking antibodies.  
Pfs48/45 has been produced in different eukaryotic expression systems including baculovirus (Sf9) 
cells [61], vaccinia virus [62], Saccharomyces cervesiae [63], Pichia pastoris [63], Chlamydomonas 
reinhardtii [64], Escherichia coli [65-68] and Nicotiana benthamiana [69]. However, these systems 
have led to disappointing results with low yields of properly folded recombinant proteins or 
minimal reactivity with monoclonal antibodies against conformational transmission-blocking 
epitopes. In contrast, the Lactococcus lactis expression system has been more successful, with the 
R0.10C [70-72] and R0.6C [73] proteins demonstrating good yields, correct folding and functional 
activity. 
Several lines of evidence from pre-clinical studies suggest Pfs48/45 as a good TBV candidate. For 
instance, studies demonstrate that monocolonal antibodies directed against Pfs48/45 lead to 
prevention of parasite transmission to mosquitoes in SMFAs [36, 74]. In addition, antibody-
prevalence studies in endemic areas show that natural antibodies against Pfs48/45 correlate with 
TRA [75]. Furthermore, several rodent studies show that properly folded fragments of Pfs48/45 
are able to elicit high levels of transmission-blocking antibodies in the host [65, 70, 73, 76]. 

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3.7 Clinical trials of Pfs48/45-based vaccines  
Only one Pfs48/45-based vaccine has reached clinical trials. R0.6C is a multistage chimera 
consisting of a transmission blocking (Pfs48/45-6C) and a blood-stage (GLURP-R0) antigen. In pre-
clinical studies using the Lactoccus lactis expression system, the GLURP-R0 antigen facilitated 
synthesis of a correctly folded Pfs48/45 C-terminal fragment [70]. Then, a series of novel fusion 
proteins were generated and tested in rats with aluminium hydroxide as an adjuvant [73]. 
Functional transmission-blocking antibodies were generated with transmission-blocking activity 
associated with specific IgG levels [73].  
The safety and efficacy of this R0.6C vaccine is currently being tested in the Netherlands in healthy 
adults as a Phase I clinical trial. The STOP-TRANS study [NCT04862416] commenced recruitment 
in May 2021 and aims to recruit 32 participants across four groups. The single domain R0.6C 
vaccine will be adjuvanted with Alhydrogel alone, or combined with Matrix-M.  
Of note is the fact that R0.6C consists of just a single domain of Pfs48/45 whereas the vaccine we 
are studying in this proposed trial contains all three domains of the protein. Transmission blocking 
epitopes have been found within all three domains of Pfs48/45, hence a full length Pfs48/45 
vaccine offers more targets to elicit transmission blocking antibodies against, compared to R0.6C. 
[65, 76-79].  
 
 
 

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4 PFS48/45 IN MATRIX-M VACCINE DEVELOPMENT 
4.1 Characteristics of Pfs48/45 in Matrix-M 
Pfs48/45 is a soluble protein vaccine based on the P. falciparum transmission-blocking target 
antigen. The Pfs48/45 protein is based on the sequence from the 3D7 P. falciparum strain. The 
recombinant protein is expressed in, and secreted from, the insect Schneider 2 (S2) cell line derived 
from Melanogaster drosophila. 
Matrix-M is a potent, saponin-based adjuvant, comprising defined purified fractions from bark 
extracts of the bark of the Quillaja saponaria Molina tree, phosphatidylcholine and cholesterol, 
formulated as a 40nm-sized complex. Matrix-M adjuvant has been shown to efficiently activate 
and recruit immune cells to the draining lymph node, including T-cells, B-cells, NK-cells, dendritic 
cells and granulocytes, which may lead to enhanced antigen presentation [80]. Matrix-M has 
demonstrated a favourable safety profile, as well as the enhancement of cellular and humoral 
immune responses to a range of vaccine [80-82].  
Pfs48/45 and Matrix-M adjuvant will be mixed immediately prior to administration for co-
administration. 
4.2 Vaccine development strategy 
The clinical development of Pfs48/45 in Matrix-M adjuvant is aimed towards the production of an 
effective transmission-blocking malaria vaccine for individuals in malaria endemic regions. This 
vaccine would be used in a three-dose vaccination regimen (Table 1). 
Group 
Group 
size 
D0 
D28 
D56 
Group 1 
 
8-10 
Pfs48/45 10 µg 
Matrix-M 50 µg 
Pfs48/45 10 µg 
Matrix-M 50 µg 
Pfs48/45 10 µg 
Matrix-M 50 µg 
Group 2 
8-10 
Pfs48/45 50 µg 
Matrix-M 50 µg 
Pfs48/45 50 µg 
Matrix-M 50 µg 
Pfs48/45 50 µg 
Matrix-M 50 µg 
Group 3 
 
8-10 
Pfs48/45 50 µg 
Matrix-M 50 µg 
Pfs48/45 50 µg 
Matrix-M 50 µg 
Pfs48/45 10 µg 
Matrix-M 50 µg 
Table 1: Vaccination protocol for study groups 1, 2 and 3 
 
This first-in-human, dose-escalation study aims to provide an initial assessment of the frequency 
and magnitude of AEs following vaccination with Pfs48/45 in the Matrix-M adjuvant for evaluation 
of safety, as well as immunogenicity and ex vivo efficacy via membrane feeding assays. In addition, 
the optimal dose of Pfs48/45 in Matrix-M1 will be determined from this trial. Data generated from 
this study will inform progression to a Phase Ib trial in an endemic region to evaluate the safety 
and immunogenicity of Pfs48/45 in Matrix-M at various doses, in a similar dose-escalation study, 
to establish the optimal dose and schedule in an endemic setting.  
 
 

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4.3 Pfs48/45 immunogenicity and ex vivo efficacy in pre-clinical studies  
The Pfs48/45 soluble protein vaccine being studied in this proposed trial is the full-length protein 
consisting of all three protein domains. In pre-clinical studies involving mice, the immunogenicity 
of different proteins (as measured by anti-Pfs48/45 IgG titres) was assessed by ELISA. Here, three 
5 µg doses of different forms of the protein was administered to each group three weeks apart 
with AddaVax as an adjuvant. Figure 4 shows that mice immunised with both versions of the full-
length protein had high levels of anti-Pfs48/45 IgG titres. 
 
Figure 4: Anti-Pfs48/45 IgG titres in mice immunised with different forms of the Pfs48/45 protein. 
CD1 outbred mice were immunised with three 5 µg doses of vaccine three weeks apart with 
AddaVax as an adjuvant and IgG titres measured at two weeks following the third dose. The 
following protein were assessed: Full-length Pfs48/45 protein (AA: 27-428) with all seven N-glycans 
sites (Pfs48/45 + N-Gly), full-length Pfs48/45 protein with all seven N-glycans sites mutated out 
(Pfs48/45 – N-Gly Mutant). OVA=ovalbumin (negative control). 
In addition, SMFA showed that full-length Pfs48/45 induces transmission reducing activity (TRA) 
when IgG antibodies from immunised mice was diluted into blood containing stage V NF54 
gametocytes and fed to 3-6 day old starved female Anopheles stephensi mosquitoes (Figure 5). 
Here, there were decreased oocyst numbers per mosquito when mice were immunised with full-
length Pfs48/45 compared with controls.  
OVA
Pfs48/45 + N-Gly
Pfs48/45 - N-Gly Mutant
0
1
3
4
5
6
7
8
Pfs48/45 Specifc IgG Titres
Endpoint titre (Log10)
   2
Pfs48/45 + N-Gly
Pfs48/45 - N-Gly Mutant
OVA

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Figure 5: Standard membrane feeding assay (SMFA) involving mice immunized with different 
forms of the Pfs48/45 protein at a concentration of 750 g/ml of purified IgG. Red numbers above 
each column denote percentage transmission reducing activity (%TRA) for each protein. OVA = 
ovalbumin (negative control); 4B7 = Pfs25 specific mouse control monoclonal antibody (positive 
control); Pfs48/45 + N-Gly = full-length Pfs48/45 with all seven N-glycans sites present; s48/45-FL 
– N-Gly Mutant = full-length Pfs48/45 with all seven N-glycans sites absent. 
Finally, data suggests that the combination of Pfs48/45 with Matrix-M resulted in higher 
immunogenicity in mice when compared to Pfs48/45 with Alhydrogel (Figure 6). Here, CD1 
outbred mice were immunised with two doses of 0.1 µg or 1 µg Pfs48/45 three weeks apart with 
Alhydrogel or Matrix-M. They were bled at three weeks following the second dose and 
immunogenicity assessed by anti-Pfs48/45 IgG ELISA and SMFA. The ELISA data suggests that the 
Matrix-M adjuvant potentiated increased levels of anti-Pfs48/45 IgG titres when compared to the 
Alhydrogel adjuvant, with minimal difference between the 0.1 µg and 1 µg dose.  
OVA
4B7
Pfs48/45 + N-Gly
Pfs48/45 - N-Gly Mutant
0
10
20
30
40
50
60
70
80
Oocyst number per mosquito
             99.7                100              100         %TRA

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Figure 6: Comparison of Alhydrogel and Matrix-M as adjuvants for immunisation with Pfs48/45. 
CD1 outbred mice were immunized with either two doses of 0.1 µg or 1 µg of Pfs48/45 with either 
adjuvant at three-week intervals and bled at day 20 and day 42. A: Pfs48/45 specific IgG endpoint 
titres of immunized mice. B: SMFA of IgG purified from D42 of immunized mice. Normal mouse 
sera acts as a negative control and the Pfs25 specific mAb 4B7 as positive control. 
 4.4 Clinical experience with Pfs48/45 in Matrix-M 
There is no prior clinical experience with the Pfs48/45 protein specifically in the Matrix-M 
adjuvant.  
Matrix-M adjuvant, has been administered to tens of thousands of individuals in clinical trials 
across the USA, Europe, Africa and Australia. In addition to trials involving vaccines to respiratory 
syncytial virus, influenza and SARS-CoV-2, two malaria pre-erythrocytic candidate vaccines 
(ChAd63/MVA ME-TRAP and VLP R21) have been administered with Matrix-M adjuvant in four 
phase I and phase I/II trials conducted in Oxford and Burkina Faso (ClinicalTrials.gov identifiers: 
NCT02572388, NCT02925403, NCT01669512, NCT02905019). A phase III trial involving 4800 
children has recently started in Africa with sites in Burkina Faso, Mali, Kenya and Tanzania. 
Available safety data demonstrates that the Matrix-M adjuvant is well-tolerated with acceptable 
short-term reactogenicity and unremarkable long-term safety profile in COVID-19 vaccine clinical 
trials conducted by Novavax collaborators in subjects who received Matrix-M [83]. 
The profile of AEs following vaccination with Pfs48/45 may be partially predicted from previous 
studies assessing protein vaccines using the Matrix-M adjuvant. Local AEs are likely to include 
injection site pain, erythema, swelling, itching and warmth. Forseeable systemic AEs would include 
headache, fatigue, myalgia, arthralgia, malaise, feverishness, fever and nausea. 
 
0
20
40
60
Oocyst number per mosquito
59.6%
89.6%
%TRA
1µg Pfs48/45
in Alhydrogel
1µg Pfs48/45
in Matrix M
4B7 positive
control
Normal mouse sera
negative control
90.8%

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5 INVESTIGATIONAL PRODUCTS 
5.1 Pfs48/45 
5.1.1 Vaccine supply 
Pfs48/45 is manufactured by GenIbet (Oeiras, Portugal), where the vaccines are formulated and 
vialed. Xerimis Ltd (Reading, UK) will perform certification and QP release, labelling and storage of 
the Pfs48/45 for investigational use only, and later supply to the clinical site (CCVTM). 
5.1.2 Vaccine formulation and packaging 
The Pfs48/45 protein drug product is formulated in 20 mM Tris Buffered Saline (TBS), 150mM NaCl, 
pH 7.4 (adjusted by sodium hydroxide). The final 0.2 µm filtered drug product was dispensed 
manually using an electronic dispenser preset to deliver 500 µl into type 1, 2 ml glass vials. Filling 
is performed by Apotex Production and Laboratory. The vials were sterilized and depyrogenated 
with a 13 mm grey bromobutyl rubber stopper supplied by Adelphi Healthcare Packaging 
(Haywards Heath, UK) and a 13 mm complete tear off, clear lacquered aluminium seal. The 
containers and closures were tested for compliance with defined specifications. 
5.1.3 Storage of Vaccines  
All vaccines will be stored at -80C (nominal). All movements of the study vaccines will be 
documented. Vaccine accountability, storage, shipment and handling will be in accordance with 
relevant local Standard Operating Procedures (SOPs) and forms. 
5.2 Matrix-M 
5.2.1 Matrix-M adjuvant supply 
Matrix-M adjuvant will be supplied to Xerimis by Novavax AB (Uppsala, Sweden), a subsidiary of 
Novavax, Inc (Gaithersburg, USA), where the adjuvant is formulated and vialed. Matrix-M will be 
labelled for investigational use only by Xermis Ltd before supplying the adjuvant to the clinical site. 
All adjuvants will be certified for release by a qualified person (QP) at Xerimis Ltd.  
5.2.2 Matrix-M adjuvant formulation and packaging  
Matrix-M is formulated at a concentration of 0.375mg/ml in PBS. The drug product is filled into 
sterile 3 mL glass vials. Matrix-M adjuvant is a slightly brown to colourless, slightly opalescent, non-
viscous liquid. 
5.2.3 Matrix-M adjuvant storage and Handling  
Matrix-M adjuvant is stored refrigerated at 2 to 8°C. All movements of the adjuvant will be 
documented. Accountability, storage, shipment and handling of Matrix-M adjuvant will be in 
accordance with relevant local SOPs and forms. 
5.3 Administration of Pfs48/45 in Matrix-M adjuvant 
A mixture of Pfs48/45 at a dose of 10 μg or 50 μg, with 50 μg of Matrix-M adjuvant will be 
administered to all volunteers. Matrix-M and Pfs48/45 will be mixed immediately prior to 
administration and will be administered intramuscularly within one hour of thawing of Pfs48/45.  
All vaccines will be administered intramuscularly in the deltoid muscle of the non-dominant arm 
preferentially, according to SOP VC002 Vaccination. The vaccinating investigator will wear gloves 

VAC085 Clinical Trial Protocol, V2.0 11 January 2023, University of Oxford 
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and eye protection. During administration of the vaccines, Advanced Life Support (ALS) drugs and 
resuscitation equipment will be immediately available for the management of anaphylaxis. On 
vaccination day, vaccines will be allowed to thaw to room temperature and administered within 
one hour. After insertion of the needle but before injection, aspiration should be attempted to 
ensure that the injection is not intravascular. A single vial of vaccine will be used per dose 
administered and any remaining vaccine not used will be discarded. 
 

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6 OBJECTIVES AND ENDPOINTS 
6.1 Primary Objectives  
To assess safety and tolerability of the Pfs48/45 with Matrix-M vaccine at various doses in healthy 
adult volunteers. 
6.1.1 Primary Outcome Measures 
The specific endpoints for safety and reactogenicity will be actively and passively collected data on 
AEs. The following parameters will be assessed:  
• Occurrence of solicited local reactogenicity signs and symptoms for 7 days following each 
vaccination  
• Occurrence of solicited systemic reactogenicity signs and symptoms for 7 days following 
each vaccination  
• Occurrence of unsolicited adverse events (AEs) for 28 days following the vaccination  
• Change from baseline for safety laboratory measures for 28 days following vaccination 
• Occurrence of serious adverse events (SAEs) during the whole study duration  
• Occurrence of AEs of special interest during the whole study duration 
Solicited and unsolicited AE data will be collected at each clinic visit. It will be collected from e-
diaries, clinical review, clinical examination (including observations) and laboratory results. This AE 
data will be tabulated, detailing frequency, duration and severity of AEs. 
Haematological and biochemical laboratory values will be presented according to local grading 
scales. SAEs, AEs of special interest and withdrawal due to AE(s)/SAE(s) will be described in detail. 
Volunteers will be followed for approximately 8 months following initial dose of Pfs48/45 with 
Matrix-M and approximately 6 months following the third dose.  
6.2 Secondary Objectives 
To assess the humoral and cellular immunogenicity of the Pfs48/45 with Matrix-M vaccine, when 
administered to healthy adult volunteers. 
To assess ex vivo efficacy of the Pfs48/45 with Matrix-M vaccine, when administered to healthy 
adult volunteers. 
6.2.1 Secondary Immunological Outcome Measures 
Pfs48/45-specific immunogenicity will be assessed by a variety of immunological assays, with 
comparison before and after vaccination. The main outcome measures will be humoral and B cell 
responses to the Pfs48/45 protein – total IgG, isotypes and avidity; and T cell responses to Pfs48/45 
by ex vivo enzyme-linked immunospot assay (ELISpot). 
Ex vivo functional blocking activity of purified IgG against P. falciparum will be assessed by 
membrane feeding assays .  
Other established and exploratory immunology assays may be carried out, which may include 
collaboration with other specialist laboratories within or beyond Europe. This would involve 
transfer of serum/plasma, but samples would be anonymised. Volunteers will be consented for 
this. 
The secondary timepoints will be days 1, 29, 57, 140 and 240. 

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7 DESCRIPTION AND JUSTIFICATION OF STUDY DESIGN  
7.1 
Study rationale  
As outlined above, the currently available methods for preventing and treating malaria remain 
inadequate. Although focused efforts have led to substantial reductions in morbidity and mortality 
worldwide, progress has now stalled. An effective vaccine against P. falciparum would contribute 
substantially to disease control and elimination efforts.  
Pre-clinical studies suggest Pfs48/45 as a good TBV candidate. In rodent studies, properly folded 
fragments of Pfs48/45 are able to elicit high levels of transmission-blocking antibodies in the host 
[65, 70, 73, 76, 84]. The Pfs48/45 protein we are testing in this study differs from previously studied 
versions of the antigen in that the vaccine candidate being tested is full-length, offering a wider 
variety of blocking epitopes than the 6C-based counterparts. In addition, combining Pfs48/45 with 
the Matrix-M adjuvant, which has been shown to be an effective adjuvant in combination with a 
range of different vaccines, offers a promising strategy. 
This small, first-in-human study will provide initial data on the safety profile of vaccination with 
Pfs48/45 in the Matrix-M adjuvant, as well as immunogenicity and ex vivo efficacy via membrane 
feeding assays.  
7.2  Rationale for selected doses 
The two doses of 10 μg and 50 μg Pfs48/45 in 50 μg Matrix-M were selected on the basis of 
experience with other candidate protein vaccines which have been tested at the same dose in 
humans with the same adjuvant. These include the VAC077 and VAC082 trial of Pfs25-IMX313 in 
Matrix-M1 as well as the VAC084 trial of Pvs25-IMX313 in Matrix-M1. 
Mouse studies suggest that a vaccine dose of up to 250 μg/kg is safe. In this study we are testing 
a maximum dose of 50 μg Pfs48/45 in 50 μg Matrix-M. This is equivalent to a dose of 0.8 μg/kg in 
humans. This means that there is a safety margin based on body weight of 250/0.8 = 312-fold in 
humans as compared to mice.  
7.3  Study overview 
This is an open-label, single-centre, non-randomised, first-in-human Phase Ia study to assess the 
safety and immunogenicity of the Pfs48/45 vaccine, administered with Matrix-M adjuvant.  
Up to 30 healthy, malaria-naïve adults aged between 18 and 45 years will be recruited into one of 
three groups (n=8-10 per group) at the Centre for Clinical Vaccinology and Tropical Medicine 
(CCVTM), Oxford over approximately 12 months.  
All volunteers will receive three doses of Pfs48/45 at 10 µg or 50 µg, in 50 µg Matrix-M, 
administered intramuscularly and given four weeks apart. Enrolment will be staggered. Initially, a 
single volunteer from Group 1 will receive the first vaccination alone, followed by clinical review 
on the day after vaccination and further monitoring via a diary card for at least a further 24 hours. 
Provided there are no safety concerns, a further two volunteers from Group 1 may then be 
vaccinated, at least 1 hour apart. Following review of the second and third volunteers at the 48-
hour timepoint, an internal safety review will be conducted. If no safety concerns are identified, 
the remaining volunteers in the group will be vaccinated. The staggered enrolment process will be 
repeated for Group 2 for the first three volunteers. Following this, recruitment to Groups 2 and 3 
will occur in parallel.  

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Volunteers will attend regular follow-up visits after each of the three vaccinations and will self-
report AEs in the post-vaccination period via a diary card. Blood will be taken at regular intervals 
to assess safety outcomes via laboratory adverse events, as well as the immune response to 
vaccination. Volunteers will be followed until approximately 8 months from the time of enrolment. 
All vaccinations and follow-up visits will be performed at the CCVTM in Oxford.  
7.4 
Study groups 
There will be three study groups (Group 1, Group 2 and Group 3), each comprising 8-10 volunteers. 
There will be staggered enrolment for Groups 1 and 2 as described below in Section 7.3.1. Any 
volunteer who is withdrawn or withdraws from the study may be replaced at the discretion of the 
Investigators.  
7.4.1 First volunteers 
The first volunteer from Group 1 to receive Pfs48/45 in Matrix-M will be enrolled and vaccinated 
alone. Providing there are no safety concerns at 48 hours after the first volunteer has been 
vaccinated, the second and third volunteers from Group 1 will be vaccinated. Following a further 
48 hour period, an internal safety review will be conducted involving the review of participant e-
diaries by the Chief Investigator with the Lead Clinician. If at this point there are no safety concerns, 
the rest of Group 1 may be immunised. This staggered approach will be repeated for the second 
dose. After at least eight volunteers from Group 1 have received two out of three doses of 10 µg 
Pfs48/45 in 50 µg Matrix-M and had seven days of follow-up, there will be a formal safety review 
(see below) prior to dose escalation (i.e. recruitment to Groups 2 and 3). The staggered enrolment 
process will continue for the third vaccination in Group 1 and for Group 2 for the first three 
volunteers at each of the three doses. Following this, recruitment to Groups 2 and 3 will occur in 
parallel.   
7.4.2 Dose escalation 
A minimum of data from eight volunteers recruited to Group 1 will be reviewed prior to dose 
escalation (i.e. recruitment to Groups 2 or 3). The data to be reviewed will include but is not limited 
to: physical observations, patient e-diary cards and safety bloods (full blood count, urea and 
electrolytes, liver function tests). Data up to and including the first seven days following 
vaccination will be required for each of the eight volunteers prior to dose escalation. E-diary cards 
will be reviewed on a daily basis for the first eight volunteers recruited to Group 1 for the first 
seven days following vaccination. A formal safety review will be undertaken by the LSM prior to 
dose escalation. This would take the form of a safety report incorporating the relevant safety data 
described above, which would need to be reviewed and approved by the CI and LSM prior to 
recruitment into Groups 2 and 3. 
7.4.3 Group 1 
Eight to ten volunteers will be recruited into Group 1 (Table 1) and each volunteer will attend for 
three vaccinations. On the day of enrolment, Pfs48/45 will be administered at a dose of 10 µg in 
50 µg Matrix-M adjuvant. After approximately 4 weeks, a second dose will be administered, 
followed by a third and final vaccination approximately 4 weeks later. Second and third 
vaccinations will be administered at the same dose of both vaccine and adjuvant as at the initial 
vaccination. Volunteers will be followed until 8 months (approximately) from the time of 
enrolment.   

VAC085 Clinical Trial Protocol, V2.0 11 January 2023, University of Oxford 
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7.4.4 Group 2 
Eight to ten volunteers will be recruited into Group 2 (Table 1) and each volunteer will attend for 
three vaccinations. On the day of enrolment, Pfs48/45 will be administered at a dose of 50 µg in 
50 µg Matrix-M adjuvant. After approximately 4 weeks, a second dose will be administered, 
followed by a third and final vaccination approximately 4 weeks later. Second and third 
vaccinations will be administered at the same dose of both vaccine and adjuvant as at the initial 
vaccination. Volunteers will be followed until 8 months (approximately) from the time of 
enrolment.   
7.4.5 Group 3 
Eight to ten volunteers will be recruited into Group 3 (Table 1) and each volunteer will attend for 
three vaccinations. On the day of enrolment, Pfs48/45 will be administered at a dose of 50 µg in 
50 µg Matrix-M adjuvant. After approximately 4 weeks, a second dose will be administered, 
followed by a third and final vaccination approximately 4 weeks later. The second vaccination will 
be at the same dose as the first vaccination whilst the third vaccination will be administered at a 
lower dose of Pfs48/45 10 µg in 50 µg Matrix-M adjuvant. Volunteers will be followed until 8 
months (approximately) from the time of enrolment.   
 
7.5 
Duration of study 
7.5.1 Definition of the start and end of the trial  
The start of the trial is defined as the date of enrolment of the first volunteer. The end of the trial 
is the date of the last follow-up of the last volunteer. 
7.5.2 Duration of volunteer participation 
Volunteers will be considered to be enrolled in the trial on the receipt of the first vaccination. Total 
duration of follow-up will be approximately 8 months from enrolment. 
7.6 
Potential risks for volunteers 
7.6.1 Phlebotomy  
The maximum estimated volume of blood drawn (942mL) over the study period should not 
compromise otherwise healthy volunteers, however, volunteers will be monitored for anaemia. 
There may be minor bruising, local tenderness, pre-syncopal symptoms or syncope (rarely) 
associated with venepuncture, which will not be documented as AEs if they occur. The actual total 
blood volume may vary depending on any requirement for repeat clinical tests, e.g. in the event 
of a clinically significant abnormal result.  
7.6.2 Vaccinations 
Potential foreseeable risks from vaccination can be partially predicted from previous studies assessing 
protein vaccines, administered with the Matrix-M adjuvant, which include local and systemic 
reactions. Generally, an increase in solicited AEs is seen following administration of investigational 
vaccines with Matrix-M adjuvant, compared to the antigen alone or placebo, however, 
reactogenicity is transient with the vast majority of adverse events resolving within 2-7 days.  
Local AEs are likely to include injection site pain, erythema, swelling, itching and warmth. 
Forseeable systemic adverse events would include: headache, fatigue, myalgia, arthralgia, malaise, 

VAC085 Clinical Trial Protocol, V2.0 11 January 2023, University of Oxford 
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feverishness, fever and nausea. The majority of local and systemic AEs are foreseen to be mild to 
moderate in nature, however, it is anticipated that rarely, these may be reported as severe. Of the 
systemic AEs reported as severe following receipt of other investigational vaccines administered 
with Matrix-M adjuvant, myalgia is the most common.  
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 observed for an hour after each vaccination and vaccinated in a clinical area where ALS trained 
physicians, equipment and drugs are immediately available for the management of any SARs. 
7.7 
Potential benefits for volunteers 
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 P. 
falciparum transmission-blocking vaccine. Volunteers will also receive information about their 
general health status. 

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8 RECRUITMENT AND WITHDRAWAL FOR TRIAL VOLUNTEERS  
Volunteers may be recruited by use of an advertisement +/- registration form formally approved 
by the ethics committee(s) and distributed or posted in the following places: 
- 
In public places, including buses and trains, with the agreement of the owner/proprietor. 
- 
In newspapers or other literature for circulation. 
- 
On a website operated by the Investigators’ clinical trials group or with the agreement of 
the owner or operator (including online recruitment through our website).  
- 
As a post on Twitter, Facebook or other similar account owned and operated by the 
Investigators’ clinical trials group. 
- 
By email distribution to a group or list only with the express agreement of the network 
administrator or with equivalent authorisation, including those who have already 
expressed an interest in taking part in any clinical trial at the Oxford Vaccine Centre. This 
will include a link to the website advertisement. 
- 
On stalls or stands at exhibitions or fairs. 
- 
Via presentations (e.g. presentations at lectures or invited seminars). 
- 
Oxford Vaccine Centre databases: We may contact individuals from databases of groups 
within the CCVTM (including the Oxford Vaccine Centre database) of previous trial 
participants who have expressed an interest in receiving information about all future 
studies for which they may be eligible. 
8.1 
Informed consent 
The information sheet will be made available to the volunteer at least 24 hours prior to the 
screening visit, thus they will have a minimum of 24 hours to consider study participation. At the 
screening visit, the volunteer will be fully informed of all aspects of the trial, the potential risks and 
their obligations. The following will be emphasised: 
• Participation in the study is entirely voluntary. 
• Refusal to participate involves no penalty or loss of medical benefits. 
• The volunteer may withdraw from the study at any time.  
• The volunteer is free to ask questions at any time to allow him or her to understand the 
purpose of the study and the procedures involved. 
• There is no direct benefit from participating. 
• The volunteer’s general practitioner (GP) will be contacted to corroborate their medical 
history. Volunteers will only be enrolled in the study if written information regarding the 
volunteer’s medical history is obtained from the GP.   
• The volunteer will be registered on the TOPS database (The Overvolunteering Prevention 
System; www.tops.org.uk).  
• With volunteer consent, blood samples will be taken and any leftover samples stored 
indefinitely for use in other, ethically approved research.  
The aims of the study and all tests to be carried out will be explained. The volunteer will be given 
the opportunity to ask about details of the trial and will then have time to consider whether or not 
to participate before completing the written consent form. In addition to the consent form, 
volunteers will be asked to complete an additional separate form regarding consent for the GP to 
release medical records, a copy of which will be sent to the GP. 
The participant must personally sign and date the latest approved version of the Informed Consent 
Form before any trial specific procedures are performed. 

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Written and verbal versions of the Participant Information Sheet and Informed Consent Form will 
be presented to the participants detailing no less than: the exact nature of the trial; what it will 
involve for the participant; the implications and constraints of the protocol; the known side effects 
and any risks involved in taking part. It will be clearly stated that the participant is free to withdraw 
from the trial at any time without prejudice to future care, without affecting their legal rights and 
with no obligation to give the reason for withdrawal. 
The participant will be allowed the opportunity to question the Investigator, their GP or other 
independent parties to decide whether they will participate in the trial. Written informed consent 
will then be obtained by means of participant dated signature and dated signature of the person 
who presented and obtained the informed consent. 
The person who obtained the consent must be suitably qualified and experienced, and have been 
authorised to do so by the Chief Investigator. A copy of the signed Informed Consent Form will be 
given to the participant. The original signed form will be retained at the trial site. 
8.2  Inclusion and exclusion criteria  
8.2.1 Inclusion criteria 
The volunteer must satisfy all the following criteria to be eligible for the study: 
• Healthy adult aged 18 to 45 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 GP. 
• Volunteers with the potential to become pregnant only: must practice continuous effective 
contraception for the duration of the study (see section 10.10). 
• Agreement to refrain from blood donation for the duration of the study. 
• Able and willing to provide written informed consent to participate in the trial. 
8.2.2 Exclusion criteria 
The volunteer may not enter the study if any of the following apply: 
• History of clinical malaria (any species). 
• Travel to a clearly malaria endemic locality during the study period or within the preceding 
six months. 
• Use of immunoglobulins or blood products (e.g., blood transfusion) in the last three 
months. 
• Receipt of any vaccine in the 30 days preceding enrolment, or planned receipt of any other 
vaccine within 30 days following each study vaccination, with the exception of COVID-19 
vaccines, which should not be received between 14 days before to 7 days after any study 
vaccination. 
• Receipt of an investigational product in the 30 days preceding enrolment, or planned 
receipt during the study period. 
• Concurrent involvement in another clinical trial or planned involvement during the study 
period. 
• Prior receipt of an investigational vaccine likely to impact on interpretation of the trial data, 
as assessed by the Investigator. 
• Any confirmed or suspected immunosuppressive or immunodeficient state, including HIV 
infection; asplenia; recurrent, severe infections and chronic (more than 14 days) 

VAC085 Clinical Trial Protocol, V2.0 11 January 2023, University of Oxford 
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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 reaction to vaccinations. 
• Pregnancy, lactation or 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 that may 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 
25 standard UK units every week. 
• Suspected or known injecting drug abuse in the 5 years preceding enrolment. 
• Hepatitis B surface antigen (HBsAg) detected in serum. 
• Seropositive for hepatitis C virus (antibodies to HCV) at screening (unless volunteer has 
taken part in a prior hepatitis C vaccine study with confirmed negative HCV antibodies prior 
to participation in that study, and negative HCV ribonucleic acid (RNA) PCR at screening for 
this study). 
• Volunteers unable to be closely followed for social, geographic or psychological reasons. 
• Any clinically significant abnormal finding on biochemistry or haematology blood tests, 
urinalysis or clinical examination. In the event of abnormal test results, confirmatory repeat 
tests will be requested. Procedures for identifying laboratory values meeting exclusion 
criteria are shown in SOP VC027.  
• 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 
8.2.3 Prevention of ‘Over Volunteering’ 
Volunteers will be excluded from the study if they are concurrently involved in another trial. In 
order to check this, volunteers will be asked to provide their National Insurance or Passport 
number (if they are not entitled to a NI number) and will be registered on a national database of 
participants in clinical trials (www.tops.org.uk). 
8.2.4 Vaccination and re-vaccination exclusion criteria 
The following events associated with vaccine immunisation constitute absolute contraindications 
to further administration of vaccine. If any of these events occur during the study, the participant 
must be withdrawn and followed until resolution of the event, as with any adverse event:  
• Anaphylactic reaction following administration of vaccine.  
• Pregnancy.  
The following AEs constitute contraindications to administration of vaccine at that point in time; if 
any one of these AEs occurs at the time scheduled for vaccination, the participant may be 
vaccinated at a later date, or withdrawn at the discretion of the Investigator. The participant 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 

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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.  
• Current COVID-19 infection, defined as ongoing symptoms with positive COVID-19 PCR 
swab test taken during current illness or positive COVID-19 PCR swab or rapid antigen test 
within preceding 7 days without symptoms. Vaccinations will be delayed by a minimum of 
7 days from the date of the first positive COVID-19 PCR swab or rapid antigen test, as long 
as symptoms are improving or resolved. It will be at the discretion of the Investigator to 
withdraw a participant if they develop severe COVID-19 disease. 
8.2.5 Concomitant medications  
As set out by the exclusion criteria, volunteers may not enter the study if they have received any 
vaccine in the 30 days prior to enrolment or there is planned receipt of any other vaccine within 
30 days of each vaccination, any investigational medicinal product within 30 days prior to 
enrolment or if receipt is planned during the study period, or any chronic (more than 14 days) 
immunosuppressant medication within 6 months prior to enrolment or if receipt is planned at any 
time during the study period (inhaled and topical steroids are permitted). One exception is COVID-
19 vaccines, which should not be received between 14 days before to 7 days after any study 
vaccinations. 
8.3 
Withdrawal of volunteers 
In accordance with the principles of the most recent revision of the Declaration of Helsinki (2013) 
and any other applicable regulations, a volunteer has the right to withdraw from the study at any 
time and for any reason and is not obliged to give his or her reasons for doing so. In addition, the 
volunteer may withdraw/be withdrawn from further study procedures at any time in the interests 
of the volunteer’s health and wellbeing, or for any of the following reasons: 
• Administrative decision by the Investigator. 
• Ineligibility (either arising during the study or retrospectively, having been overlooked at 
screening). 
• Significant protocol deviation. 
• Volunteer non-compliance with study requirements. 
• An AE, which requires discontinuation of the study involvement or results in inability to 
continue to comply with study procedures. 
The Local Safety Monitor (LSM) may recommend withdrawal of volunteers. The reason for 
withdrawal from further study procedures will be recorded in the Case Report Form (CRF) where 
available. For all AEs, appropriate follow-up visits or medical care will be arranged, with the 
agreement of the volunteer, until the AE has resolved, stabilised or a non-trial related causality 
has been assigned.  
Any volunteer who fails to attend for two or more follow-up visits will be deemed to have 
withdrawn from the study.  
If a volunteer withdraws from the study, blood samples collected before their withdrawal from the 
trial will be used/stored unless the volunteer specifically requests otherwise. Similarly, all data 
collected up to the point of withdrawal will be stored, unless they specifically request for it to be 
destroyed. Volunteers are free to request that their blood samples be destroyed any time during 
or after the study.  

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In all cases of participant withdrawal, excepting those of complete consent withdrawal, long-term 
safety data collection, including some procedures such as safety bloods, will continue as 
appropriate if participants have received one or more vaccine doses.  
8.4 
Pregnancy 
Should a volunteer become pregnant during the trial, they will be followed up as other volunteers 
and in addition will be followed until pregnancy outcome, with their permission. Depending on the 
timings of pregnancy outcome relative to planned study follow-up timepoints, this may involve 
additional telephone consultations. We will not routinely perform venepuncture on such 
volunteers. 
 
 

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9. TREATMENT OF TRIAL VOLUNTEERS 
This section describes the clinical procedures for evaluating study participants, vaccination and 
follow-up after vaccination.  
9.1 
Trial sites 
Volunteers will be recruited and undergo all visits at the CCVTM, Oxford, this is inclusive of 
screening, vaccinations and follow-up visits.   
9.2 
Study procedures 
Procedures will be performed at the time points indicated in the schedule of events (Table 2). 
Additional procedures or laboratory tests may be performed, at the discretion of the Investigators 
if clinically necessary.  
9.2.1 Observations 
Pulse, blood pressure and temperature will be measured at the time points indicated in the 
schedule of events (Table 2). In addition, weight and height will be measured at screening. 
9.2.2 Blood tests 
Blood will be drawn at the time points indicated in the schedule of events (Table 2) and the 
following laboratory assays performed: 
At Oxford University Hospitals NHS Foundation Trust, using NHS standard procedures: 
• Haematology: Full blood count.  
• Biochemistry: Sodium, potassium, urea, creatinine, albumin and liver function tests. 
• Diagnostic serology: HBsAg, HCV antibodies, HIV antibodies. 
• Immunology: Human Leukocyte Antigen (HLA) typing at the day of enrolment (D0). 
At the Jenner Institute research laboratories: 
• Immunology:  
o Pfs48/45-specific immunogenicity will be assessed by a variety of immunological 
assays. These will include ex vivo ELISpot assays for interferon gamma and antibody 
ELISAs for measuring total IgG, isotype and avidity.  
o The functional activity of the vaccine-induced antibodies will be tested using 
membrane-feeding assays. The functional activity of the purified IgG will be tested 
against P. falciparum by membrane feeding assay.  
o Other exploratory immunological assays including cytokine analysis, other antibody 
assays (plasma cell and memory B cell 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. B cells, plasma and/or 
serum may also be analysed and used to produce human monoclonal antibodies. 
o Samples may also be sent to collaborating laboratories within and outside the UK 
(including outside of Europe) for other immunological assays as required. This would 
involve the transfer of serum, plasma or blood cells to these laboratories, but these 
would remain pseudo-anonymised. Informed consent for this will be obtained from 

VAC085 Clinical Trial Protocol, V2.0 11 January 2023, University of Oxford 
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volunteers. Immunological assays will be conducted according to the procedures 
established in the test laboratories.  
With the volunteers’ informed consent, any leftover cells, plasma, serum (or their purified 
components) will be stored indefinitely in the Oxford Vaccine Centre Biobank for future 
immunological analysis of malaria-specific or vaccine-related responses. This may include human 
DNA and RNA analysis. If a participant elects not to permit this, all of that participant’s leftover 
samples will be discarded after the required period of storage to meet Good Clinical Practice (GCP) 
and regulatory requirements. 
9.2.3  Urinalysis 
Urine will be tested for the presence of clinically significant proteinuria, glycosuria or haematuria 
(as defined in Appendix A) at the screening visits. For volunteers with the potential to become 
pregnant (see section 10.10 for definition), urine will be tested for beta-human chorionic 
gonadotrophin (β-HCG) at screening, immediately prior to each vaccination, at days 0, 28 and 56 
and on day 84.  
9.2.4 Vaccinations  
Before each vaccination, the ongoing eligibility of the volunteer will be reviewed. The vaccine will 
be administered as described above in section 5.3. The injection site will be covered with a sterile 
dressing and the volunteer will stay in the local trial site for observation for 1 hour, in case of 
immediate AEs. 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 
after vaccination (+/- 10 minutes).  
An oral thermometer and tape measure 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.  
9.2.5 Management of post-vaccination fevers 
Post-vaccination fevers may cause diagnostic uncertainty as concurrent COVID-19 infection is a 
possibility. If a participant develops a fever within the first 24 hours after vaccination and no other 
symptoms associated with COVID-19 disease (new onset cough, anosmia or ageusia), then post-
vaccination fever is most likely.  
If a participant develops a fever post-vaccination, which is associated with either new onset cough 
or anosmia or ageusia, they will be advised to self-isolate as per UK Health Security Agency (UK 
HSA) guidance and directed to local community based COVID-19 testing. Whilst awaiting their 
COVID-19 PCR test results, participants will be advised not to attend the clinic for study visits.  
If a participant tests positive for COVID-19 at any stage of the study, they will be advised to follow 
current UK HSA guidance in place. During clinic visits protective equipment (PPE) will be worn as 
per the infection control SOP that covers all PPE requirements for clinical trials at the Oxford 
Vaccine Centre in the era of the COVID-19 pandemic.  
Any study visits which are due whilst a participant is self-isolating, should be rescheduled to occur 
when self-isolation ends, if this falls within the visit window. If a participant remains in self-
isolation during the period of the visit window, then the study visit will be conducted by telephone 
and no blood tests will be taken. Study  visit windows have been increased in order to 
accommodate these scenarios.  

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If a participant is self-isolating for a prolonged period post-vaccination and hence would miss all 
three initial post vaccination visits, then it will be at the Investigator’s discretion to conduct these 
as physical visits at CCVTM to allow full assessment including blood tests. These study visits would 
be conducted in a side room with study staff wearing level 1 PPE. 
9.2.6 E-diary 
Following each vaccination, volunteers will be asked to complete an e-diary. Volunteers will be 
asked about foreseeable local and systemic AEs for 7 days (solicited AEs). After this, volunteers will 
be asked to record any AE daily, for 28 days (unsolicited AEs). E-diaries will be reviewed with 
volunteers at each post-vaccination clinic visit.  
An electronic PDF copy of the e-diary or hard copy will be issued to participants if there are 
technical issues with the e-diary or if the participant has internet/accessibility issues. 
9.2.7 Photographs 
Photographs of the injection site may be taken in the event of severe pain, redness and/or swelling 
following vaccination to enable assessment of any improvement/deterioration. Severe is defined 
as a grading of 3 in any one of the categories detailed in Table 6. Photographs will not include the 
participant’s face. 
9.3 
Study visits  
All clinical reviews and procedures will be undertaken by one of the clinical team. The procedures 
to be included in each visit are documented in Table 2. Each review is assigned a time point and a 
window period within which the review will be conducted. 
9.3.1 Screening visits  
There 
will 
be 
an 
initial 
pre-screening 
questionnaire 
that 
is 
completed 
online 
(https://www.onlinesurveys.ac.uk) to assess eligibility. Use of this tool has been approved by the 
University of Oxford. If the prospective participant meets initial eligibility criteria, we will seek 
consent via email to subsequently contact individuals by telephone. During this pre-screening 
telephone call with a member of the clinical team to discuss the study, we will provide the 
opportunity for interested individuals to discuss the study, eligibility criteria and study 
requirements. If individuals continue to express an interest in taking part in the trial following the 
pre-screening call, and if they meet eligibility criteria, a screening visit will be scheduled.  
Screening visits may take place up to 90 days prior to enrolment. Informed consent will be gained 
at screening. If consent is given, the screening procedures indicated in the schedule of events 
(Table 2) will be undertaken.  
The potential participant’s GP will be contacted with the written permission of the participant after 
screening using the GP screening letter. This will enable the study team to ascertain any significant 
medical history and act as notification that the participant has volunteered for the study. 
During the screening the volunteers will be asked to provide their National Insurance or passport 
number so that this can be entered on to a national database which helps prevent volunteers from 
participating in more than one clinical trial simultaneously or over-volunteering for clinical trials 
(https://www.tops.org.uk). 
Where more than 90 days has lapsed between the initial screening visit and date of enrolment, a 
re-screening visit will be conducted to ascertain if there have been any changes in the medical 
history or physical examination findings. Urinalysis, biochemistry and haematology blood tests will 

VAC085 Clinical Trial Protocol, V2.0 11 January 2023, University of Oxford 
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be repeated. Other blood tests will not be repeated routinely. Where a reply from the potential 
participant’s GP has already been received, the GP will not be re-contacted unless there is any 
need to obtain futher information where appropriate (e.g. regarding a new medical event or 
finding) and at the discretion of the Investigator. 
Hepatitis B, Hepatitis C and HIV status will be checked via blood tests at the screening visit. Some 
individuals may test positive for Hepatitis C virus due to their previous involvement in a Hepatitis 
C vaccine study. If this applies to an individual and they wish to take part in our study, with their 
consent, we will contact the team responsible for the Hepatitis C vaccine study to check the 
participant’s Hepatitis C status prior to enrolment in this malaria vaccine study. A copy of the 
consent will be held by both study teams. 
9.3.2 Enrolment and first vaccination (V1) with Pfs48/45 in Matrix-M adjuvant (all groups) 
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 first dose of vaccine and be enrolled in the trial.  
At the day 0 visit, any new medical issues or symptoms that have arisen will be assessed. Physical 
observations, urinary β-hCG test in volunteers with the potential to become pregnant and 
venepuncture for immunology and safety bloods will be undertaken according to Table 3. The 
inclusion and exclusion criteria for the study will be reviewed. If the volunteer remains eligible and 
there are no contraindications to vaccination, the Pfs48/45 vaccine, formulated in Matrix-M 
adjuvant will be administered according to the doses described in Table 2 and as described in 
Section 5.3. The visit will be carried out as described in above Section 9.2.4.  
9.3.3 Reviews post-vaccination on days 1, 7 and 14 (all groups) 
On subsequent visits, the volunteers will be assessed for local and systemic AEs, using the e-diary, 
interim history, physical examination and blood tests at the time points indicated in the schedule 
of events (Table 2). Blood will also be taken for exploratory immunology analyses.  
Volunteers will be asked to record all AEs via the e-diary. E-diaries will be reviewed with volunteers 
at the clinic visits on days 1 and 7. Volunteers will be asked directly about foreseeable local and 
systemic AEs for 7 days (solicited AEs). They will also be asked daily about any new symptoms from 
day 7 until day 28 via the diary but will not be asked directly about foreseeable symptoms. E-diaries 
will be reviewed for details of solicited and unsolicited AEs for 28 days after each vaccination, and 
any new or previously undocumented medical issues or symptoms that have arisen will be 
assessed. Physical observations and venepuncture for immunology and safety bloods will be 
undertaken according to Table 2. At any post-vaccination visit, where valid written, informed 
consent is in place, photographs may be taken for clinical comparison. This will enable qualitative 
as well as quantitative assessment of redness and swelling at the injection site. Confidentiality will 
be maintained as described in Section 13.5. 
9.3.4 Second vaccination (V2) with Pfs48/45 in Matrix-M adjuvant on day 28 (all groups) 
This visit will include a follow-up visit for the first vaccination and administration of the second 
vaccine. Any new medical issues or symptoms that have arisen will be assessed. Physical 
observations, urinary β-hCG test in volunteers with the potential to become pregnant and 
venepuncture for immunology and safety bloods will be undertaken according to Table 2. The 
inclusion and exclusion criteria for the study will be reviewed. If the volunteer remains eligible and 
there are no contraindications to vaccination, the Pfs48/45 vaccine formulated in Matrix-M 
adjuvant will be administered intramuscularly and preferentially into the opposite arm (if 

VAC085 Clinical Trial Protocol, V2.0 11 January 2023, University of Oxford 
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possible)to that used for the first vaccination and as described in sections 5.3 and 9.2.4. Dosing 
will be determined by group allocation (see Table 1). 
9.3.5 Reviews post-vaccination on days 29, 35 and 42 (all groups) 
On subsequent visits, the volunteers will be assessed for local and systemic AEs, using the e-diary, 
interim history, physical examination and blood tests at the time points indicated in the schedule 
of events (Table 2). Blood will also be taken for exploratory immunology analysis.  
Volunteers will be asked to record all AEs via the e-diary. E-diaries will be reviewed with volunteers 
at the clinic visits on days 29 and 35. Volunteers will be asked directly about foreseeable local and 
systemic AEs for 7 days (solicited AEs). They will also be asked daily about any new symptoms from 
day 35 until day 56 via the diary but will not be asked directly about foreseeable symptoms. E-
diaries will be reviewed for details of solicited and unsolicited AEs for 28 days after each 
vaccination, and any new or undocumented medical issues or symptoms that have arisen will be 
assessed. Physical observations and venepuncture for immunology and safety bloods will be 
undertaken according to Table 2. At any post-vaccination visit, where valid written, informed 
consent is in place, photographs may be taken for clinical comparison. Confidentiality will be 
maintained as described in Section 13.5. 
9.3.6 Third vaccination (V3) with Pfs48/45 in Matrix-M adjuvant on day 56 (all groups) 
This visit will include a follow-up visit for the second vaccination and administration of the third 
vaccine. Any new medical issues or symptoms that have arisen will be assessed. Physical 
observations, urinary β-hCG test in volunteers with the potential to become pregnant and 
venepuncture for immunology and safety bloods will be undertaken according to Table 2. The 
inclusion and exclusion criteria for the study will be reviewed. If the volunteer remains eligible and 
there are no contraindications to vaccination, the Pfs48/45 vaccine, formulated in Matrix-M 
adjuvant will be administered intramuscularly and preferentially into the opposite arm (if possible) 
to that used for the second vaccination and as described in sections 5.3 and 9.2.4. Dosing will be 
determined by group allocation (see Table 1). 
9.3.7 Reviews post-vaccination on days 57, 63, 70 and 84  
On subsequent visits, the volunteers will be assessed for local and systemic adverse events, using 
the e-diary, interim history, physical examination and blood tests at the time points indicated in 
the schedule of attendances (Table 2). Blood will also be taken for exploratory immunology 
analysis.  
Volunteers will be asked to record all AEs via the e-diary and at visits on days 57 and 63, during 
which e-diaries will be reviewed with volunteers. Volunteers will be asked directly about 
foreseeable local and systemic AEs for 7 days (solicited AEs). Volunteers will be asked to continue 
to record any new symptoms from day 63 until day 84, on a daily basis via the diary but will not be 
asked directly about foreseeable symptoms. E-diaries will be reviewed for details of solicited and 
unsolicited AEs for 28 days after each vaccination, and any new or undocumented medical issues 
or symptoms that have arisen will be assessed. Physical observations, procedures (including a 
urinary pregnancy test at D84) and venepuncture for immunology and safety bloods will be 
undertaken according to Table 3. At any post-vaccination visit, where valid written, informed 
consent is in place, photographs may be taken for clinical comparison. Confidentiality will be 
maintained as described in Section 13.5. 

VAC085 Clinical Trial Protocol, V2.0 11 January 2023, University of Oxford 
Page 44 of 66 
 
9.3.8 Long-term follow up post-vaccination on days 140 and 240  
On days 140 and 240, volunteers will be assessed for serious adverse events, by interim history 
and physical examination, as deemed necessary by the Investigator. Blood tests will be performed 
as indicated in the schedule of attendances for these timepoints (Table 2). Blood will also be taken 
for exploratory immunology analysis. At any post-vaccination visit, where valid written, informed 
consent is in place, photographs may be taken for clinical comparison. Confidentiality will be 
maintained as described in Section 13.5. 
 
 
 
 
 
 
 
 
 
 

VAC085 Clinical Trial Protocol, V2.0 11 January 2023, University of Oxford 
Page 45 of 66 
 
  
S 
V1 
  
  
  
V2 
  
  
  
V3 
  
  
  
  
  
  
Attendance number 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13 
14 
15 
16 
Timeline (days)  
  
0 
1 
7 
14 
28 
29 
35 
42 
56 
57 
63 
70 
84 
140 
240 
Window (days) 
-90 
  
±1 
±2 
-2/+4 
-7/+14 
±1 
±2 
-2/+4 
-7/+14 
±1 
±2 
-2/+4 
-7/+14 
±14 
±14 
Inclusion/Exclusion criteria 
X 
X 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
Informed consent 
X 
X 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
Physical observations* 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Medical history/ examination 
X 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
Photographs 
 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
Urinalysis 
X 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
Urine β-hCG** 
X 
X 
  
  
  
X 
  
  
  
X 
  
  
  
X  
  
  
Review contraindications 
  
X 
  
  
  
X 
  
  
  
X 
  
  
  
  
  
  
Review AEs & medications 
  
  
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Vaccination 
  
X 
  
  
  
X 
  
  
  
X 
  
  
  
  
  
  
Diary card issued 
  
X 
  
  
  
X 
  
  
  
X 
  
  
  
  
  
  
Diary card collected 
  
  
  
  
  
X 
  
  
  
X 
  
  
  
X 
  
  
Immunology plasma (mL) 
  
40 
10 
10 
30 
30 
10 
60 
30 
30 
10 
60 
110 
80 
30 
30 
Immunology serum (mL) 
  
40 
  
  
20 
40 
  
  
40 
40 
  
  
30 
40 
20 
20 
RNA preservation (mL) 
  
3 
3 
  
  
3 
3 
  
  
3 
3 
  
  
  
  
  
Haematology (mL)# 
2 
2 
  
2 
2 
2 
  
2 
2 
2 
  
2 
2 
2 
  
  
Biochemistry (mL) ## 
3 
3 
  
3 
3 
3 
  
3 
3 
3 
  
3 
3 
3 
  
  
HLA typing (mL) 
  
4 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
HIV, HBV, HCV serology (mL) 
5 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
Total Blood Volume (mL) 
10 
92 
13 
15 
55 
78 
13 
65 
75 
78 
13 
65 
145 
125 
50 
50 
Cumulative blood volume (mL) 
10 
102 
115 
130 
185 
263 
276 
341 
416 
494 
507 
572 
717 
842 
892 
942$ 
Table 2: Schedule of events  
S = screening visit; V = vaccination; (X) = If considered necessary, emphasising any acute complaints. *Physical observations will include blood pressure, heart rate and temperature, plus 
height and weight at screening only. **For volunteers with the potential to become pregnant. # Haematology will comprise full blood count. ## Biochemistry will include sodium, potassium, 
urea, creatinine, albumin, liver function tests. $ Cumulative blood volume if blood taken as per schedule and excluding any repeat safety blood tests that may be necessary.

VAC085 Clinical Trial Protocol, V2.0 11 January 2023, University of Oxford 
Page 46 of 66 
 
10 ASSESSMENT OF SAFETY 
Safety will be assessed by the frequency, incidence and nature of AEs and SAEs arising during the 
study. The safety profile will be assessed on an ongoing basis by the Investigators during the safety 
reporting window for each participant (from provision of consent to their last study visit). The CI 
and relevant Investigators (as per the trial delegation log) will also review safety issues and SAEs 
as they arise. 
10.1 Definitions 
10.1.1 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. 
Each participant-reported AE will be graded by the participant according to the table for grading 
severity of adverse events (see Table 3). Severity gradings may be reviewed and discussed with the 
participants at the clinic visits. 
10.1.2 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 ARs. 
10.1.3 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 or prolongation of hospitalisation, regardless of length of stay, even if it is 
a precautionary measure for continued observation. Hospitalisation (including inpatient or 
outpatient hospitalisation for an elective procedure) for a pre-existing condition that has 
not worsened unexpectedly does not constitute an SAE. 
• An important medical event (that may not cause death, be life-threatening, or require 
hospitalisation) that may, based upon appropriate medical judgment, jeopardise the 
volunteer and/or require medical or surgical intervention to prevent one of the outcomes 
listed above. 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. 

VAC085 Clinical Trial Protocol, V2.0 11 January 2023, University of Oxford 
Page 47 of 66 
 
• Congenital anomaly or birth defect. 
10.1.4 Serious Adverse Reaction (SAR) 
An AE (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. 
10.1.5 Suspected Unexpected Serious Adverse Reaction (SUSAR) 
All SAEs at least possibly related to Pfs48/45 in Matrix-M will be considered unexpected and be 
reported as SUSARs. 
10.1.6 Foreseeable Adverse Reactions 
The foreseeable ARs following vaccination with Pfs48/45 in Matrix-M adjuvant are: injection site 
pain, erythema, warmth, swelling, pruritus, myalgia, arthralgia, headache, fatigue, fever, 
feverishness, malaise and nausea. These AEs will be listed as ‘solicited AEs’ providing they occur 
within 7 days of the day of vaccination. ‘Unsolicited AEs’ are AEs other than the foreseeable ARs 
occurring within the first 7 days, or any AEs occurring after the first 7 days after vaccination. 
10.2 Causality Assessment 
For every unsolicited AE, an assessment of the relationship of the event to the administration of 
the vaccine will be undertaken by the CI or the CI-delegated clinician at the coordinating site 
(Oxford). An intervention-related AE refers to an AE for which there is a possible, 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 3).  
Causality assessment will take place during planned safety reviews, interim analyses (e.g. if a 
holding or stopping rule is activated) and at the final safety analysis, except for SAEs, for which 
causality should be assigned by the clinically trained reporting Investigator. 
 
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. 

VAC085 Clinical Trial Protocol, V2.0 11 January 2023, University of Oxford 
Page 48 of 66 
 
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 3: Guidelines for assessing the relationship of vaccine administration to an AE. 
10.3 Reporting Procedures for all AEs  
All AEs occurring in the 28 days following each vaccination observed by the Investigator or reported 
by the volunteer, whether or not attributed to the study vaccine, will be recorded. Recording and 
reporting of all AEs will take place as detailed in SOP VC027. We will not record bruising due to 
blood-taking or cannulation.  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). SAEs will be collected throughout the entire trial period. For 
each participant, this means the safety reporting window per participant (from provision of study 
consent to final follow-up visit). 
Each AE will be graded by the participant according to the table for grading severity of AEs (see 
Table 4). Severity gradings may be reviewed and discussed with the participants at the clinic visits. 
Volunteers will be instructed on how to self-assess the severity of these AEs. There will also be 
space on the e-diary to self-document unsolicited AEs, and whether medication was taken to 
relieve the symptoms. 
The severity of clinical AEs will be assessed by the Investigators according to the scales in Tables 4-
6. Severity grading for laboratory AEs are dependent on the Oxford University Hospitals 
laboratory’s reference and will be graded according to the scales in the site-specific table that can 
be found in the Trial Master File (TMF). These ranges will be based on FDA guidance relative to 
local laboratory reference ranges [https://www.fda.gov/media/73679/download].  
 
 
 
 
 

VAC085 Clinical Trial Protocol, V2.0 11 January 2023, University of Oxford 
Page 49 of 66 
 
Grade 
Severity 
GRADE 0 
None 
GRADE 1 
Mild: 
Transient 
or 
mild 
discomfort 
(< 
48 
hours); 
no 
medical 
intervention/therapy required 
GRADE 2 
Moderate: Mild to moderate limitation in activity – some assistance may be 
needed; no or minimal medical intervention/therapy required 
GRADE 3 
Severe: Marked limitation in activity, some assistance usually required; may 
require medical intervention/therapy  
Table 4: Severity grading criteria for AEs. 
 
Physical Observations 
Grade 1 
Grade 2 
Grade 3 
Tachycardia – beats per 
min* 
101-115 
116-130 
>130 
Hypotension (systolic) 
mm Hg 
85-89 
80-84 
<80 
Hypertension (systolic) 
mm Hg** 
141-159 
160-179 
180 
Hypertension (diastolic) 
mm Hg** 
91-99 
100-109 
110 
Fever °C 
37.6 – 38.0 
38.1-39.0 
>39.0 
Table 5: Severity grading criteria for clinically significant abnormal physical observations. All 
observations should be measured at rest. *Only applies when resting heart rate is between 60 and 
100 beats per minute. Use clinical judgement when characterising bradycardia in some healthy 
participant populations (e.g. conditioned athletes). **Systolic or diastolic hypertension may only 
be confirmed as clinically significant (and therefore an AE) if persistently present when 
observations are repeated (i.e. isolated measurements of hypertension are not clinically 
significant). 
 
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 

VAC085 Clinical Trial Protocol, V2.0 11 January 2023, University of Oxford 
Page 50 of 66 
 
Erythema at injection site 
1 
>3 - ≤50 mm 
2 
>50 - ≤100 mm 
3 
>100 mm 
Swelling at injection site* 
1 
>3 - ≤20 mm 
2 
>20 - ≤50 mm 
3 
>50 mm 
Table 6: Severity grading criteria for local adverse events. *Erythema or swelling ≤3mm is an 
expected consequence of skin puncture and will therefore not be considered an adverse event. 
10.4 Reporting Procedures for SAEs  
This will be conducted in accordance with the SOP OVC005 Safety Reporting. In order to comply 
with current regulations on SAE 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 after the Investigators become aware of their occurrence, 
as described in SOP OVC005. Copies of all reports will be forwarded for review to the CI (as the 
Sponsor’s representative) within 24 hours of the Investigator being aware of the suspected SAE. 
The Local Safety Monitor (LSM) will be notified of SAEs which are deemed possibly, probably or 
definitely related to study interventions; the LSM will be notified immediately (within 24 hours) of 
the Investigators’ being aware of their occurrence. SAEs will not normally be reported immediately 
to the REC 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 CI 
and/or LSM. In addition to the reporting above, the Investigator shall include all SAEs in the annual 
Development Safety Update Report (DSUR) report. 
10.4.1 Reporting Procedures for SUSARS 
The CI will report all SUSARs to the MHRA and REC within required timelines (15 days for all 
SUSARs, unless life-threatening or fatal. Fatal and life-threatening SUSARs will be reported to the 
MHRA and REC no later than 7 calendar days after the Sponsor or delegate is first aware of the 
reaction. Any additional relevant information will be reported within 8 calendar days of the initial 
report. All other SUSARs will be reported within 15 calendar days of awareness. The CI 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. 
10.5 Development Safety Update Report 
The CI will submit (in addition to the expedited reporting above) DSURs once a year throughout 
the clinical trial, or on request, to the Competent Authority (MHRA in the UK, Ethics Committee, 
HRA, Host NHS Trust and Sponsor). 

VAC085 Clinical Trial Protocol, V2.0 11 January 2023, University of Oxford 
Page 51 of 66 
 
10.6 AEs of Special Interest 
AEs of special interest will be reported as SAEs. These are: 
• Severe hypersensitivity reactions (e.g. anaphylaxis) 
• Any new, suspected autoimmune or chronic inflammatory disease 
10.7 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 
SOP VC027. Abnormal clinical findings from medical history, examination or blood tests will be 
assessed as to their clinical significance throughout the trial. Laboratory AEs 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. 
10.8 Local Safety Monitor 
The LSM will be appointed to provide real-time safety oversight. The LSM will review SAEs deemed 
possibly, probably or definitely related to study interventions. The LSM will be notified within 24 
hours of the Investigators’ being aware of their occurrence. The LSM has the power to terminate 
the study if deemed necessary following a study intervention-related SAE. At the time of writing, 
the LSM is Prof Brian Angus, a Clinical Tutor in Medicine, Honorary Consultant Physician and 
Director, Centre for Tropical Medicine at the University of Oxford. All correspondence between 
the Investigator and the LSM will be conveyed by the Investigator to the trial Sponsor on their 
request. 
The LSM may be contacted for advice and independent review by the Investigator or trial Sponsor 
in the following situations: 
• Following any SAE deemed to be possibly, probably, or definitely related to a study 
intervention 
• Any other situation where the Investigator or trial sponsor feels independent advice or 
review is important. 
The study can be put on hold upon advice of the LSM, CI, study sponsor or Ethical Committee(s), 
for any single event or combination of multiple events which, in their professional opinion, 
jeopardise the safety of the participants or the reliability of the data. If the study is placed on hold 
it may only be restarted following discussion with and approval from the LSM, the ethics 
committee(s), the trial Sponsor, the MHRA and CI. 
10.9 Safety Stopping/Holding Rules  
Safety holding rules have been developed considering the fact that this is a first-in-human study.  
10.9.1 Group holding rules (applies to Groups 1 and 2/3) 
If a holding rule is activated, then further vaccinations in the group (Group 1 and/or Group 2/3) 
will not occur until an internal safety review has been conducted and it is deemed appropriate to 
restart dosing. Note that as Groups 2 and 3 will be recruited in parallel, if a group holding rule is 
activated for one of the two groups, then further vaccinations in both groups would go on hold. 
The regulatory authority must be informed of the hold via substantial amendment and a request 

VAC085 Clinical Trial Protocol, V2.0 11 January 2023, University of Oxford 
Page 52 of 66 
 
to restart dosing with pertinent data must be submitted as a further, subsequent 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. 
• 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 programmes on the various 
components of the vaccine (i.e. the Matrix-M adjuvant). 
The local ethics committee and adjuvant manufacturers (Novavax) will also be notified if a holding 
rule is activated or released to ensure that each Party is obliged to comply with the performance 
and activities related to the clinical trial (there is a confidential Safety Data Exchange Agreement, 
SDEA, in place). All vaccinated volunteers will be followed for safety until resolution or stabilisation 
(if determined to be chronic sequelae) of their AEs. Novaxax will be provided with SAE and 
pregnancy reports and any safety concerns causing a suspension as per the SDEA beween Oxford 
and Novavax 
The group holding rules are as follows: 
• Solicited local AEs: 
o If two or more doses of a vaccine are followed by a Grade 3 solicited local AE beginning 
within 2 days after vaccination (day of vaccination and one subsequent day) and 
persisting at Grade 3 for > 72 hrs. 
• Solicited systemic AEs:  
o If two or more doses of a vaccine are followed by a Grade 3 solicited systemic AE 
beginning within 2 days after vaccination (day of vaccination and one subsequent day) 
and persisting at Grade 3 for > 48 hrs.  
• Unsolicited AEs: 
o If two or more volunteers develop a Grade 3 unsolicited AE (including the same 
laboratory AE) that is considered possibly, probably or definitely related to vaccination. 
o However, if a study participant has a Grade 3 unsolicited (or laboratory) AE considered 
‘possibly’ related to vaccination which persists at Grade 3 < 48 hours that, in the opinion 
of the Investigator, is of non-clinical significance and where a different cause is judged 
as likely, the event will not be counted as part of the group holding AEs. 
• A serious AE considered possibly, probably or definitely related to vaccination occurs 
10.9.2 Individual stopping rules 
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). 
• 
Local reactions: Injection site ulceration, abscess or necrosis. 

VAC085 Clinical Trial Protocol, V2.0 11 January 2023, University of Oxford 
Page 53 of 66 
 
• 
Laboratory AEs: 
o The volunteer develops a Grade 3 laboratory AE considered possibly, probably or 
definitely related within 7 days after vaccination and persisting continuously at Grade 
3 for >72hrs. 
• Solicited systemic AEs:  
o The volunteer develops a Grade 3 systemic solicited AE 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 AEs:  
o The volunteer has a Grade 3 AE, considered possibly, probably or definitely related to 
vaccination, persisting continuously at Grade 3 for >72hrs.  
o The volunteer has an acute allergic reaction or anaphylactic shock following the 
administration of the vaccine investigational product. 
• A serious AE considered possibly, probably or definitely related to vaccination occurs 
10.10 Contraception and pregnancy 
Volunteers with the potential to become pregnant are defined as those who are fertile and able 
to become pregnant following menarche until becoming post-menopausal, unless permanently 
sterile. A post-menopausal state is defined as no menstruation for 12 months without a known 
cause. 
Volunteers with the potential to become pregnant 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 and there is 
currently no information about the effect of this vaccine on a foetus.  
Acceptable forms of contraception for volunteers with the potential to become pregnant are: 
• Established use of oral, injected or implanted hormonal methods of contraception. 
• Placement of an intrauterine device or intrauterine system. 
• Male sterilization: if the vasectomised partner is the sole partner for the participant. 
• True abstinence from sex with sperm-producing partners, when this is in line with the 
preferred and usual lifestyle of the participant (periodic abstinence and withdrawal are not 
acceptable methods of contraception). 
Pregnancy, lactation or intention to become pregnant during the study is an exclusion criteria. 
 
 

VAC085 Clinical Trial Protocol, V2.0 11 January 2023, University of Oxford 
Page 54 of 66 
 
11 STATISTICS 
11.1  Sample size 
This is an observational and descriptive safety study, where healthy adult volunteers living in the 
UK will be vaccinated with Pfs48/45, in formulation with the Matrix-M adjuvant. Up to a total of 
30 volunteers across three equally divided groups will be vaccinated. Each volunteer will receive 
three doses of Pfs48/45, formulated in 50 µg Matrix-M, on days 0, 28 and 56 (see Table 2 for exact 
dosing regimen depending on group allocation). This sample size should allow estimation and 
comparison of the frequency and magnitude of the outcome measures, at and between the three 
different dosing regimens. As this is the first study involving the Pfs48/45 in Matrix-M vaccine, 
there is no available data to enable more precise modelling/power calculations. However, it is 
considered that eight to ten volunteers in each group will be sufficient to enable detection of 
statistically significant differences in the primary and secondary outcome measures (see Section 
6) between the groups. Specifically, we will be able to assess the optimal dose for future studies 
based on the safety/tolerability as well as immunogenicity of each of the three dosing regimens. 
 
 
 
 

VAC085 Clinical Trial Protocol, V2.0 11 January 2023, University of Oxford 
Page 55 of 66 
 
12 QUALITY CONTROL AND QUALITY ASSURANCE PROCEDURES 
12.1  Investigator procedures 
Approved site-specific SOPs will be used at all clinical and laboratory sites. 
12.2 Monitoring 
Monitoring will be performed by Appledown external monitors using established procedures, 
according to the principles of Good Clinical Practice (GCP).  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 and GCP. The site will provide direct access to all trial-related source 
data/documents and reports for the purpose of auditing by the Sponsor and inspection by local 
and regulatory authorities. 
12.3 Modification to protocol 
No amendments to this protocol will be made without consultation with, and the agreement of, 
the Sponsor. Any amendments to the trial that become 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 CI and will be made 
a formal part of the protocol following ethical and regulatory approval, as applicable.  
An administrative change to the protocol is one that modifies administrative and logistical aspects 
of a protocol but does not affect the participant’s safety, the objectives of the trial and its progress. 
An administrative change may not require UK ethical committee approval; however, the REC will 
be notified in the event of any such change.  
The Investigator is responsible for ensuring that changes to an approved trial, during the period 
for which ethical committee(s) approval has already been given, are not initiated without review 
and approval from the ethics committee(s) and regulatory authorities except to eliminate 
apparent immediate hazards to the participant (Urgent Safety Measures only). Protocol adherence 
is a fundamental part of the conduct of a clinical study.  Changes to the approved protocol need 
prior approval unless for urgent safety reasons. Investigators must not request a protocol waiver 
to enter a patient who does not satisfy the selection criteria. 
12.4 Protocol deviation  
Any deviations from the protocol will be documented in a protocol deviation form and filed in the 
TMF.  
12.5 Audit & inspection  
The Quality Assurance manager performs systems-based internal audits to check that trials are 
being conducted, data recorded, analysed and accurately reported according to study protocols, 
departmental SOPs and in compliance with GCP. The audit schedule includes laboratory activities. 
The internal audits will supplement the independent monitoring process and will review processes 
not covered by the independent monitor. 
The Sponsor, trial site, ethical committee(s), and authorised individuals may carry out audit to 
ensure compliance with the protocol, GCP and appropriate regulations. GCP inspections may also 
be undertaken by the regulatory authority to ensure compliance with protocol and national 
regulations. The Sponsor will assist in any inspections. 
 

VAC085 Clinical Trial Protocol, V2.0 11 January 2023, University of Oxford 
Page 56 of 66 
 
12.6 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 participants of the trial; or 
(b) the scientific value of the trial. 
In the event that a serious breach is suspected the Sponsor must be contacted within 1 working 
day. In collaboration with the CI the serious breach will be reviewed by the Sponsor and, if 
appropriate, the Sponsor will report it to the REC, regulatory authority and the relevant NHS host 
organisation within seven calendar days. 
12.7 Publication policy 
The Investigators will be involved in reviewing drafts of the manuscripts, abstracts, press releases 
and any other publications arising from the study.  Authorship will be determined in accordance 
with the International Committee of Medical Journal Editors guidelines and other contributors will 
be acknowledged.  
12.8 Intellectual Property 
Ownership of intellectual property generated by employees of the University vests in the 
University.  The protection and exploitation of any new intellectual property is managed by the 
University’s technology transfer office, Oxford University Innovation. 

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13 ETHICS 
13.1 Declaration of Helsinki 
The Investigator will ensure that this study is conducted according to the principles of the current 
revision of the Declaration of Helsinki 2008. 
13.2 GCP guidelines 
The Investigator will ensure that this study is conducted in full conformity to the Medicines for 
Human Use (Clinical Trials) Regulations 2004 (SI 2004 No. 1031) and its amendments and with the 
guidelines for GCP (CPMP/ICH/135/95) July 1996.  
13.3 Approvals 
Following Sponsor approval, a copy of the protocol, proposed informed consent form, other 
written volunteer information and the proposed advertising material will be submitted to an 
appropriate REC and the MHRA for written approval. The Investigator will submit and, where 
necessary, obtain approval from the REC and the MHRA for all subsequent amendments to the 
protocol and associated trial documents. A non-substantial amendment does not require REC 
approval; however, the REC will be notified in the event of any such change. The Investigator will 
notify all deviations from the protocol or SAEs occurring at the site to the Sponsor and will notify 
the REC(s) and regulatory authorities of these if necessary and in accordance with procedures. 
13.4 
Reporting  
The CI shall submit once a year throughout the study, or on request, an Annual Progress Report to 
the REC, HRA (where required) and Sponsor. In addition, an End of Trial notification and final report 
will be submitted to the MHRA, the REC, host organisation and Sponsor. 
13.5 Volunteer confidentiality 
The research data will be pseudoanonymised; participant data will be identified by a unique study 
number in the CRF and database. Separate confidential files containing identifiable information 
and links to the study number will be stored in secured locations. These secured locations are 
either physical premises or web servers owned by the University of Oxford or in the case of the 
eCRF on the OpenClinica database. The OpenClinica database has undergone the Third Party 
Security Assessment process and been officially approved as a processor of data for which the 
University of Oxford is the controller. Only the Sponsor, Investigators, the clinical monitor, the 
ethical committee(s) and the regulatory authorities will have access to the records.  
Photographs taken (if required, with the volunteer’s written, informed consent) will not include 
the volunteer’s face and will be identified by the volunteer’s trial specific identification number 
only. Once developed, photographs will be stored as confidential records, as above. This material 
may be shown to other professional staff, used for educational purposes, or included in a scientific 
publication.  
 

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14 DATA HANDLING AND RECORD KEEPING 
14.1 Data handling 
The CI will be the data custodian with responsibility for delegating the receiving, entering, cleaning, 
querying, analysing and storing all data that accrues from the study. Data will be entered into the 
volunteers’ CRFs in a paper and/or electronic format (using the OpenClinica™ database). Electronic 
data will be stored on secure servers which are outsourced by OpenClinica™. OpenClinica™ meets 
FDA part 11B standards and is a system approved by the University of Oxford. This includes safety 
data, laboratory data and outcome data. Data are entered in a web browser on personal 
computers and then transferred to the OpenClinica Database by encrypted (https) transfer. Safety 
data will also be collected through an electronic diary, which is stored on a third party server (see 
Section 13.5).  
14.2 Record keeping 
The Investigators will maintain and retain appropriate medical and research records and essential 
documents for this trial in compliance with GCP and regulatory and institutional requirements for 
the protection of confidentiality of volunteers. The CI, co-investigators, clinical research nurses and 
authorised personnel will have access to records. The Investigators will permit authorised 
representatives of the Sponsor, ethical committee(s), regulatory agencies, and the monitors to 
examine (and when required by applicable law, to copy) clinical records for the purposes of quality 
assurance reviews, audits and evaluation of the study safety and progress. 
14.3 Source data and electronic case report forms (eCRFs) 
All protocol-required information will be collected in the electronic diaries and eCRFs designed by 
the Investigator. All source documents will be filed in the participants’ notes. Source documents 
are original documents, data, and records from which the volunteer’s eCRF data are obtained. For 
this study these will include, but are not limited to: the volunteer consent form, blood results, GP 
letters, laboratory records and correspondence. In the majority of cases, electronic diaries and 
eCRF entries will be considered source data as these are the site of the original recording (i.e. there 
is no other written or electronic record of data). In this study this will include, but is not limited to: 
medical history, medication records, vital signs, physical examination records, urinalysis results, 
blood results, AE data and details of study interventions. All source data and volunteer CRFs will 
be stored securely. 
14.4 Data protection  
The trial will comply with the UK General Data Protection Regulation (UK GDPR) and Data 
Protection Act 2018. The study protocol, documentation, data and all other information generated 
will be held in strictest confidence. No information concerning the study or the data will be 
released to any unauthorised third party, without prior written approval of the Sponsor. 
14.5 Storage and use of data 
The University of Oxford will keep identifiable information from participants collected during the 
study initially for 5 years after the study has finished. This will be limited to the minimum personal 
data necessary. In addition, we will securely store the anonymised research data and any research 
documents with personal information, such as consent forms, initially for 5 years after the end of 
the study. The need to store this information for longer, in relation to licensing of the vaccine will 
be reviewed every 5 years. Once the study has been completed, all documents would be archived 

VAC085 Clinical Trial Protocol, V2.0 11 January 2023, University of Oxford 
Page 59 of 66 
 
in a secure facility. Files will be confidentially destroyed if storage is no longer required. For 
effective vaccines that may be licensed, secure storage of research data may be required for at 
least 15 years after the end of the study, subject to adjustments in clinical trials regulations. 
Financial information will be stored for seven years in line with University of Oxford financial policy.  
 
Where specific written consent is provided, personal details may be used to contact participants 
about future related research. These details and clinical information will be held separately. Note 
that the informed consent form from this study will be retained, separately, for as long as the 
participant is on the database. If participants elect to take part in another study carried out by the 
Jenner Institute, personal information and medical information including blood test results may 
be accessed to avoid unnecessary repetition.  
Where specific written consent is provided, personal details may be used to contact participants 
about future related research. If participants elect to take part in another study carried out by the 
Jenner Institute, personal information and medical information including blood test results may 
be accessed to avoid unnecessary repetition.  
 

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15 FINANCING AND INSURANCE 
15.1 Financing 
The study is funded through a grant from OptiMalVax, a European Commission Horizon 2020 
funded project, as well as through internal funds from the Nuffield Department of Medicine, 
University of Oxford. The OptiMalVax grant aims to funds work to develop candidate multi-stage 
malaria vaccines through innovative trial designs in malaria-endemic areas.  
15.2 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).  
15.3 Compensation 
Volunteers will be compensated for their time and for the inconvenience caused by procedures as 
below.   
- Screening visit  
 
 
 
 
 
 
 
£25  
- Subsequent visits: 
 
- Travel expenses 
 
 
 
 
 
 
£15 per visit 
- Inconvenience of blood tests: 
 
 
 
 
£10 per blood donation 
- Time required for visit: 
 
 
 
 
 
£20 per hour 
Where travel expenses are greater than £15 per visit because the volunteer lives outside the city 
of the trial site, the volunteer may be given further reimbursement to meet the cost of travel 
necessary for study visits at the Investigator’s discretion.  
 
Table 7:  Estimated compensation amounts. 
Time in trial 
(approx.) 
Maximum visits Maximum volume of 
blood taken (mls) 
Compensation 
Groups 1-3 
1 year 
16 
942 
£790 

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16 APPENDICES 
Appendix A: laboratory values for exclusion 
Laboratory parameters for inclusion/exclusion in the trial will be considered on an individual basis, 
with Investigator discretion for interpretation of results and the need for repeated or further tests. 
In general, volunteers will be excluded if a result at screening constitutes what would qualify as a 
grade 1 (or higher) laboratory adverse event, according to the laboratory adverse event tables 
(filed in the TMF), on repeat of an abnormal test result. Urinalysis at screening will be assessed as 
per the table below: 
 
URINE ANALYSIS (using MULTISTIX) 
Protein* 
2+ or Protein creatinine ratio of ≥ 50 mg/mmol 
Blood£ 
2+ on two dipstick tests  
Glucose 
1+ 
Table 8: Urinalysis assessment.  
*In the event of the dipstick testing positive for protein with ≥1+ protein urine should be sent for 
a protein creatinine ratio. 
£ In the event of urine dipstick testing positive for ≥1+ blood with, or without, protein, a repeat 
dipstick test will be carried out to confirm haematuria. In volunteers of child-bearing potential, a 
menstrual history will be taken to elicit whether the participant is currently menstruating and if 
they are, urine dipstick will be repeated after 1 - 2 weeks. If blood and/or proteinuria persist in any 
volunteer, an interpretation of the results will be undertaken by the Investigator on an individual 
basis to determine if they will be excluded from the trial, and the appropriate follow-up arranged. 
 
 
 
 
 
 
 
 
 
 
 
 
 

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17 REFERENCES 
1. 
Organization, W.H. Malaria.  22/06/2021]; Available from: 
https://www.who.int/data/gho/data/themes/malaria. 
2. 
Kochar, D.K., et al., Plasmodium vivax malaria. Emerg Infect Dis, 2005. 11(1): p. 132-4. 
3. 
Organisation, W.H. World malaria report 2020. 2020  22/06/2021]; Available from: 
https://www.who.int/publications/i/item/9789240015791. 
4. 
White, N.J., et al., Malaria. Lancet, 2014. 383(9918): p. 723-35. 
5. 
WHO. WHO Fact Sheets: Malaria. 2021; Available from: https://www.who.int/news-
room/fact-sheets/detail/malaria. 
6. 
Cibulskis, R.E., et al., Malaria: Global progress 2000 - 2015 and future challenges. Infect 
Dis Poverty, 2016. 5(1): p. 61. 
7. 
WHO. Global Technical Strategy for Malaria 2016 - 2030. 2015  25/06/2021]; Available 
from: https://www.who.int/docs/default-source/documents/global-technical-strategy-for-
malaria-2016-2030.pdf?sfvrsn=c82afcc_0. 
8. 
Bhatt, S., et al., The effect of malaria control on Plasmodium falciparum in Africa between 
2000 and 2015. Nature, 2015. 526(7572): p. 207-211. 
9. 
Visser, T., et al., Rapid diagnostic tests for malaria. Bull World Health Organ, 2015. 
93(12): p. 862-6. 
10. 
Mbonye, A.K., et al., A Cluster Randomised Trial Introducing Rapid Diagnostic Tests into 
Registered Drug Shops in Uganda: Impact on Appropriate Treatment of Malaria. PLoS 
One, 2015. 10(7): p. e0129545. 
11. 
Ansah, E.K., et al., The impact of providing rapid diagnostic malaria tests on fever 
management in the private retail sector in Ghana: a cluster randomized trial. BMJ, 2015. 
350: p. h1019. 
12. 
Smith Paintain, L., et al., Community health workers and stand-alone or integrated case 
management of malaria: a systematic literature review. Am J Trop Med Hyg, 2014. 91(3): 
p. 461-470. 
13. 
Kayentao, K., et al., Intermittent preventive therapy for malaria during pregnancy using 2 
vs 3 or more doses of sulfadoxine-pyrimethamine and risk of low birth weight in Africa: 
systematic review and meta-analysis. JAMA, 2013. 309(6): p. 594-604. 
14. 
Desai, M., et al., Impact of Sulfadoxine-Pyrimethamine Resistance on Effectiveness of 
Intermittent Preventive Therapy for Malaria in Pregnancy at Clearing Infections and 
Preventing Low Birth Weight. Clin Infect Dis, 2016. 62(3): p. 323-333. 
15. 
Cairns, M., et al., Estimating the potential public health impact of seasonal malaria 
chemoprevention in African children. Nat Commun, 2012. 3: p. 881. 
16. 
WHO. Updated WHO Policy Recommendation (October 2012): Single dose Primaquine 
as a gaemtocytocide in Plasmodium falciparum malaria. 2012; Available from: 
https://www.who.int/malaria/pq_updated_policy_recommendation_en_102012.pdf. 
17. 
Recht, J., E.A. Ashley, and N.J. White, Use of primaquine and glucose-6-phosphate 
dehydrogenase deficiency testing: Divergent policies and practices in malaria endemic 
countries. PLoS Negl Trop Dis, 2018. 12(4): p. e0006230. 
18. 
WHO. World malaria report 2020.  [cited 2021 22/06]; Available from: 
https://www.who.int/publications/i/item/9789240015791. 
19. 
WHO. High burden to high impact. A targeted malaria response. 2018; Available from: 
https://apps.who.int/iris/bitstream/handle/10665/275868/WHO-CDS-GMP-2018.25-
eng.pdf. 

VAC085 Clinical Trial Protocol, V2.0 11 January 2023, University of Oxford 
Page 63 of 66 
 
20. 
Wongsrichanalai, C. and C.H. Sibley, Fighting drug-resistant Plasmodium falciparum: the 
challenge of artemisinin resistance. Clin Microbiol Infect, 2013. 19(10): p. 908-16. 
21. 
WHO. Malaria Vaccine Roadmap.  22/06/2021]; Available from: 
https://www.who.int/immunization/topics/malaria/vaccine_roadmap. 
22. 
Sinden, R.E., A biologist's perspective on malaria vaccine development. Hum Vaccin, 
2010. 6(1): p. 3-11. 
23. 
Meibalan, E. and M. Marti, Biology of Malaria Transmission. Cold Spring Harb Perspect 
Med, 2017. 7(3). 
24. 
Sinden, R.E., The cell biology of sexual development in plasmodium. Parasitology, 1983. 
86 (Pt 4): p. 7-28. 
25. 
Joice, R., et al., Plasmodium falciparum transmission stages accumulate in the human 
bone marrow. Sci Transl Med, 2014. 6(244): p. 244re5. 
26. 
Billker, O., et al., Identification of xanthurenic acid as the putative inducer of malaria 
development in the mosquito. Nature, 1998. 392(6673): p. 289-92. 
27. 
Centers for Disease Control and Prevention.  [cited 2021 16 July]; Available from: 
https://www.cdc.gov/malaria/about/biology/index.html. 
28. 
RTS S Clinical Trials Partnership, Efficacy and safety of RTS,S/AS01 malaria vaccine with 
or without a booster dose in infants and children in Africa: final results of a phase 3, 
individually randomised, controlled trial. Lancet, 2015. 386(9988): p. 31-45. 
29. 
Olotu, A., et al., Seven-Year Efficacy of RTS,S/AS01 Malaria Vaccine among Young 
African Children. N Engl J Med, 2016. 374(26): p. 2519-29. 
30. 
Arevalo-Herrera, M., et al., Protective Efficacy of Plasmodium vivax Radiation-Attenuated 
Sporozoites in Colombian Volunteers: A Randomized Controlled Trial. PLoS Negl Trop 
Dis, 2016. 10(10): p. e0005070. 
31. 
mal, E.R.A.C.G.o.V., A research agenda for malaria eradication: vaccines. PLoS Med, 
2011. 8(1): p. e1000398. 
32. 
Duffy, P.E. and J. Patrick Gorres, Malaria vaccines since 2000: progress, priorities, 
products. NPJ Vaccines, 2020. 5: p. 48. 
33. 
Huff, C.G., D.F. Marchbank, and T. Shiroishi, Changes in infectiousness of malarial 
gametocytes. II. Analysis of the possible causative factors. Exp Parasitol, 1958. 7(4): p. 
399-417. 
34. 
Carter, R. and D.H. Chen, Malaria transmission blocked by immunisation with gametes of 
the malaria parasite. Nature, 1976. 263(5572): p. 57-60. 
35. 
Ponnudurai, T., et al., The production of mature gametocytes of Plasmodium falciparum in 
continuous cultures of different isolates infective to mosquitoes. Trans R Soc Trop Med 
Hyg, 1982. 76(2): p. 242-50. 
36. 
Rener, J., et al., Target antigens of transmission-blocking immunity on gametes of 
plasmodium falciparum. J Exp Med, 1983. 158(3): p. 976-81. 
37. 
Nikolaeva, D., S.J. Draper, and S. Biswas, Toward the development of effective 
transmission-blocking vaccines for malaria. Expert Rev Vaccines, 2015. 14(5): p. 653-80. 
38. 
Delves, M.J., F. Angrisano, and A.M. Blagborough, Antimalarial Transmission-Blocking 
Interventions: Past, Present, and Future. Trends Parasitol, 2018. 34(9): p. 735-746. 
39. 
Duffy, P.E. and D.C. Kaslow, A novel malaria protein, Pfs28, and Pfs25 are genetically 
linked and synergistic as falciparum malaria transmission-blocking vaccines. Infect 
Immun, 1997. 65(3): p. 1109-13. 

VAC085 Clinical Trial Protocol, V2.0 11 January 2023, University of Oxford 
Page 64 of 66 
 
40. 
Hisaeda, H., et al., Antibodies to Plasmodium vivax transmission-blocking vaccine 
candidate antigens Pvs25 and Pvs28 do not show synergism. Vaccine, 2001. 20(5-6): p. 
763-70. 
41. 
van Dijk, M.R., et al., A central role for P48/45 in malaria parasite male gamete fertility. 
Cell, 2001. 104(1): p. 153-64. 
42. 
Eksi, S., et al., Malaria transmission-blocking antigen, Pfs230, mediates human red blood 
cell binding to exflagellating male parasites and oocyst production. Mol Microbiol, 2006. 
61(4): p. 991-8. 
43. 
Miura, K., et al., Functional comparison of Plasmodium falciparum transmission-blocking 
vaccine candidates by the standard membrane-feeding assay. Infect Immun, 2013. 
81(12): p. 4377-82. 
44. 
Atkinson, S.C., et al., The Anopheles-midgut APN1 structure reveals a new malaria 
transmission-blocking vaccine epitope. Nat Struct Mol Biol, 2015. 22(7): p. 532-9. 
45. 
Niu, G., et al., The fibrinogen-like domain of FREP1 protein is a broad-spectrum malaria 
transmission-blocking vaccine antigen. J Biol Chem, 2017. 292(28): p. 11960-11969. 
46. 
Roeffen, W., et al., Association between anti-Pfs48/45 reactivity and P. falciparum 
transmission-blocking activity in sera from Cameroon. Parasite Immunol, 1996. 18(2): p. 
103-9. 
47. 
Carter, R., et al., Restricted or absent immune responses in human populations to 
Plasmodium falciparum gamete antigens that are targets of malaria transmission-blocking 
antibodies. J Exp Med, 1989. 169(1): p. 135-47. 
48. 
Healer, J., et al., Transmission-blocking immunity to Plasmodium falciparum in malaria-
immune individuals is associated with antibodies to the gamete surface protein Pfs230. 
Parasitology, 1999. 119 ( Pt 5): p. 425-33. 
49. 
Jones, S., et al., Naturally acquired antibody responses to recombinant Pfs230 and 
Pfs48/45 transmission blocking vaccine candidates. J Infect, 2015. 71(1): p. 117-27. 
50. 
Ouedraogo, A.L., et al., Naturally acquired immune responses to Plasmodium falciparum 
sexual stage antigens Pfs48/45 and Pfs230 in an area of seasonal transmission. Infect 
Immun, 2011. 79(12): p. 4957-64. 
51. 
Stone, W.J.R., et al., Unravelling the immune signature of Plasmodium falciparum 
transmission-reducing immunity. Nat Commun, 2018. 9(1): p. 558. 
52. 
Roeffen, W., et al., Transmission blocking immunity as observed in a feeder system and 
serological reactivity to Pfs 48/45 and Pfs230 in field sera. Mem Inst Oswaldo Cruz, 1994. 
89 Suppl 2: p. 13-5. 
53. 
Bousema, J.T., et al., Rapid onset of transmission-reducing antibodies in javanese 
migrants exposed to malaria in papua, indonesia. Am J Trop Med Hyg, 2006. 74(3): p. 
425-31. 
54. 
Bousema, T., et al., The dynamics of naturally acquired immune responses to 
Plasmodium falciparum sexual stage antigens Pfs230 & Pfs48/45 in a low endemic area 
in Tanzania. PLoS One, 2010. 5(11): p. e14114. 
55. 
Niederwieser, I., I. Felger, and H.P. Beck, Limited polymorphism in Plasmodium 
falciparum sexual-stage antigens. Am J Trop Med Hyg, 2001. 64(1-2): p. 9-11. 
56. 
Cheru, L., et al., The IC(50) of anti-Pfs25 antibody in membrane-feeding assay varies 
among species. Vaccine, 2010. 28(27): p. 4423-9. 
57. 
Talaat, K.R., et al., Safety and Immunogenicity of Pfs25-EPA/Alhydrogel(R), a 
Transmission Blocking Vaccine against Plasmodium falciparum: An Open Label Study in 
Malaria Naive Adults. PLoS One, 2016. 11(10): p. e0163144. 

VAC085 Clinical Trial Protocol, V2.0 11 January 2023, University of Oxford 
Page 65 of 66 
 
58. 
Brod, F., et al., Combination of RTS,S and Pfs25-IMX313 Induces a Functional Antibody 
Response Against Malaria Infection and Transmission in Mice. Front Immunol, 2018. 9: p. 
2780. 
59. 
Sherrard-Smith, E., et al., Synergy in anti-malarial pre-erythrocytic and transmission-
blocking antibodies is achieved by reducing parasite density. Elife, 2018. 7. 
60. 
Simon, N., et al., Adhesion protein complexes of malaria gametocytes assemble following 
parasite transmission to the mosquito. Parasitol Int, 2016. 65(1): p. 27-30. 
61. 
Kocken, C.H., et al., Cloning and expression of the gene coding for the transmission 
blocking target antigen Pfs48/45 of Plasmodium falciparum. Mol Biochem Parasitol, 1993. 
61(1): p. 59-68. 
62. 
Milek, R.L., et al., Plasmodium falciparum: heterologous synthesis of the transmission-
blocking vaccine candidate Pfs48/45 in recombinant vaccinia virus-infected cells. Exp 
Parasitol, 1998. 90(2): p. 165-74. 
63. 
Milek, R.L., H.G. Stunnenberg, and R.N. Konings, Assembly and expression of a synthetic 
gene encoding the antigen Pfs48/45 of the human malaria parasite Plasmodium 
falciparum in yeast. Vaccine, 2000. 18(14): p. 1402-11. 
64. 
Jones, C.S., et al., Heterologous expression of the C-terminal antigenic domain of the 
malaria vaccine candidate Pfs48/45 in the green algae Chlamydomonas reinhardtii. Appl 
Microbiol Biotechnol, 2013. 97(5): p. 1987-95. 
65. 
Outchkourov, N.S., et al., Correctly folded Pfs48/45 protein of Plasmodium falciparum 
elicits malaria transmission-blocking immunity in mice. Proc Natl Acad Sci U S A, 2008. 
105(11): p. 4301-5. 
66. 
Chowdhury, D.R., et al., A potent malaria transmission blocking vaccine based on codon 
harmonized full length Pfs48/45 expressed in Escherichia coli. PLoS One, 2009. 4(7): p. 
e6352. 
67. 
Milek, R.L., et al., Immunological properties of recombinant proteins of the transmission 
blocking vaccine candidate, Pfs48/45, of the human malaria parasite Plasmodium 
falciparum produced in Escherichia coli. Parasite Immunol, 1998. 20(8): p. 377-85. 
68. 
Pritsch, M., et al., Comparison of Intranasal Outer Membrane Vesicles with Cholera Toxin 
and Injected MF59C.1 as Adjuvants for Malaria Transmission Blocking Antigens AnAPN1 
and Pfs48/45. J Immunol Res, 2016. 2016: p. 3576028. 
69. 
Prokhnevsky, A., et al., Development of a single-replicon miniBYV vector for co-
expression of heterologous proteins. Mol Biotechnol, 2015. 57(2): p. 101-10. 
70. 
Theisen, M., et al., A multi-stage malaria vaccine candidate targeting both transmission 
and asexual parasite life-cycle stages. Vaccine, 2014. 32(22): p. 2623-30. 
71. 
Roeffen, W., et al., Transmission-blocking activity of antibodies to Plasmodium falciparum 
GLURP.10C chimeric protein formulated in different adjuvants. Malar J, 2015. 14: p. 443. 
72. 
Baldwin, S.L., et al., Synthetic TLR4 agonists enhance functional antibodies and CD4+ T-
cell responses against the Plasmodium falciparum GMZ2.6C multi-stage vaccine antigen. 
Vaccine, 2016. 34(19): p. 2207-15. 
73. 
Singh, S.K., et al., A Plasmodium falciparum 48/45 single epitope R0.6C subunit protein 
elicits high levels of transmission blocking antibodies. Vaccine, 2015. 33(16): p. 1981-6. 
74. 
Vermeulen, A.N., et al., Sequential expression of antigens on sexual stages of 
Plasmodium falciparum accessible to transmission-blocking antibodies in the mosquito. J 
Exp Med, 1985. 162(5): p. 1460-76. 
75. 
Stone, W.J., et al., Naturally acquired immunity to sexual stage P. falciparum parasites. 
Parasitology, 2016. 143(2): p. 187-98. 

VAC085 Clinical Trial Protocol, V2.0 11 January 2023, University of Oxford 
Page 66 of 66 
 
76. 
Lennartz, F., et al., Structural basis for recognition of the malaria vaccine candidate 
Pfs48/45 by a transmission blocking antibody. Nat Commun, 2018. 9(1): p. 3822. 
77. 
Roeffen, W., et al., Plasmodium falciparum: production and characterization of rat 
monoclonal antibodies specific for the sexual-stage Pfs48/45 antigen. Exp Parasitol, 
2001. 97(1): p. 45-9. 
78. 
Targett, G.A., et al., Plasmodium falciparum sexual stage antigens: immunogenicity and 
cell-mediated responses. Immunol Lett, 1990. 25(1-3): p. 77-81. 
79. 
Kundu, P., et al., Structural delineation of potent transmission-blocking epitope I on 
malaria antigen Pfs48/45. Nat Commun, 2018. 9(1): p. 4458. 
80. 
Bengtsson, K.L., et al., Matrix-M adjuvant enhances antibody, cellular and protective 
immune responses of a Zaire Ebola/Makona virus glycoprotein (GP) nanoparticle vaccine 
in mice. Vaccine, 2016. 34(16): p. 1927-35. 
81. 
Cox, R.J., et al., Evaluation of a virosomal H5N1 vaccine formulated with Matrix M™ 
adjuvant in a phase I clinical trial. Vaccine, 2011. 29(45): p. 8049-59. 
82. 
Venkatraman, N., et al., Safety and immunogenicity of heterologous prime-boost 
immunization with viral-vectored malaria vaccines adjuvanted with Matrix-M. Vaccine, 
2017. 35(45): p. 6208-6217. 
83. 
Heath, P.T., et al., Safety and Efficacy of NVX-CoV2373 Covid-19 Vaccine. N Engl J Med, 
2021. 385(13): p. 1172-1183. 
84. 
Mamedov, T., et al., A Plant-Produced in vivo deglycosylated full-length Pfs48/45 as a 
Transmission-Blocking Vaccine Candidate against malaria. Sci Rep, 2019. 9(1): p. 9868.