Clinical trial NCT05252845
Conditions: Malaria,Falciparum
Keywords: Malaria vaccine Immunogenicity of the malaria vaccine Immunologic response to malaria vaccine
Sponsor: University of Oxford
Start date: 2023-01-04; Completion date: 2023-09-18
Enrollment: 127 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
Malaria remains one of the leading causes of morbidity and mortality worldwide. Plasmodium falciparum is a complex pathogen with numerous immune evasion mechanisms which has added layers of complexity to the development of safe and protective vaccines. There remains an urgent need to identify and develop more protective and more affordable vaccine candidates that could achieve the World Health Organization (WHO) goal of 75% efficacy against clinical malaria.

R21 is a novel pre-erythrocytic candidate malaria vaccine. R21 includes Hepatitis B surface antigen (HBsAg) fused to the C-terminus and central repeats of the circumsporozoite protein of P. falciparum (CSP), which self-assemble into virus-like particles in yeast. R21 lacks the excess HBsAg found and comprises only fusion protein moieties.

R21/MatrixM (MM) had a favourable safety profile and was well tolerated. The majority of adverse events were mild, with the most common event being fever. None of the serious adverse events were attributed to the vaccine. At one year, vaccine efficacy remained high, at 77%. Participants vaccinated with R21/MM showed high titres of malaria-specific anti- Asn-Ala-Asn-Pro (NANP) antibodies 28 days after the third vaccination, which were almost doubled with the higher adjuvant dose. Titres waned but were boosted to levels similar to peak titres after the primary series of vaccinations after a fourth dose administered one year later.

Currently, there are no safety and immunogenicity data for the use of R21/MatrixM in Asian populations. This trial will generate the required data for the use of this vaccine in Asia. For integration with the current targeted malaria elimination (TME) activities, which provide mass drug administrations at months M0, M1, and M2, it would be most efficient and practical to provide the vaccine at the same intervals.

In summary: The investigators propose to conduct a safety and immunogenicity trial of R21/MatrixM in Thai adults. The major aims of this study are to 1) assess the safety and immunogenicity of R21/MatrixM in Thai adults 2) confirm that the co-administration of antimalarial drugs with the malaria vaccine R21/MatrixM does not reduce the immunogenicity of the vaccine and 3) assess the absorption and pharmacokinetics of antimalarial drugs piperaquine, and a single low dose of primaquine (SLDPQ) when co-administered with R21/MatrixM.

This is a randomized, open label, single centre, Phase 2 study. 120 healthy non-pregnant Thai adults, aged 18-55 years, inclusive, will be recruited.

Each participant will be randomized into one of the following study arms in a ratio of 5:5:2, as follows:

1. R21/MatrixM + Dihydroartimisinin (DHA)-Piperaquine (PIP)+ primaquine (PQ) (Group 1, n=50) will receive R21/MatrixM + 3 doses DHA-PIP+PQ at Month 0, Month 1 and Month 2
2. R21/MatrixM only (Group 2, n=50) will receive R21/MatrixM standard dose at Month 0, Month 1 and Month 2
3. DHA-PIP+PQ only (Group 3, n=20) will receive 3 doses DHA-PIP+PQ at Month 0, Month 1 and Month 2

Detailed description:
This is a randomized, open label, single centre, Phase 2 study.

Screening and eligibility assessment (Screening visit) All potential volunteers will have a screening visit, which may take place up to 7 days prior to enrolment. Once informed consent is given, a screening number will be assigned in sequential order. Screening numbers will be issued consecutively (e.g. R21-001, R21-002, R21-003, …)

Enrolment, baseline assessment, regimen allocation, and first vaccination (Month 0 / Day 0 visit; Baseline visit) All inclusion and exclusion criteria will be checked before enrolment in the study. Physical examination will be performed. Any new medical issues or symptoms that have arisen will be assessed. Blood will be collected for baseline P. falciparum testing, haemoglobin and biochemistry. Participants with parasitaemia or anaemia will be treated according to national guidelines. Blood will be collected and stored for measurement of antibodies against P. falciparum circumsporozoite (anti-circumsporozoite antibody) until shipment to the reference laboratory. Urine will be collected from women of child-bearing age for immediate pregnancy test.

Enrolment, baseline assessment, regimen allocation, and first vaccination (Month 0 / Day 0 visit; Baseline visit)

All inclusion and exclusion criteria will be checked before enrolment in the study. Physical examination will be performed. Any new medical issues or symptoms that have arisen will be assessed. Blood will be collected for baseline P. falciparum testing, haemoglobin and biochemistry. Blood will be collected and stored for measurement of antibodies against P. falciparum circumsporozoite (anti-circumsporozoite antibody) until shipment to the reference laboratory. Urine will be collected from women of child-bearing age for immediate pregnancy test.

If all inclusion criteria are fulfilled and none of the exclusion criteria apply, the patient will be enrolled into the study and a case record form (CRF) specific to each participant completed. Regimen allocation and administration of the vaccine(s) will be on Day 0. The randomization lists will be prepared by Mahidol-Oxford Tropical Medicine Research Unit (MORU).

Randomization numbers will be generated in blocks, for the 3 study arms in a ratio of 5:5:2, as follows:

* R21/MatrixM + DHA-PIP+PQ (Group 1)
* R21/MatrixM alone (Group 2)
* DHA-PIP+PQ alone (Group 3)

Study participants will be assigned the next available randomization number on the list, and thus will be randomly allocated to Group 1, 2, or 3. This is an open-label study. Participants and clinical investigators will not be blinded to group allocation.

Subjects (in group 1 and 2) will then be vaccinated by intramuscular (IM) needle injection into the deltoid region of the arm. Subjects in Groups 1 and 3 will also receive anti-malarial medications.

The study participants will be observed closely for at least 30 minutes following the administration of each study vaccine, with appropriate medical treatment readily available in case of an anaphylactic reaction.

Subsequent vaccination visits (Month 1 / Day 0 and Month 2 / Day 0 visits)

Physical examination will be performed. Any new medical issues or symptoms that have arisen will be assessed. Blood will be collected for P. falciparum testing. Participants with parasitaemia or anaemia will be treated according to national guidelines. Blood will be collected and stored for measurement of antibodies against P. falciparum circumsporozoite (anti-circumsporozoite) until shipment to the reference laboratory. Urine will be collected from women of child-bearing age for immediate pregnancy test.

Before vaccination, the on-going eligibility of the volunteer will be reviewed. All participants will attend the clinic for vaccination visits, will be observed closely for at least 30 minutes following the administration of each study vaccine.

Follow up assessments

Follow-up assessments (visits not involving vaccination) will be done at month 3 and month 6 after the first vaccination.

Blood tests

Blood will be drawn at the time points indicated in the schedule of procedures and the following laboratory assays performed:

At screening:

* Haematology: haemoglobin (Hb), leukocytes (white blood cells \[WBC\]) and platelets (PLT).
* Biochemistry: alanine aminotransferase (ALT), aspartate aminotransferase (AST) and creatinine (CREA).
* Diagnostic serology: HBsAg, HCV antibodies, HIV antibodies (Counselling will be given prior to testing blood for these blood-borne viruses)
* P. falciparum testing

At M0, D0: P. falciparum testing, Haematology: Hb, WBC, PLT, Biochemistry: ALT, AST, CREA, anti-circumsporozoite antibody; Pharmacokinetics (piperaquine and primaquine drug levels will be assessed if additional data are required) At M0, D1: Pharmacokinetics (piperaquine and primaquine drug levels) At M0, D2: Pharmacokinetics (piperaquine drug levels) At M0, D7: Haematology: Hb, WBC, PLT; Biochemistry: ALT, AST, CREA; Pharmacokinetics (piperaquine drug levels) At M1, D0: P. falciparum testing, anti-circumsporozoite antibody; Pharmacokinetics (piperaquine drug levels) At M1, D1: Pharmacokinetics (piperaquine and primaquine drug levels) At M1, D2: Pharmacokinetics (piperaquine drug levels) At M1, D7: Haematology: Hb, WBC, PLT; Biochemistry: ALT, AST, CREA; Pharmacokinetics (piperaquine drug levels) At M2, D0: P. falciparum testing, anti-circumsporozoite antibody; Pharmacokinetics (piperaquine drug levels) At M2, D1: Pharmacokinetics (piperaquine and primaquine drug levels) At M2, D2: Pharmacokinetics (piperaquine drug levels) At M2, D7: Haematology: Hb, WBC, PLT; Biochemistry: ALT, AST, CREA; Pharmacokinetics (piperaquine drug levels) At M3: P. falciparum testing, Haematology: Hb, WBC, PLT; Biochemistry: ALT, AST, CREA; anti-circumsporozoite antibody At M6: P. falciparum testing, anti-circumsporozoite antibody

Arms: arm 1: The R21/Matrix-M vaccine (IM injection) + co-formulated Dihydroartemisinin/Piperaquine tablets + one single low dose of Primaquine arm 2: The R21/Matrix-M vaccine (IM injection) only arm 3: Co-formulated Dihydroartemisinin/Piperaquine tablets + one single low dose of Primaquine
Interventions: intervention 1: R21/Matrix-M vaccination intervention 2: DHA-PIP intervention 3: PQ
Intervention descriptions: intervention 1: R21/Matrix-M is the standard formulation of the vaccine (Once prepared, a standard dose of R21/Matrix-M contains approximately 5 µg of R21 antigen, in 0.5 mL of liquid adjuvant containing 50 µg MatrixM). The reconstituted vaccine will be administered by slow IM injection on Day 0 at Study Months 0, 1 and 2. intervention 2: Dihydroartemisinin/piperaquine tablets for adult patients each contain 40 mg dihydroartemisinin and 320 mg piperaquine with a therapeutic dose range between 2-10 mg/kg/day dihydroartemisinin and 16-26 mg/kg/dose piperaquine. Participants will receive three rounds of antimalarial drugs - each round starting on the day of vaccination (Day 0) at Study Months 0, 1 and 2. Each round consists of three daily doses of co-formulated dihydroartemisinin/piperaquine on Day 0, 1, and 2. intervention 3: Each participant will receive a single low dose primaquine on the day of vaccination (Day 0) at Month 0, Month 1, and Month 2. One single low dose primaquine of approximately 0.25 mg/kg will be administered.

Protocol documents:
Prot_000.pdf: 







































































































SAP_001.pdf: 
Page 1 of 17 
Statistical Analysis Plan version 1.0 date 10 Mar 2023 
 
CONFIDENTIAL 
 
 
 
STATISTICAL ANALYSIS PLAN 
 
 
 
A Randomized, Open label, Single Centre, Phase 2 Trial of the Malaria Vaccine, R21/Matrix-M™, to 
Assess Safety and Immunogenicity of the vaccine in Thai Adults 
 
 
 
 
 
 
 
 
Version Number 1.0 
 
 
 
 
Protocol number: MAL22001 
Study reference: VAC081 
OxTREC Ref: 9-22 
 
 
Date and Version No: 10/03/2023 version 1.0 
 
 
 
Prepared by: Prof Mavuto Mukaka, Head of Statistics, MORU 
 
 
Reviewed by: 
 
Program Manager: Dr Pongphaya Pongsuwan 
 
Principal Investigator: Prof Lorenz von Seidlein, Principal Investigator, MORU 
 
 
Mahidol-Oxford Tropical Medicine Research Unit 




Page 3 of 17 
Statistical Analysis Plan version 1.0 date 10 Mar 2023 
 
CONTENTS 
1. INTRODUCTION................................................................................................................... 4 
2. BACKGROUND INFORMATION ............................................................................................. 4 
2.1 
OBJECTIVES ....................................................................................................................... 4 
2.1.1 
PRIMARY OBJECTIVES....................................................................................................... 4 
2.1.2 
SECONDARY OBJECTIVES .................................................................................................. 4 
2.1.3 
STUDY DESIGN .................................................................................................................. 4 
2.1.4 
PATIENT POPULATION ...................................................................................................... 5 
2.1.5 
SAMPLE SIZE ..................................................................................................................... 5 
2.1.6 
RANDOMISATION ............................................................................................................. 6 
3. ANALYSIS OF PRIMARY AND SECONDARY OUTCOMES .......................................................... 7 
3.1.1 
INCLUSION IN ANALYSIS ................................................................................................... 7 
3.1.2 
PRIMARY OUTCOME ........................................................................................................ 7 
4. STATISTICAL METHODS ....................................................................................................... 7 
4.1 
GENERAL CONSIDERATIONS ............................................................................................. 7 
4.1.1 
DATA CHECKS ................................................................................................................... 7 
4.1.2 
BASELINE DATA ................................................................................................................ 8 
4.1.3 
SCREENING AND RECRUITMENT FLOWCHART ................................................................. 8 
4.1.4 
ANALYSIS POPULATION .................................................................................................... 8 
4.1.5 
ANALYSES TO ADDRESS PRIMARY AND SECONDARY OBJECTIVES ................................... 8 
4.1.6 
DESCRIPTIVE ANALYSES .................................................................................................... 8 
4.1.7 
ANALYSIS OF IMMUNOGENICITY.................................................................................... 10 
5. STATISTICAL ANALYSES OF PRIMARY SAFETY ENDPOINTS ................................................... 16 


Page 4 of 17 
Statistical Analysis Plan version 1.0 date 10 Mar 2023 
 
1 
INTRODUCTION 
This study is a randomized, open-label, single centre, Phase 2 trial of R21/Matrix-M™. A total of 120 
healthy non-pregnant Thai adults, aged 18-55 years, inclusive, will be enrolled and randomised to one 
of three study groups: 
● 
Arm 1: R21/Matrix-M™ in subjects who concurrently receive co-formulated 
dihydroartemisinin/piperaquine + single low dose of primaquine (SLDPQ). 
● 
Arm 2: R21/Matrix-M™ alone. 
 
● 
Arm 3: Co-formulated dihydroartemisinin/piperaquine + SLDPQ 
 
We propose to conduct a safety and immunogenicity trial of R21/Matrix-M™. The major aims of this 
study are to: (1) assess the safety and immunogenicity of R21/Matrix-M™ in healthy Thai adults, (2) 
confirm that the co-administration of antimalarial drugs with R21/Matrix-M™ does not reduce the 
immunogenicity of the vaccine, and (3) assess the pharmacokinetics of antimalarial drug: piperaquine in 
subjects who concurrently receive R21/Matrix-M™. This study will contribute to our understanding of 
the immunogenicity of a vaccine, which could potentially be used to help stop the spread of malaria and 
anti-malarial resistance in the region. 
 
2 
BACKGROUND INFORMATION 
 
2.1 
OBJECTIVES 
 
2.1.1 
PRIMARY OBJECTIVES 
● To assess the safety of the R21/Matrix-M™ vaccine, with and without concurrent anti-malarial 
administration, in Thai adults. 
2.1.2 
SECONDARY OBJECTIVES 
● To compare the immunologic response to R21/Matrix-M™ vaccine when concurrently given with 
DHA-PIP + SLD-PQ (Arm 1) with R21/Matrix-M™ vaccine alone (Arm 2) in Thai adults. 
● To compare the piperaquine levels of participants who received the antimalarial drugs alone (Arm 
3) with participants who received the antimalarial drugs combined vaccine (Arm 1). 
2.1.3 
STUDY DESIGN 
This study is a randomized, open-label, single centre, Phase 2 trial of R21/Matrix-M™. A total of 120 
healthy non-pregnant Thai adults, aged 18-55 years, inclusive, will be enrolled and randomised to one 
of three study groups: 
● 
Arm 1: R21/Matrix-M™ in subjects who concurrently receive co-formulated 
dihydroartemisinin/piperaquine + single low dose of primaquine (SLDPQ). 
● 
Arm 2: R21/Matrix-M™ alone. 
 
● 
Arm 3: Co-formulated dihydroartemisinin/piperaquine + SLDPQ 


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Statistical Analysis Plan version 1.0 date 10 Mar 2023 
 
2.1.4 
PATIENT POPULATION 
 
Healthy Thai adults aged 18-55 years. 
The inclusion and exclusion are detailed in the protocol section B5. 
 
2.1.5 
SAMPLE SIZE 
The sample size calculations are based on the objective of comparing the serologic response to 
R21/Matrix-M™ vaccine concurrently given with DHA-PIP + SLD-PQ with R21/Matrix-M™ vaccine alone 
in adults instead of basing it on safety outcome. This is a Phase II trial. Sample sizes for Phase II trials 
are usually small, as safety is still of paramount importance. Because of this limitation, these phase II 
studies are usually powered on the immunogenicity/efficacy endpoints because safety events are rare 
and a sample size based on safety would lead to a very high sample size that would not be acceptable 
from ethical standpoint. Another reason why powering it on immunogenicity/efficacy (a co-primary 
objective) is important, is that if the vaccine has a poor immunogenicity profile then the safety question 
becomes redundant unless the study is a non-inferiority trial. We are optimistic that this sample size will 
be able to reveal important safety signals between arms which can be further explored in a phase III 
trial. The efficacy endpoint (Immunogenicity objective) is a co-primary objective to the safety objective. 
We include both “safety” and “immunogenicity” terms in the study title because both of them are key 
endpoints. Valid sample sizes for comparing sample sizes between two groups are usually obtained using 
exact test methods such as the Fisher’s exact test power calculations. This is done using simulations as 
sample size formulas are not well developed. To estimate the sample size we use a four-fold increase in 
anti –CS antibody titre as an assumed correlate of protection. For the sample size calculation, we assume 
that the background rate of response in the reference groups will be 5% and the true rate of anti-CS 
antibody response in the vaccine groups is 80%. We wish to exclude, for the vaccine, a one-tailed lower 
95% CI for the reference-vaccine group difference of lower than 30%. The study aims to detect this 
difference with at least 90% power (because the detectable difference is big) and testing at 5% 
significance level. Based on these assumptions and using the method of Blackwelder for precision-based 
sample size calculations, a total of 20 participants per arm would be needed for arm 3 to test against 
each of arms 1 and 2 separately because the anticipated difference is too big. However, for arms 1 and 
2 (Table 1 below), we will need more participants per group because a smaller detectable effect is 
anticipated. Using the Fisher’s exact test power simulations, a difference in serologic response of 30% 
(say 50% vs 80%) gives more than 80% power (i.e. 85% power) with a sample size of 50 participants in 
each of arms 1 and 2 testing at 5% significance level. Therefore, the total sample size for all the 3 arms 
will be 120 participants. The size of study groups is relatively small hence it is imperative to minimise 
potential losses. Whenever possible an attempt will be made to replace study participates should a 
participant be withdrawn or choose to withdraw. The stata command for obtaining the Fishers exact 
test power simulations is: power two proportions 0.50 0.80, test(fisher) n1(50) n2(50). This has been 
performed in Stata 16. The required sample sizes are summarized as follows: 


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Statistical Analysis Plan version 1.0 date 10 Mar 2023 
 
Table 1. Sample size and vaccine vials required for each arm 
 
Group # Participants 
Vaccine 
Dose 
M0 
Dose 
M1 
Dose 
M2 
Vaccine 
vials 
required 
1 
50 
R21/Matrix- 
M™ 
Single 
standard 
dose 
(0.75mL) 
Single 
standard 
dose 
(0.75mL) 
Single 
standard 
dose 
(0.75mL) 
300 
2 
50 
R21/Matrix- 
M™ 
Single 
standard 
dose 
(0.75mL) 
Single 
standard 
dose 
(0.75mL) 
Single 
standard 
dose 
(0.75mL) 
300 
3 
20 
No vaccine 
No 
vaccine 
No vaccine No vaccine 
0 
Total 
120 
600 
Remark: R21/Matrix-M™ = adult formulation containing 10 µg of R21 and 50 μg Matrix-M in the 
standard dose of 0.75mL. 
 
2.1.6 
RANDOMISATION 
Randomization numbers will be generated in blocks, for the 3 study arms in a ratio of 5:5:2, as follows: 
 
● R21/Matrix-M™ + DHA-PIP+PQ (Arm 1) 
● R21/Matrix-M™ alone (Arm 2) 
● DHA-PIP+PQ alone (Arm 3) 
Study participants will be assigned the next available randomization number on the list, and thus will be 
randomly allocated to Group 1, 2, or 3. This is an open-label study. Participants and clinical investigators 
will not be blinded to group allocation. 


Page 7 of 17 
Statistical Analysis Plan version 1.0 date 10 Mar 2023 
 
3 
ANALYSIS OF PRIMARY AND SECONDARY OUTCOMES 
3.1 
INCLUSION IN ANALYSIS 
 
All patients who received at least one dose of vaccine will be included in the safety analyses. Patients 
lost to follow-up before the completion of the follow-up period assessments will be censored at the last 
day seen. 
 
 
Exclusion 
 
Given the incidence of malaria in the study area, it is highly unlikely that any volunteer became infected 
with any Plasmodium species during the trial. However, to exclude the remote possibility that a 
participant becomes infected we will screen participants for malaria at the time screening, Month 0, 
Month 1, Month 2, Month 3, and Month 6. The malaria screen consists of a RDT. The result will be 
available in <30 min. Subjects positive for malaria at screening or Month 0 will be excluded from 
participation in the study and will not be enrolled or vaccinated. Subjects positive for malaria at Month 
1 or Month 2 would be withdrawn from further vaccinations. In the remote scenario that a volunteer 
becomes parasitaemic during the study results obtained from that point in time onward will be 
confounded by the immune response to the natural infection. Immunology results obtained during and 
after an episode of parasitaemia will be excluded from the immunology analysis. The results will be 
included in the safety analysis. 
 
The results will be reported in two ways: a) excluding results for the out-of-window visit as specified in 
the protocol (analogous to the per protocol approach PP) and b) including the results analogous to an 
intention to treat ITT approach. 
 
3.2 
PRIMARY OUTCOME 
 
The primary outcome measures will be: 
 
● The safety of the investigational treatment in each group: 
o Occurrence of serious adverse events (SAEs) from the date of the first vaccination to 28 days 
after the last vaccination, according to the MedRA classification. 
o Occurrence of SAEs during the whole study period, i.e. during a 6 month follow up period 
from the receipt of first vaccination, according to the MedRA classification. 
 
● 
For all the 3 groups, the concentration of antibodies against Plasmodium falciparum 
circumsporozoite (anti-CS antibody), one month after the first dose (at Study month 1), one month 
after the second dose (at Study month 2). 
 
4 
STATISTICAL METHODS 
4.1 
GENERAL CONSIDERATIONS 
4.1.1 
DATA CHECKS 
Data for each variable including calculated changes from baseline will be summarised using descriptive 
statistics such as n, mean, median, minimum and maximum, to check for any values that are out of range 


Page 8 of 17 
Statistical Analysis Plan version 1.0 date 10 Mar 2023 
 
or implausible. Missing data will be identified and queried when preparing data for analysis. 
 
4.1.2 
BASELINE DATA 
Patient demographic characteristics (age, gender, weight), and all other baseline information will be 
summarised for each treatment group. 
 
Numbers (with percentages) for binary and categorical variables. The mean (standard deviation) will be 
presented for continuous normally distributed data. The median (interquartile or full range) for 
continuous variables will be presented. Highly skewed data that take a wide range of values (including 
very large values) will be log transformed for meaningful analyses. 
 
There will be no tests of statistical significance conducted for differences between randomised groups 
on any baseline variables. 
 
4.1.3 
SCREENING AND RECRUITMENT FLOWCHART 
A study flowchart showing numbers of participants screened, randomised and those who are available 
for the final analysis populations will be constructed. Reasons for drop-out or exclusions will be shown 
at each stage. 
 
4.1.4 
ANALYSIS POPULATION 
Primary outcome analyses will be carried out on both the according to protocol (PP) and the intention 
to treat (ITT) population. The PP analyses will be the main strategy for the immunogenicity analyses. The 
safety outcomes will be ITT. In the event of some subjects not meeting the in-/exclusion criteria at 
baseline being erroneously enrolled in the study, a modified intention to treat analysis (mITT) will be 
considered. In that case, reasons for exclusions will be clearly documented and justified. In the intention- 
to-treat analysis, every participant randomized in the study (who receive the correct or incorrect study 
agent, one or more doses, and complete or incomplete doses) will be analysed, except if he/she did not 
receive any dose of the study vaccine or if no post-randomization data was collected for this participant. 
The per-protocol analysis will compare participants according to the study agent/treatment actually 
received and will include only those participants who satisfied the inclusion/exclusion criteria, followed 
the protocol, and received complete, correct doses. The following non-compliant participants will be 
excluded: 
• 
Participants included without meeting at least one inclusion criterion 
• 
Participants included despite meeting at least one exclusion criterion 
• 
Participants found non-compliant with the blood sampling schedule 
• 
Participants vaccinated with the wrong study agent (non-compliance with the randomization 
code) 
• 
Participants excluded from the intention-to-treat (mITT) analysis. 
 
4.1.5 
ANALYSES TO ADDRESS PRIMARY AND SECONDARY OBJECTIVES 
 
4.1.6 
DESCRIPTIVE ANALYSES 
For each study measure descriptive statistics within each randomised group and overall will be 
presented for data at each study time point. 


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Statistical Analysis Plan version 1.0 date 10 Mar 2023 
 
Table 2. –Characteristics (M0 Day 0 measurements) of the participants by vaccine group 
 
 
Characteristic 
R21/Matrix-M™ 
+ DHA-PIP + SLD-PQ 
n=X 
R21/Matrix-M™ 
n=X 
No Vaccine 
n=X 
Gender 
n (% male) 
Age (years): 
median (range) 
Weight (kg): 
median (range) 
QTc int (msec): 
median (range) 
Fever n (%) 
Hb 
mean (sd) 
WBC 
median (range) 
Platelets 
median (range) 
Creatinine 
median (range) 
AST 
median (range) 
ALT 
median (range) 
Temperature 
mean (sd) 


Page 10 of 17 
Statistical Analysis Plan version 1.0 date 10 Mar 2023 
 
4.1.7 
ANALYSIS OF IMMUNOGENICITY 
 
The serum anti-CS antibody titre and avidity responses for each treatment arm will be compared to 
the titre before vaccine administration as outlined in tables 2a, 2b, 3a and 3b below. In addition, 
immunology will be analyzed and compared in terms of the potential parameters using appropriate 
statistics according to the data structure in which data will be presented to the statistician. While the 
final choice of immunologic assays will be determined at a later stage the may include the following: 
 
● Plasmodium falciparum. Circumsporozoite Protein. (NANP)6 Ab.IgG 
● Plasmodium falciparum. Circumsporozoide Protein. (NANP)6 Ab.IgG Avidity 
● Plasmodium falciparum. anti-C-Term Circumsporozoide Ab.IgG 
● Plasmodium falciparum. anti-C-Term Circumsporozoide Ab.IgG avidity 
● Plasmodium falciparum. Circumsporozoite Full length (N+C-Terminal) 
● Plasmodium falciparum. anti-full length CSP Ab.IgG avidity 
The serum anti-CS antibody titre -Proportion in study group with >4-fold rise in titre from baseline to: 
One month after first dose; One month after second dose; One month after the third dose; Six 
Months after the first dose for all groups. 


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Statistical Analysis Plan version 1.0 date 10 Mar 2023 
 
Table 3ai. Plasmodium falciparum. Circumsporozoite Protein.(NANP)6 Ab.IgG 
 
ARM 
1 month after 
1st dose 
n (%) 
1 month after 
2nd dose 
n (%) 
1 month after 
3rd dose 
n (%) 
6 months after 
1st dose 
n (%) 
R21/Matrix-M™ 
+ DHA-PIP + SLD-PQ 
R21/Matrix-M™ 
No vaccine 
 
 
Table 3aii. Plasmodium falciparum. anti-C-Term Circumsporozoide Ab.IgG 
 
ARM 
1 month after 
1st dose 
n (%) 
1 month after 
2nd dose 
n (%) 
1 month after 
3rd dose 
n (%) 
6 months after 
1st dose 
n (%) 
R21/Matrix-M™ 
+ DHA-PIP + SLD-PQ 
R21/Matrix-M™ 
No vaccine 
 
 
Table 3aiii. Plasmodium falciparum. Circumsporozoite Full length (N+C-Terminal) 
 
ARM 
1 month after 
1st dose 
n (%) 
1 month after 
2nd dose 
n (%) 
1 month after 
3rd dose 
n (%) 
6 months after 
1st dose 
n (%) 
R21/Matrix-M™ 
+ DHA-PIP + SLD-PQ 
R21/Matrix-M™ 
No vaccine 


Page 12 of 17 
Statistical Analysis Plan version 1.0 date 10 Mar 2023 
 
AVIDITY 
 
Avidity -Proportion in study group with >4-fold rise from baseline to: One month after first dose; One 
month after second dose; One month after the third dose; Six months after the first dose for all the 
three groups. These will be summarised in tables 2bi to 2bii as follows: 
 
 
Table 3bi. Plasmodium falciparum. Circumsporozoide Protein.(NANP)6 Ab.IgG Avidity 
 
ARM 
1 month after 
1st dose 
n (%) 
1 month after 
2nd dose 
n (%) 
1 month after 
3rd dose 
n (%) 
6 months after 
1st dose 
n (%) 
R21/Matrix-M™ 
+ DHA-PIP + SLD-PQ 
R21/Matrix-M™ 
No vaccine 
 
 
Table 3bii. Plasmodium falciparum. anti-C-Term Circumsporozoide Ab.IgG avidity 
 
ARM 
1 month after 
1st dose 
n (%) 
1 month after 
2nd dose 
n (%) 
1 month after 
3rd dose 
n (%) 
6 months after 
1st dose 
n (%) 
R21/Matrix-M™ 
+ DHA-PIP + SLD-PQ 
R21/Matrix-M™ 
No vaccine 
 
 
Table 3biii. P falciparum. anti-full length CSP Ab.IgG avidity 
 
ARM 
1 month after 
1st dose 
n (%) 
1 month after 
2nd dose 
n (%) 
1 month after 
3rd dose 
n (%) 
6 months after 
1st dose 
n (%) 
R21/Matrix-M™ 
+ DHA-PIP + SLD-PQ 
R21/Matrix-M™ 
No vaccine 
The frequency and the proportions of participants who are seropositive* for antibodies at each 
timepoint will be summarized in the table below 


Page 13 of 17 
Statistical Analysis Plan version 1.0 date 10 Mar 2023 
 
Table 3ci. Proportion seropositive* for antibodies at each timepoint: Plasmodium falciparum. 
Circumsporozoite Protein.(NANP)6 Ab.IgG 
 
ARM 
1 month after 
1st dose 
n (%) 
1 month after 
2nd dose 
n (%) 
1 month after 
3rd dose 
n (%) 
6 months after 
1st dose 
n (%) 
R21/Matrix-M™ 
+ DHA-PIP + SLD-PQ 
R21/Matrix-M™ 
No vaccine 
* The percentage of subjects seroconverting after each immunization based on a value greater than 
the mean titer at baseline (before immunization # 1) plus 2 standard deviations for all subjects 
included in the analysis. 
 
 
Table 3cii. Proportion seropositive* for antibodies at each timepoint: Plasmodium falciparum. 
anti-C-Term Circumsporozoide Ab.IgG 
 
ARM 
1 month after 
1st dose 
n (%) 
1 month after 
2nd dose 
n (%) 
1 month after 
3rd dose 
n (%) 
6 months after 
1st dose 
n (%) 
R21/Matrix-M™ 
+ DHA-PIP + SLD-PQ 
R21/Matrix-M™ 
No vaccine 
* The percentage of subjects seroconverting after each immunization based on a value greater than 
the mean titer at baseline (before immunization # 1) plus 2 standard deviations for all subjects 
included in the analysis. 
 
 
Table 3ciii. Proportion seropositive* for antibodies at each timepoint: Plasmodium falciparum. 
Circumsporozoite Full length (N+C-Terminal) 
 
ARM 
1 month after 
1st dose 
n (%) 
1 month after 
2nd dose 
n (%) 
1 month after 
3rd dose 
n (%) 
6 months after 
1st dose 
n (%) 
R21/Matrix-M™ 
+ DHA-PIP + SLD-PQ 
R21/Matrix-M™ 
No vaccine 
* The percentage of subjects seroconverting after each immunization based on a value greater than 
the mean titer at baseline (before immunization # 1) plus 2 standard deviations for all subjects 
included in the analysis. 


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Statistical Analysis Plan version 1.0 date 10 Mar 2023 
 
The serum anti-CS antibody titre 
The serum anti-CS antibody titre -The geometric mean-in titre from baseline to: One month after first 
dose; One month after second dose; One month after the third dose; Six months after the first dose 
for all groups. 
 
Table 4ai. Plasmodium falciparum. Circumsporozoite Protein.(NANP)6 Ab.IgG 
 
ARM 
1 month after 
1stdose 
Geometric Mean 
(95 % CI) 
1 month after 
2nddose 
Geometric Mean 
(95 % CI) 
1 month after 
3rd dose 
Geometric Mean 
(95 % CI) 
6 months after 
1st dose 
Geometric Mean 
(95 % CI) 
R21/Matrix-M™ 
+DHA-PIP + SLD-PQ 
R21/Matrix-M™ 
No vaccine 
 
 
Table 4aii. Plasmodium falciparum. anti-C-Term Circumsporozoide Ab.IgG 
 
ARM 
1 month after 
1st dose 
Geometric Mean 
(95 % CI) 
1 month after 
2nd dose 
Geometric Mean 
(95 % CI) 
1 monthafter 
3rd dose 
Geometric Mean 
(95 % CI) 
6 months after 
1st dose 
Geometric Mean 
(95 % CI) 
R21/Matrix-M™ 
+ DHA-PIP + SLD-PQ 
R21/Matrix-M™ 
No vaccine 
 
 
Table 4aiii. Plasmodium falciparum. Circumsporozoite Full length (N+C-Terminal) 
 
ARM 
1 month after 
1st dose 
Geometric Mean 
(95 % CI) 
1 month after 
2nd dose 
Geometric Mean 
(95 % CI) 
1 month after 
3rd dose 
Geometric Mean 
(95 % CI) 
6 months after 
1st dose 
Geometric Mean 
(95 % CI) 
R21/Matrix-M™ 
+ DHA-PIP + SLD-PQ 
R21/Matrix-M™ 
No vaccine 


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Statistical Analysis Plan version 1.0 date 10 Mar 2023 
 
AVIDITY (Geometric means) 
 
Avidity -The geometric mean in titre from baseline to: One month after first dose; One month after 
second dose; One month after the third dose; Six months after the first dose for all groups. 
 
Table 4bi. Plasmodium falciparum. Circumsporozoide Protein.(NANP)6 Ab.IgG Avidity 
 
ARM 
1 month after 
1st dose 
Geometric Mean 
(95 % CI) 
1 month after 
1st dose 
Geometric Mean 
(95 % CI) 
1 month after 
3rd dose 
Geometric Mean 
(95 % CI) 
6 months after 
1st dose 
Geometric Mean 
(95 % CI) 
R21/Matrix-M™ 
+ DHA-PIP + SLD-PQ 
R21/Matrix-M™ 
No vaccine 
 
 
Table 4bii. Plasmodium falciparum. anti-C-Term Circumsporozoide Ab.IgG avidity 
 
ARM 
1 month after 
1st dose 
Geometric Mean 
(95 % CI) 
1 month after 
2nd dose 
Geometric Mean 
(95 % CI) 
1 month after 
3rd dose 
Geometric Mean 
(95 % CI) 
6 months after 
1st dose 
Geometric Mean 
(95 % CI) 
R21/Matrix-M™ 
+ DHA-PIP + SLD-PQ 
R21/Matrix-M™ 
No vaccine 
 
 
Table 4biii. P falciparum. anti-full length CSP Ab.IgG avidity 
 
ARM 
1 month after 
1st dose 
Geometric Mean 
(95 % CI) 
1 month after 
2nd dose 
Geometric Mean 
(95 % CI) 
1 month after 
3rd dose 
Geometric Mean 
(95 % CI) 
6 months after 
1st dose 
Geometric Mean 
(95 % CI) 
R21/Matrix-M™ 
+ DHA-PIP + SLD-PQ 
R21/Matrix-M™ 
No vaccine 


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Statistical Analysis Plan version 1.0 date 10 Mar 2023 
 
5. 
STATISTICAL ANALYSES OF PRIMARY SAFETY ENDPOINTS 
Safety analysis will be performed on patients allocated to receive vaccination. The percentage of 
subjects with at least one local AEs with at least one general AE (solicited and unsolicited) and with any 
AE during the 7-day or total follow-up period up to 28 days after the last vaccine dose and overall will 
be tabulated with exact 95% CI. The percentage of doses followed by at least one local AE (solicited and 
unsolicited), by at least one general AE (solicited and unsolicited) and by any AE during the 7-day or the 
total follow up period up to 28 days after the last vaccine will be tabulated with exact 95% CI. The same 
computations will be done for Grade 3 AEs, for any AEs considered related to vaccination and for any 
Grade 3 AEs considered related to vaccination. The percentage of subjects reporting each individual 
solicited local AE (any grade and Grade 3) and solicited general AE (any grade, Grade 3, any related, 
Grade 3 related, resulting in medically attended visit) during the 7-day follow-up period (Day 0-6) after 
each vaccine dose and overall will be tabulated for each group. Similarly, the percentage of doses 
followed by each individual solicited local and general AE will be tabulated, overall vaccination course, 
with exact 95% CI. For fever, the number and percentage of subjects reporting fever by half degree (°C) 
cumulative increments during the first seven days (Day 0-6) after each vaccine dose and overall will be 
tabulated. Similar tabulations will be performed for any fever with a causal relationship to vaccination 
and Grade 3 (> 39.5°C) causally related fever. The percentage of subjects reporting unsolicited AEs from 
the date of the first vaccine dose up to 28 days after the last vaccine dose will be tabulated by group 
and by MedDRA preferred term with exact 95% CI. Similar tabulation will be done for Grade 3 unsolicited 
AEs, for any causally related unsolicited AEs and for Grade 3 causally related unsolicited AEs. The 
percentage of subjects reporting SAEs and pregnancies will be described in detail. The percentage of 
vaccinated subjects reporting AEs of specific interest (meningitis and pIMDs) will be described in detail. 
Thus, in summary, the safety analysis will be as follows: 
● Frequency (%) of occurrence of solicited local and general AEs within seven days (day of 
vaccination and six subsequent days) after each vaccination with exact 95% CI by group and 
severity. 
● Frequency (%) of occurrence of unsolicited AEs from the date of the first vaccination to 28 days 
after the last vaccination, according to the Medical Dictionary for Regulatory Activities 
(MedDRA) classification with exact 95% CI, by group and severity. 
● Frequency (%) of occurrence of SAEs (all, fatal, related to investigational vaccine) within 28 days 
(day of vaccination and 28 subsequent days) after each vaccination, according to the MedDRA 
classification with 95% CI. 
● Frequency (%) of occurrence of SAEs (all, fatal, related to investigational vaccine) during the 
whole study period according to the MedDRA classification with exact 95% CI. 
● Frequency (%) of occurrence of AEs and SAEs leading to withdrawal from further vaccination 
from Dose 1 (Day 0) up to study conclusion (Day 180), according to the MedDRA classification, 
for each vaccinated subject exact 95% CI. 
● Frequency (%) of occurrence of pIMDs from Dose 1 (Day 0) up to study conclusion (Day 180), 
according to MedDRA classification, for each vaccinated subject with exact 95% CI. 
● Frequency (%) of occurrence of meningitis from Dose 1 (Day 0) up to study conclusion (Day 180), 
according to the MedDRA classification, for each vaccinated subject exact 95% CI. 
These will be summarised in table 4 below: 


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Statistical Analysis Plan version 1.0 date 10 Mar 2023 
 
Table 4 Safety analysis 
 
Safety parameter 
R21/Matrix-M™ 
+ DHA-PIP + SLD-PQ 
n=X 
R21/Matrix-M™ 
n=X 
No Vaccine 
n=X 
Solicited local and general AEs within seven 
days n(%), (95% CI) 
Unsolicited AEs: date of 1st vaccine to 28 days 
after last vaccine n(%), (95% CI) 
SAEs within 30 days after each vaccine n (%), 
(95% CI) 
SAEs for whole period n (%), (95% CI) 
AEs+SAEs leading to withdrawal from further 
vaccination from Dose 1 to study conclusion 
n(%), (95% CI) 
pIMDs from Dose 1 to study conclusion n(%), 
(95% CI) 
 
 
Biochemistry and haematological safety assessment. 
 
Biochemistry (ALT, AST and creatinine) and haematological (haemoglobin, WBC, and platelets) 
laboratory values will be presented according to toxicity grading scales and tabulated by group. The 
normal ranges and toxicity grading for laboratory safety parameters those will be used in this study are 
in Appendix section (Table A1): 
 
The following definitions will be used for clinical adverse events in this report: The intensity should be 
assigned to one of the following categories: 
 
 
1 (mild) 
= 
An AE which is easily tolerated by the subject, causing minimal 
discomfort and not interfering with everyday activities. 
2 (moderate) 
= 
An AE which is sufficiently discomforting to interfere with normal 
everyday activities. 
3 (severe) 
= 
An AE which prevents normal, everyday activities. In adults, such an AE 
would, for example, prevent attendance at work/school and would 
necessitate the administration of corrective therapy. 
 
An AE that is assessed as Grade 3 (severe) will not be confused with a SAE. Grade 3 is a category used 
for rating the intensity of an event; and both AEs and SAEs can be assessed as Grade 3. A grade 3 AE 
may not necessarily be an SAE in this study. 
***