Seasonal Vaccination With the RTS,S/AS01 Malaria Vaccine Given With or Without Seasonal Malaria Chemoprevention: Extension of a Randomised, Double-blind Phase 3 Trial Until Children Reach the Age of Five Years
Phase: PHASE3
Conditions: Malaria,Falciparum, Child, Only
Sponsor: London School of Hygiene and Tropical Medicine

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This supplement contains the following items.  
1. Final RTS,S + SMC Extension study protocol, dated 27 January 2022. 
2. Statistical analysis plan, dated 23 June 2022. 
 
Clinical trials registration no. NCT04319380 
 

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Version 8 
 27.01.22 
 
 
 
 
Seasonal vaccination with the RTS,S/AS01E malaria vaccine 
given with or without seasonal malaria chemoprevention:  
extension of a randomised, double-blind Phase 3 trial until 
children reach the age of five years. 
 
 
 
Brief Title: A trial of RTS,S/AS01E and SMC: extension study 
 
Protocol Identifying Number - PATH: CVIA 083 
 
Protocol Identifying Number - GSK: MALARIA-106 EXT:099 Y4-5 SUPP 
 
Lead Principal Investigators: Daniel Chandramohan & Brian Greenwood 
 
Site Study Directors: Jean Bosco Ouedraogo & Alassane Dicko  
 
Sponsor: London School of Hygiene & Tropical Medicine, 
 
      Keppel Street, WC1E 7HT, 
 
      London, UK 
 
 
 
 

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TABLE OF CONTENTS 
 
TABLE OF CONTENTS ..................................................................................................................................... 3 
LIST OF ABBREVIATIONS ............................................................................................................................... 6 
STATEMENT OF COMPLIANCE ...................................................................................................................... 7 
PROTOCOL SUMMARY .................................................................................................................................. 8 
SCHEMATIC OF STUDY DESIGN ................................................................................................................... 12 
1. 
KEY ROLES ....................................................................................................................................... 12 
2. 
INTRODUCTION: BACKGROUND INFORMATION AND SCIENTIFIC RATIONALE .............................. 14 
2.1 
The RTS,S/AS01E Vaccine ........................................................................................................ 15 
2.2 
Seasonal Malaria Chemoprevention (SMC) ............................................................................ 15 
2.3 
A trial of seasonal vaccination with the RTS,S/AS01E malaria vaccine with or without SMC . 16 
2.3.1 
Outline ............................................................................................................................. 16 
2.3.2 
Progress with the trial ..................................................................................................... 17 
2.3.3 
Rationale for continuation of the trial for a further two years ....................................... 17 
2.4 
Potential risks and benefits ..................................................................................................... 18 
2.4.1 
Risks of receipt of RTS,S/AS01E or tetanus toxoid or tetanus/diphtheria vaccine ......... 18 
2.4.2 
Risks of receipt of SP and AQ .......................................................................................... 19 
2.4.3 
Risks of accidental disclosure of private medical information ....................................... 19 
2.4.4 
Risks of participation in the study ................................................................................... 19 
2.4.5 
Risks of phlebotomy ........................................................................................................ 19 
2.4.6 
Known potential benefits ................................................................................................ 19 
3. 
OBJECTIVES AND PURPOSE ............................................................................................................. 20 
4. 
STUDY DESIGN AND ENDPOINTS .................................................................................................... 21 
4.1 
Study areas .............................................................................................................................. 21 
4.2 
Study endpoints ...................................................................................................................... 22 
4.2.1 
Primary endpoint ............................................................................................................ 22 
4.2.2 
Secondary endpoints ...................................................................................................... 22 
4.2.3 
Exploratory endpoints ..................................................................................................... 23 
5. 
STUDY RECRUITMENT, ENROLLMENT AND WITHDRAWAL ............................................................ 23 
5.1 
Participant inclusion criteria ................................................................................................... 23 
5.2 
Participant exclusion criteria .................................................................................................. 23 
5.3 
National and community consent ........................................................................................... 24 

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5.4 
Recruitment ............................................................................................................................ 24 
5.5 
Participant withdrawal or termination ................................................................................... 24 
6. 
STUDY VACCINE............................................................................................................................... 25 
6.1 
Study vaccines and antimalarial medications ......................................................................... 25 
6.1.1 
Product storage and handling ......................................................................................... 25 
6.1.2 
Preparation ..................................................................................................................... 26 
6.1.3 
Route of administration .................................................................................................. 26 
6.2 
Study vaccine accountability procedures ............................................................................... 26 
7. 
STUDY Interventions ....................................................................................................................... 26 
7.1 
Insecticide-treated bednets (LLIN) .......................................................................................... 26 
7.2 
The RTS,S/AS01E vaccine ......................................................................................................... 26 
7.3 
Comparator vaccines .............................................................................................................. 27 
7.4 
Vaccine administration ........................................................................................................... 27 
7.5 
Seasonal Malaria Chemoprevention (SMC) ............................................................................ 27 
7.6 
Follow-up and measurement of endpoints ............................................................................ 28 
7.6.1 
Passive surveillance for uncomplicated and severe malaria. ......................................... 28 
7.6.2 
Active surveillance for malaria ........................................................................................ 29 
7.6.3 
Prevalence of malaria parasitaemia and anaemia. ......................................................... 29 
7.6.4 
Evaluation of mutations in the P.falciparum csp gene ................................................... 29 
7.6.5 
Immune response to the RTS,S/AS01E vaccine ............................................................... 29 
7.6.6 
Malaria endemicity in the study area ............................................................................. 29 
7.6.7 
Drug resistance ............................................................................................................... 29 
7.6.8 
In vivo measurement of drug sensitivity to SP+AQ......................................................... 30 
7.6.9 
Investigation of possible rebound malaria ..................................................................... 30 
8. 
ASSESSMENT OF SAFETY ................................................................................................................. 30 
8.1 
Safety assessments ................................................................................................................. 30 
9. 
Laboratory Procedures.................................................................................................................... 31 
9.1 
Detection of malaria. .............................................................................................................. 31 
9.2 
Detection of markers of resistance to SP and AQ. .................................................................. 32 
9.3 
Differentiation of malaria re-infections and recrudescences ................................................. 32 
9.4 
Detection of mutations in the csp gene of P. falciparum ....................................................... 32 
9.5 
Measurement of haemoglobin concentration ........................................................................ 32 
9.6 
Measurement of anti-CSP concentration ............................................................................... 32 

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10. 
STATISTICAL CONSIDERATIONS ................................................................................................... 32 
10.1 
Statistical analysis plan ........................................................................................................... 32 
10.2 
Sample size and analysis of the primary efficacy endpoint .................................................... 33 
10.3 
Sample size and analysis of  secondary endpoint(s) ............................................................... 34 
10.3.1 
Sample size for measurement of prevalence of parasitaemia in school children. ......... 34 
10.3.2 
Sample size for the serological assays ............................................................................ 35 
10.3.3 
Sample size for the in vivo study of anti-malarial drug resistance ................................. 35 
10.3.4 
Sample size for the rebound study ................................................................................. 35 
11. 
SOCIO-ECONOMIC STUDIES ........................................................................................................ 35 
12. 
Assessment of the IMPACT OF THE COVID-19 PANDEMIC ON CONSULTATIONS AND 
HOSPITALISATIONS in the study sites ..................................................................................................... 35 
13. 
DATA MANAGEMENT and CLINICAL MONITORING .................................................................... 36 
14. 
ETHICS/PROTECTION OF HUMAN SUBJECTS .............................................................................. 37 
14.1 
Future Use of Stored Specimens ............................................................................................. 37 
14.2 
Compensation ......................................................................................................................... 37 
15. 
TRIAL MANAGEMENT AND ROLES OF THE INVESTIGATORS AND COLLABORATORS ................. 37 
16. 
FUNDING ..................................................................................................................................... 38 
17. 
DISSEMINATION PLANS ............................................................................................................... 38 
18. 
REFERENCES ................................................................................................................................ 39 
19. 
TIME AND EVENT TABLE ............................................................................................................. 41 
20. 
APPENDICES ................................................................................................................................ 42 
 
  
 
 

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LIST OF ABBREVIATIONS 
 
 
ACT 
Artemisinin combination therapy 
AE 
Adverse event 
AESI 
Adverse event of special interest 
A/L 
Artemether/lumefantrine 
anti-CS 
Antibody to P. falciparum circumsporozoite protein repeat region 
anti-HBs 
Antibody to hepatitis B surface antigen 
AS01 
GSK’s proprietary adjuvant system containing QS21, MPL, and liposomes 
AQ 
Amodiaquine 
CSP 
Circumsporozoite protein 
DBS 
Dried blood spot 
DSMB 
Data Safety and Monitoring Board 
EMA 
European Medicines Agency 
GCP 
Good Clinical Practice 
HBsAg 
Hepatitis B surface antigen 
IB 
Investigator's Brochure 
ICH-GCP 
International Committee on Harmonisation – Good Clinical Practise 
ID 
Identification 
IRB 
Institutional Review Board 
ICH 
International Council for Harmonisation  
IRSS 
Institut de Recherche en Sciences de la Santé 
ITN 
Insecticide-treated bed net 
LLIN 
Long-lasting insecticde-treated bednets 
LDH 
Lactate dehydrogenase 
LSHTM 
London School of Hygiene and Tropical Medicine 
MMV 
Medicines for Malaria Venture 
MPAC 
Malaria Policy Advisory Committee 
MRTC 
Malaria Research and Training Centre 
NMCP 
National Malaria Control Programme 
pIMD 
Potential immune-mediated disease 
RDT  
 
Rapid diagnostic test for malaria 
RTS,S 
Protein comprising CS and hepatitis B surface antigen 
SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
SOP 
Standard Operating Procedure 
SP 
Sulphadoxine/pyrimethamine 
SMC 
Seasonal Malaria Chemoprevention 
WHO 
World Health Organization 

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STATEMENT OF COMPLIANCE 
 
The signature below constitutes the approval of this protocol and the attachments and provides the 
necessary assurances that this trial will be conducted according to all stipulations of the protocol, 
including all statements regarding confidentiality, and according to local legal and regulatory 
requirements and ICH-GCP (E6) guidelines. 
 
Study Director in Mali 
NAME (PRINTED) 
DATE 
 
Alassane Dicko 
18.3.22 
Study Director in Burkina Faso 
NAME (PRINTED) 
DATE 
 
 Jean Bosco Ouédraogo 
22 Mar 2022 
Study Director in the UK 
NAME (PRINTED) 
DATE 
 
Brian Greenwood 
19.3.22 
 
 

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PROTOCOL SUMMARY  
 
Title 
Seasonal vaccination with the RTS,S/AS01E malaria vaccine given with or without 
seasonal malaria chemoprevention: extension until children reach the age of five years. 
Design 
A trial of seasonal vaccination with the RTS,S/AS01E malaria vaccine with or without 
Seasonal Malaria Chemoprevention (SMC) was initiated in 2017 in 5887 children aged 5-17 
months in Burkina Faso and Mali. The children were enrolled in an individually randomised, 
double-blind controlled trial to investigate (a) whether the combination of seasonal 
vaccination with RTS,S/AS01E following priming combined with SMC (sulphadoxine-
pyrimethamine (SP) plus amodiaquine (AQ)) would be superior in providing protection 
against clinical episodes of malaria in young children than either intervention given alone 
and (b) whether seasonal vaccination with RTS,S/AS01E following priming given alone 
would be non-inferior to SMC given alone in preventing clinical attacks of malaria in 
children under five years of age.  Consequently, children enrolled in the study have been 
randomised individually to one of three groups: 
 
a. Group 1 (SMC alone). Children in this group received three doses of rabies vaccine in 
April-June 2017 and one dose of hepatitis A vaccine in June 2018 and in June 2019 
together with four rounds of SMC (SP+AQ) during the period August to November of 
2017, 2018 and 2019.   
b. Group 2 (RTS,S/AS01E alone). Children in this group received three doses of 
RTS,S/AS01E vaccine in April-June 2017 and one dose of RTS,S/AS01E vaccine in June 
2018 and in June 2019 together with four rounds of SMC placebo during the period 
August to November of 2017, 2018 and 2019.   
c. Group 3 (SMC + RTS,S/AS01E vaccine). Children in this group received three doses of 
RTS,S/AS01E vaccine in April-June 2017 and one dose of RTS,S/AS01E vaccine in June 
2018 and in June 2019 together with four rounds of SMC  (SP+AQ) during the period 
August to November of 2017, 2018 and 2019.   
 
The current study design is for continuation of the trial for a further two years. SMC is 
recommended until the age of five years. Thus, if seasonal vaccination with RTS,S/AS01E, or 
with another malaria vaccine with high initial efficacy but a relatively short duration of 
protection, is to be considered as a potential supplement, or possible replacement, for 
SMC, it is important to determine the efficacy of annual seasonal vaccination in primed 
children until they reach the age of five years.  If a recommendation is made to extend SMC 
until the age of 10 years, as may shortly be the case in several countries, it might then be 
appropriate to continue seasonal vaccination until this age but only if it can be shown that 
repeated booster doses of RTS,S/AS01E are safe and retain their efficacy.  
 
If it can be shown during the first three years of the trial that adding seasonal vaccination 
with RTS,S/AS01E to SMC provides added benefit or it is shown and that vaccination with 
RTS,S/AS01E is equivalent or superior to SMC given alone it will be appropriate to continue 
the trial for a further two years but if neither of these outcomes is achieved it will not.  
However, waiting until the definitive results from the first three years of trial are obtained 

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in April/May 2020 would make it impossible to obtain the vaccine and drugs, and make the 
logistic arrangements needed to deploy them, in time for the trial to continue without 
interruption during the 2020 malaria transmission season (July-October).  Therefore,  a 
preliminary analysis of both efficacy and safety data obtained up to the end of November 
2019 was recommended by one of the trial’s donors and this review was undertaken by an 
independent expert appointed by the Bill and Melinda Gates Foundation in January 2020 to 
provide advice as to whether the trial should be continued beyond its present phase and, if 
so, whether the three study arms should be retained.   The same set of data was reviewed 
at two meetings held by the Data and Safety Management Board (DSMB) on January 16th 
and 17th 2020. Both the independent expert and members of the DSMB recommended 
continuation of the trial with retention of all three study groups as outline in this proposal.  
 
This will, therefore, be a controlled, individually randomised trial with three study groups. 
The study groups and the interventions that they will receive are  indicated below- 
 
Group1 (SMC) 
Group 2 (RTS,S) 
Group 3 
 (RTS,S + SMC) 
2020 
 
 
 
June 
Tetanus or 
Tetanus/diphtheria 
toxoids x 1* 
RTS,S/AS01E x 1 
RTS,S/AS01E x 1 
July -Oct 
SMC x 4 
SMC placebo x 4 
SMC x 4 
 
2021 
 
 
 
June 
Tetanus or 
Tetanus/diphtheria 
toxoids x 1* 
RTS,S/AS01E x 1 
RTS,S/AS01E x 1 
July -Nov  
SMC x 4 (Mali) 
SMC x 5 (Burkina 
Faso) 
SMC placebo x 4 
(Mali) 
SMC placebo x 5 
(Burkina Faso) 
SMC x 4 (Mali) 
SMC x 5 (Burkina 
Faso) 
 
All children will have received previously five doses of RTS,S/AS01E vaccine or comparator 
vaccines together with SMC or placebo. 
 
* Depending on the ability to ship tetanus toxoid vaccine from India as a result of the 
COVID-19 outbreak, children in group 1 will either receive tetanus toxoid vaccine procured 
from the Serum Institute of India, or tetanus/diphtheria toxoids vaccine provided by the 
national infant immunization programmes of Burkina Faso and Mali from the stock utilized 
routinely in their national immunization programmes. 
 
Study 
Objective 
This trial, undertaken in children aged three or four years old who have previously received 
five doses of the RTS,S/ASO1 malaria vaccine or comparator  vaccines, together with 
seasonal malaria chemoprevention (SMC) with sulphadoxine-pyrimethamine (SP) plus  
amodiaquine (AQ) or placebo seeks to determine whether : 
 

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a. Administration of further doses of RTS,S/AS01E at the beginning of the malaria 
transmission until children reach the age of five years  provides additional  protection 
against clinical episodes of malaria when given together with SMC.   
 
b. Vaccination with a booster dose of the RTS,S/AS01E malaria vaccine at the beginning of 
the malaria transmission until children reach the age of five years is non-inferior to  
SMC in preventing clinical attacks of malaria and would be easier to deliver than SMC.  
 
Endpoints 
The primary end-point for the trial is the incidence of clinical episodes of malaria, defined 
as an episode of fever (temperature >= 37.5o C) or a history of fever within the past 48 
hours, that is severe enough to require treatment at a health centre or by a community 
health worker and which is accompanied by a positive blood film with a parasite density of 
5,000 parasites per µl or more.   
 
Secondary end-points for the trial include – 
 
a. Clinical episodes of an uncomplicated febrile illness (temperature >= 37.5o C), or a history 
of fever within the past 48 hours, with a positive blood film (any level of asexual 
parasitaemia) or a positive rapid diagnostic test (RDT) for malaria. 
 
b. Hospital admissions with malaria, including cases of severe malaria who meet WHO 
criteria for a diagnosis of severe malaria.  
 
c. The prevalence of malaria infection not severe enough to warrant a clinic visit detected 
in a subset of randomly selected children during home visits. This activity is currently 
suspended due to COVID-19. 
 
d. The prevalence of malaria parasitaemia, including gametocytaemia, moderate and 
severe anaemia and malnutrition at the end of the malaria transmission season. Minor 
adverse events following repeated booster doses of RTS,S/AS01E and serious adverse 
events (SAEs), including any deaths, occurring at any time during the study with special 
reference to any cases of meningitis or cerebral malaria. 
 
Exploratory end-points for the trial include: 
 
a. Anti-CSP antibody concentrations obtained  before and after each booster dose, 
determined in a sub-sample of children. 
 
b. The prevalence of malaria parasitaemia at the end of the malaria transmission season in 
school-age children resident in the study areas, as determined during the previous three 
years,  to provide information on whether there have been any changes in the overall level 
of malaria transmission in the study area during the five years of the study.   
 
c. The prevalence of mutations in the Th2 or Th3 locus of the Plasmodium falciparum csp 
gene in children who have received repeated doses of RTS,S/AS01E.  
 
d. The presence of molecular markers of resistance to SP and AQ in parasite positive 
samples collected during the final cross-sectional survey.  

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e. The 28-day treatment outcome in children with asymptomatic malaria parasitaemia 
treated with SP+AQ identified during the final cross-sectional survey.  
 
f. Evidence of ‘rebound’ malaria in study children who have reached the age of five years at 
the time of the last year of the trial and who are no longer eligible to receive either of the 
trial interventions. 
 
g. The presence of antibodies to a broad range of malaria antigens in children who have 
received RTS,S/AS01E alone, SMC alone and RTS,S/AS01E + SMC collected after three years 
of the interventions. 
 
Population 
Children of either sex, aged three or four years old who have been enrolled in the previous 
trial of seasonal vaccination with the RTS,S/AS01E vaccine will be eligible to join this 
extension study, remaining in their original trial allocation group.   
Sample Size 
It is estimated that approximately 4,800 of the 5887 enrolled originally in the trial will be 
available for recruitment into the extension study. 
Phase 
Phase 3 
Number of 
sites 
enrolling 
participants 
Two sites: The trial will be conducted in Houndé district, Burkina Faso and in Bougouni 
district, Mali. 
 
Study 
duration 
July 2020 – June 2022  
 
 
 
 

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SCHEMATIC OF STUDY DESIGN  
Table 1. Group Description 
 
This will be a controlled, individually randomised trial with three study groups. The study groups and the 
interventions that they will receive are - 
 
Group1 (SMC) 
Group 2 
(RTS,S/AS01E) 
Group 3 
 (RTS,S/AS01E + SMC) 
2020 
 
 
 
June 
Tetanus or 
Tetanus/diphtheria 
toxoids x 1 
RTS,S/AS01E x 1 
RTS,S/AS01E x 1 
July -Oct 
SMC x 4 
SMC placebo x 4 
SMC x 4 
 
2021 
 
 
 
June 
Tetanus or 
Tetanus/diphtheria 
toxoids x 1 
RTS,S/AS01E x 1 
RTS,S/AS01E x 1 
July -Nov  
SMC x 4 (Mali) 
SMC x 5 (Burkina Faso) 
SMC placebo x 4 
(Mali) 
SMC placebo x 5 
(Burkina Faso) 
SMC x 4 (Mali) 
SMC placebo x 5 
(Burkina Faso) 
 
All children will have received previously five doses of RTS,S/AS01E vaccine or comparator vaccines ( 3 
doses of rabies and 2 doses of hepatitis A vaccines) together with SMC or placebo.   
 
 
1. KEY ROLES  
Principal Investigators 
 
Brian Greenwood 
Faculty of Infectious and Tropical Diseases  
London School of Hygiene & Tropical Medicine, Keppel St.,  
London WC1E 7HT, UK. 
Tel: + 44 207 299 4707 
E-mail: brian.greenwood@lshtm.ac.uk 
 
Daniel Chandramohan 
Faculty of Infectious and Tropical Diseases  
London School of Hygiene & Tropical Medicine, Keppel St.,  
London WC1E 7HT, UK. 
Tel:  + 44 207 927 2322 
E-mail: daniel.chandramohan@lshtm.ac.uk  
  
Site Study Directors 
Jean Bosco Ouedraogo Institut de Recherche en Sciences de la 
Santé, Direction Régionale de l’Ouest (IRSS-DRO),  

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399 Avenue de la Liberté, 01 BP 545 Bobo-Dioulasso 01, 
Burkina Faso. 
 
Or 
 
 
Institut des Sciences et Techniques (INSTech), 540 Avenue de 
Sya, 01 BP 2779, Bobo-Dioulasso, Burkina Faso. 
Tel (226) 20981880 or 20970811 
E-mail: jbouedraogo.irssbobo@fasonet.bf   
or  jbouedrago@gmail.com 
 
Alassane Dicko 
Malaria Research & Training Center; University of Sciences 
Techniques and Technologies of Bamako, P.O. Box 1805, 
Bamako, Mali.  
Tel: +223 2022 8109 
E-mail: adicko@icermali.org 
 
Clinical Investigators 
Tinto Halidou 
Unité de Recherche Clinique de Nanoro (URCN), 
Institut de Recherche en Sciences de la Santé, Direction 
Régionale de l’Ouest (IRSS-DRO), 399 Avenue de la Liberté, 
01 BP 545 Bobo-Dioulasso 01, Burkina Faso. 
Tel. +226 70346354 or +226 76541180 
E-mail: halidoutinto@gmail.com  
 
Issaka Zongo 
Institut de Recherche en Sciences de la Santé, Direction 
Régionale de l’Ouest (IRSS-DRO)  
399 Avenue de la Liberté 
 01 BP 545 Bobo-Dioulasso 01, Burkina Faso. 
Tel : (00226) 20 981 880 
E-mail: zongoissaka08@gmail.com 
 
Issaka Sagara 
Malaria Research & Training Center; Department of 
Epidemiology of Parasitic Diseases, Faculty of Medicine, 
Pharmacy and Dentistry, University of Sciences Techniques 
and Technologies of Bamako, 
P.O. Box 1805 Bamako, Mali. 
Tel: +223 2022 8109 
E-mail: isagara@icermali.org 
 
Other Investigators and 
collaborators 
Matthew Cairns 
Faculty of Epidemiology and Public Health, London School of 
Hygiene & Tropical  Medicine, Keppel St.,  

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London WC1E 7HT, UK. 
Tel:  + 44 207 958 8104  
E-mail: matthew.cairns@lshtm.ac.uk 
 
Paul Milligan 
Faculty of Epidemiology and Public Health, London School of 
Hygiene & Tropical  Medicine, Keppel St.,  
London WC1E 7HT, UK. 
Tel:  + 44 207 927 2126 
E-mail: paul.milligan@lshtm.ac.uk 
 
Paul Snell  
Faculty of Epidemiology and Public Health, London School of 
Hygiene & Tropical  Medicine, Keppel St.,  
London WC1E 7HT, UK. 
Tel:  + 44 207 927 2126 
E-mail: paul.snell@lshtm.ac.uk 
 
Jayne Webster 
Faculty of Infectious and Tropical Diseases  
London School of Hygiene & Tropical Medicine, Keppel St.,  
London WC1E 7HT, UK. 
Tel: + 44 207 299 4707 
E-mail: jayne.webster@lshtm.ac.uk 
 
Yves Daniel Compaoré 
Institut de Recherche en Sciences de la Santé, Direction 
Régionale de l’Ouest (IRSS-DRO),  
399 Avenue de la Liberté, 
 01 BP 545 Bobo-Dioulasso 01, Burkina Faso. 
tél: (00226) 20 981 880 
E-mail: yvesdaniel.co@gmail.com 
 
Abdoulaye Djimde 
Malaria Research & Training Center, Department of 
Epidemiology of Parasitic Diseases, University of  Sciences 
Techniques and Technologies of Bamako, P.O. Box 1805 
Bamako, Mali. 
Tel: +223 20220 8109 
E-mail: adjimde@icermali.org 
 
Sponsor  
London School of Hygiene & Tropical Medicine, 
Keppel Street, WC1E 7HT, 
London, UK 
 
2. INTRODUCTION: BACKGROUND INFORMATION AND SCIENTIFIC RATIONALE  

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2.1 The RTS,S/AS01E Vaccine 
The RTS,S/AS01E malaria vaccine is a recombinant protein vaccine in which the fusion protein RTS 
containing parts of the circumsporozoite protein (CSP) of Plasmodium falciparum fused to hepatitis B 
surface antigen (HBsAg) is co-expressed in yeast, together with free HBsAg (S), to form a virus like 
particle (RTS,S); it is given with the powerful adjuvant AS01 [1]. RTS,S/AS01E induces a strong antibody 
response to P. falciparum CSP and high titres of anti-CSP antibody are associated with protection [2]. 
Following a long process of development, a phase 3 trial of RTS,S/AS01E conducted in 15,439 children in 
seven countries in Africa showed that three doses of RTS,S/AS01E given with a one month interval 
between doses, followed by a fourth dose 18 months after dose 3, gave 36.5 % [95% CI 31,41%] 
protection against clinical attacks of malaria when given to  young children aged 5-17 months who were 
followed for 48 months; efficacy was less when the vaccine was given to infants at the age of 6-12 weeks 
[3]. A long term follow- up in three of the phase 3 trial sites showed that when given to children aged 5-
17 months, vaccine efficacy against severe malaria was 36.7% (95% (5% CI 14.6, 53.1) over a period of 
seven years [4].   RTS,S/AS01E provides a high level of protection during the first three months after 
vaccination,  modelled to be about 70% in the phase 3 trial, a level of initial efficacy similar to that 
observed in an earlier phase 2 trial in Gambian adults [5].  However, efficacy wanes progressively over 
the following months. A subsequent dose given 18 months after the primary series restores some but 
not all of the efficacy seen immediately after the primary series [3, 5]. In July 2015, the European 
Medicines Agency (EMA) reviewed data on the efficacy and safety of RTS,S/AS01E and concluded that 
the risk benefit balance favoured the vaccine and gave a positive opinion on its use in children aged 6 
weeks to 17 months. WHO’s SAGE committee reviewed the vaccine’s efficacy and safety in October 
2015 and made a number of recommendations on its further evaluation [6]. These included the pilot 
implementation of RTS,S/AS01E in children aged 5-17 months in three to five settings with moderate-to-
high malaria transmission intensity, with a preference for areas where SMC is not being delivered, and 
recommended evaluation of alternative approaches to deployment of the vaccine. Following the WHO 
recommendations three large RTS,S/AS01E pilot studies commenced in Ghana, Kenya and Malawi in 
2019 [7].  
2.2 Seasonal Malaria Chemoprevention (SMC) 
Seasonal malaria chemoprevention (SMC) involves monthly administration of an antimalarial drug or 
drug combination in a full therapeutic course to children on three or four occasions during the period of 
highest risk of malaria infection. Studies undertaken in several countries in West Africa, including 
Burkina Faso and Mali, showed that SMC with sulphadoxine/pyrimethamine (SP) and amodiaquine (AQ) 
is highly effective in areas where the transmission of malaria is markedly seasonal, reducing the 
incidence of severe and uncomplicated malaria by up to 80% [8-10]. SMC with a combination of SP and 
AQ is safe, with no serious drug related adverse event being reported after administration of over 
800,000 courses in Senegal [11]. Areas where SMC would be an appropriate intervention based on the 
seasonality and incidence of malaria include most of the Sahel and sub-Sahel, a population of 
approximately 200 million, and possibly other areas in southern and eastern Africa [12]. A Technical 
Expert Group of the WHO reviewed all the available evidence on the efficacy and safety of SMC in May 
2011 and recommended SMC with SP+AQ in areas of the Sahel and sub-Sahel with highly seasonal 
transmission. This recommendation was endorsed by the WHO Malaria Policy Advisory Committee 
(MPAC) in February 2012 [13]. Most countries in the Sahel and sub-Sahel region have incorporated SMC, 
along with other malaria control interventions, in their strategic malaria control plan and the 

16 
 
implementation of SMC at scale is in progress in many countries in this region with financial support 
from local governments and several international donor organisations. It is estimated that SMC will be 
delivered to about 20 million children in 2019. Preliminary evaluation suggests that SMC is providing 
about 50% protection against clinical malaria when delivered through a national programme 
(http://www.malariaconsortium.org/pages/access-smc.htm).  
 
Despite the efficacy of SMC and its deployment together with long-lasting insecticide-treated bednets 
(LLIN), malaria remains a major cause of mortality and hospital admissions among young children in 
many areas of the Sahel and sub-Sahel and new approaches to malaria control are needed in these 
areas. One potential, novel approach is combining seasonal malaria vaccination with SMC [14]. Although 
RTS,S/AS01E may be a little less effective than SMC during the malaria transmission season, this may be 
balanced by provision of protection during the dry season when some malaria transmission still occurs 
and when SMC is not given. In addition, a single seasonal dose of vaccine may be easier to administer 
than four rounds of SMC. For this reason, a trial was initiated in Burkina Faso and Mali in 2017 to test 
whether the combination of seasonal vaccination with RTS,S/AS01E combined with SMC would be more 
effective than either intervention given alone. In addition, this trial has set out to establish whether 
seasonal vaccination with RTS,S/AS01E could be used as an alternative to SMC if, for example, resistance 
emerged to the anti-malarials used for SMC. Currently, there are no other combinations of licensed 
antimalarials that could be used to replace SP and AQ for SMC and it is likely to be 5-10 years before 
novel antimalarials under development are available for this purpose.   
 
2.3 A trial of seasonal vaccination with the RTS,S/AS01E malaria vaccine with or without 
SMC 
2.3.1 Outline 
For the reasons given above, in the first quarter of 2017, 5887 children aged 5-17 months (2777  in 
Burkina Faso and 3143 in Mali) were enrolled in an individually randomised, double-blind controlled trial 
to investigate (a) whether the combination of seasonal vaccination with RTS,S/AS01E following priming 
combined with SMC with SP+AQ would be superior in providing protection against clinical episodes of 
malaria in young children than either intervention given alone and (b) whether seasonal vaccination 
with RTS,S/AS01E following priming given alone would be non-inferior to SMC given alone in preventing 
clinical attacks of malaria in young children [15].  Consequently, children enrolled in the study have been 
randomised individually to one of three groups: 
 
a. Group 1 (SMC alone). Children in this group received three doses of rabies vaccine in April-June 2017 
and one dose of hepatitis A vaccine in June 2018 and in June 2019 together with four rounds of SMC 
(SP+AQ) during the period August to November of 2017, 2018 and 2019.   
b. Group 2 (RTS,S/AS01E alone). Children in this group received three doses of RTS,S/AS01E vaccine in 
April-June 2017 and one dose of RTS,S/AS01E vaccine in June 2018 and in June 2019 together with 
four rounds of SMC placebo during the period August to November of 2017, 2018 and 2019.   
c. Group 3 (SMC + RTS,S/AS01E vaccine). Children in this group received three doses of RTS,S/AS01E 
vaccine in April-June 2017 and one dose of RTS,S/AS01E vaccine in June 2018 and in June 2019 
together with four rounds of SMC  (SP+AQ) during the period August to November of 2017, 2018 
and 2019.   

17 
 
 
The primary end-point for the trial is the incidence of clinical episodes of malaria, defined as an episode 
of illness characterised by fever (temperature >= 37.5o C), or a history of fever within the past 48 hours, 
that is severe enough to require treatment at a health centre or by a community health worker and 
which is accompanied by a positive blood film with a parasite density of 5,000 per µl or more. Secondary 
end-points include deaths, hospital admissions with malaria, the prevalence of malaria infection in 
healthy children at weekly surveys during the transmission season and the prevalence of malaria in 
study children at the end of the malaria transmission season. The economic cost of adding RTS,S/AS01E 
to SMC, its feasibility and the acceptability of each intervention are also being evaluated.  
 
The trial is scheduled to finish collection of data in March 2020 when study children will have been 
followed through three malaria transmission seasons and two and a half dry seasons. By this time, 
children in the RTS,S/AS01E groups will have received two seasonal booster doses of the RTS,S/AS01E 
vaccine following priming.    
2.3.2 Progress with the trial 
In Burkina Faso, 92% of enrolled children received all three priming doses of RTS,S/AS01E or control 
(rabies) vaccine in 2017, 90% received their first booster dose in June 2018 and 90% received their 
second booster dose in June 2019.  In Mali, 93% of children received all three priming doses of 
RTS,S/AS01E or comparator vaccine in 2017, 88% received their first booster dose of vaccine in June 
2018 and 85% their second booster dose of vaccine in June 2019.  Coverage with SMC or placebo has 
been high throughout the trial with nearly 90% of children receiving all four monthly SMC courses during 
the first and second years of the study.  Retention of children in the trial has also been high. At a census 
undertaken early in 2019, 2547 (92%) of the 2777 children enrolled in Burkina Faso and 2816 (90%) of 
the 3143 children initially enrolled in Mali were still contributing to the study.   
 
The investigators remain blind to the randomisation code. However, in March 2019 members of the 
trial’s Data and Safety Monitoring Board (DSMB) met to review safety outcomes during the first two 
years of the trial. They reviewed data on deaths, deaths attributed to malaria, hospital admissions and 
hospital admissions attributed to malaria broken down by study group. The board concluded that it was 
safe for the trial to continue for a third year retaining the three initial study groups. This 
recommendation implies that during 2018 a single booster dose of RTS,S/AS01E was not markedly 
inferior to SMC in protecting against the outcomes considered by the board and provided 
encouragement for continuation of the trial into a third year and, potentially, for longer.  
2.3.3 Rationale for continuation of the trial for a further two years 
SMC is recommended until the age of five years (10 years in Senegal). Thus, if seasonal vaccination with 
RTS,S/AS01E, or with another malaria vaccine with high initial efficacy but a relatively short duration of 
protection, is to be considered as a potential supplement, or possible replacement for SMC, it is 
important to determine the efficacy of annual seasonal vaccination in primed children until they reach 
the age of five years.  If a recommendation is made to extend SMC until the age of 10 years, as may 
shortly be the case in several countries, it might then be appropriate to continue seasonal vaccination 
until this age but only if it can be shown that repeated booster doses of RTS,S/AS01E are safe and retain 
their efficacy.  
 

18 
 
If it can be shown during the first three years of the trial that adding seasonal vaccination with 
RTS,S/AS01E to SMC provides added benefit or it is shown and that vaccination with RTS,S/AS01E is 
equivalent or superior to SMC given alone it will be appropriate to continue the trial for a further two 
years but if neither of these outcomes is achieved it will not.  However, waiting until the definitive 
results from the first three years of trial are obtained in April/May 2020 would make it impossible to 
obtain the vaccine and drugs, and make the logistic arrangements needed to deploy them, in time for 
the trial to continue without interruption during the 2020 malaria transmission season (July-October).  
Therefore, it was proposed by one of the trial’s funders that a preliminary analysis of safety and efficacy 
on data collected up to the end of November 2009 should be undertaken to determine whether it would 
be useful to continue the trial until children reached the age of five years and, if so, whether all the trial 
groups should be retained. To guide the decision-making progress during this preliminary analysis, a 
decision tree (appendix 1) was developed. Efficacy data covering three transmission seasons and two 
dry seasons were provided to an independent expert appointed by the Bill and Melinda Gates 
Foundation at the beginning of January 2020.  The same set of data was also provided to the trial’s Data 
and Safety Management Board (DSMB) which reviewed it at meetings held on January 16th and 17th 
2020. Both the independent expert and members of the DSMB recommended continuation of the trial 
with retention of all three study groups, as outline in this proposal.  
2.4 Potential risks and benefits 
Details of potential risks are provided in the Investigator’s Brochure for RTS,S/AS01. Potential risks for 
licensed products, tetanus or tetanus/diphtheria toxoid vaccine, SP, and AQ may be found in the 
package insert for the product.  
2.4.1 Risks of receipt of RTS,S/AS01E or tetanus toxoid or tetanus/diphtheria vaccine  
Recipients of  the investigational vaccine (RTS,S/AS01E) may experience pain and/or swelling at the 
injection site, fever, headache, fatigue, nausea, vomiting and/or abdominal pain, joint pain and/or 
muscle aches but these features are usually only mild. About 1:1,000 children have been reported to 
develop a febrile convulsion after receipt of RTS,S/AS01E . In the first three years of RTS,S/AS01E plus 
SMC trial only five children have experienced a febrile convulsion and it is not known if all these children 
had received RTS,S/AS01E. In the large Phase III study, MALARIA-055, an imbalance in meningitis cases of 
any etiology (i.e. including cases with confirmed etiology and cases with no etiology found), with no 
cluster in time-to-onset, was observed in children 5-17 months of age at first dose [3]. Potential 
immune-mediated disease (pIMD) is a theoretical concern with adjuvanted vaccines but no evidence of 
autoimmune disease caused by RTS,S/AS01 has been observed. Nonetheless, both meningitis and pIMDs 
will be monitored as AEs of specific interest for the duration of the study. 
 
Vaccination with tetanus or tetanus/diphtheria toxoids may cause mild local and systemic reactions and 
anaphylaxis has been recorded as a very rare adverse event following administration of this vaccine. 
Some children in the extension study will receive five doses of tetanus or tetanus/diphtheria toxoids 
during their first five years of life. Administration of a fourth dose of tetanus or 
tetanus/diphtheria/pertussis vaccine at the age of approximately 18 months is recommended in some 
routine infant immunization programmes. There is less experience of administration of a fifth dose. 
However, tetanus toxoid is recommended at each pregnancy in low income countries and many women 
must have received at least five doses of tetanus toxoid without any reports of adverse events.  

19 
 
2.4.2 Risks of receipt of SP and AQ 
SP can cause Stevens-Johnson syndrome, but this side effect has been seen very rarely when SP has 
been used for either intermittent preventive treatment of malaria in pregnancy or SMC.  Amodiaquine is 
bitter and may cause vomiting but serious side effects, which include liver damage and neurological side 
effects, are very rare.  SMC with SP+AQ has proved to be very safe.  A study conducted in Senegal in 
which the consequence of administration of 800,000 doses of SMC with SP+AQ were followed carefully 
identified only three serious potentially drug related adverse effects (two cases of hepatic damage and 
one extra-pyramidal syndrome) [11]. Enhanced pharmaco- vigilance in seven countries participating in 
the ACCESS-SMC project funded by UNITAID, through which approximately 15 and 30 million treatment 
courses were administered in 2015 and 2016 respectively has confirmed that SMC with SP+AQ is very 
safe.   
2.4.3 Risks of accidental disclosure of private medical information  
In order to ensure that all information collected on study volunteers is kept confidential, the following 
safeguards will be applied:   access to study files and personal information will be limited to study 
personnel, ethics committees, regulatory authorities, and the sponsor.  Any link between individual 
study identification number and an individual’s personal identifying information will be maintained in 
accordance with the sites SOPs to maintain each individual’s confidentiality. 
2.4.4 Risks of participation in the study  
If a participant is hurt as a direct result of participating in this study, the medical care will be provided by 
the study team at the respective research centres and the study will pay for the expenses. 
2.4.5 Risks of phlebotomy 
Venipuncture is a routine clinical procedure which the medical community commonly uses to obtain 
blood samples.  Immediate complications may be slight pain during the entry of the needle into the skin, 
very rarely dizziness and syncope.  Additionally, a haematoma may result from the venipuncture, but 
this has minimal risk.  Infection of the skin/soft tissue at the puncture site, vein, or blood stream can all 
occur, though these are very rare with both finger sticks and venous blood draws.  Late complications 
might include thrombosis of the vein due to trauma or infection.  These complications are extremely 
rare.  Participant monitoring, aseptic technique, including sterile disposable blood collection apparatus 
and adherence to standard medical precautions, reduce any risk to a minimum.   
2.4.6 Known potential benefits 
SMC is a policy established by the WHO, and provides significant benefit against clinical and severe 
malaria  in West Africa where P. falciparum transmission is highly seasonal 
(https://www.who.int/malaria/mpac/feb2012/smc_policy_recommendation.pdf). 
A booster dose of tetanus toxoid or tetanus/diphtheria/pertussis vaccine at about the age of 18 months 
is recommended by WHO but is rarely given so that a majority of the children in the control group will 
receive some benefit that they would not have obtained had they not been enrolled in the trial.  
RTS,S/AS01E has been shown in a large Phase 3 trial to provide significant protection against both 
uncomplicated and  severe malaria across different transmission settings.  

20 
 
3. OBJECTIVES AND PURPOSE  
Primary objective: 
The primary objectives of the extension study are to determine, among children aged three or four years 
of age who have previously received RTS,S/AS01E alone, SMC alone or both interventions since the age 
of 5-17 months:  
a. Whether the combination of seasonal vaccination with the malaria vaccine RTS,S/AS01E with SMC 
with SP+AQ is superior to the administration of either intervention given alone in preventing clinical 
attacks of malaria, defined as an episode of illness characterised by fever (temperature >= 37.5o C), 
or a history of fever within the past 48 hours, that is severe enough to require treatment at a health 
centre or by a community health worker and which is accompanied by a positive blood film with a 
parasite density of 5,000 parasites per µl or more.  
b. Whether seasonal vaccination with RTS,S/AS01E alone is non-inferior to SMC alone in preventing 
clinical attacks of malaria, defined as above.   
Secondary objectives: 
Secondary objectives of the extension study are to determine, among children aged three or four years 
of age who have previously received RTS,S/AS01E alone, SMC alone or both interventions since the age 
of 5-17 months: 
a. The incidence of uncomplicated clinical episodes of febrile illness (temperature ≥ 37.5◦C) or a history 
of fever within the past 48 hours with a positive blood film (any level of asexual parasitaemia) or a 
positive rapid diagnostic (RDT) for malaria. 
b. The incidence of hospital admissions with malaria including cases of severe malaria meeting the   
WHO criteria for severe malaria. 
c. The prevalence of malaria infection not severe enough to warrant a clinic visit detected in a subset 
of randomly selected children during home visits. 
d. The prevalence of malaria parasitaemia including gametocytaemia, moderate and severe anaemia 
and malnutrition at the end of malaria transmission season. 
e. The incidence of minor adverse events following repeated booster of RTS.S/AS01 or tetanus and of 
serious adverse events (SAEs), including any deaths occurring at any time during the study, with 
special reference to any cases of meningitis or cerebral malaria. 
Exploratory 0bjectives: 
Exploratory objectives include: 
a. Anti-CSP antibody concentrations obtained before and after priming and before and after each 
booster dose, determined in a sub-sample of children. 
b. The prevalence of malaria parasitaemia at the end of the malaria transmission season in school-age 
children resident in the study areas, as determined during the previous three years, to provide 
information on whether there have been any changes in the overall level of malaria transmission in 
the study area during the five years of the study.  
c. The prevalence of mutations in the Th2 or Th3 locus of the Plasmodium falciparum csp gene in 
children who have received repeated doses of RTS,S/AS01E.  
d. The presence of molecular markers of resistance to SP and AQ in parasite positive samples collected 
during the final cross-sectional survey.  

21 
 
e. The 28-day treatment outcome in children with asymptomatic malaria parasitaemia detected during 
the final cross sectional survey following treatment with SP+AQ.  
f. Evidence of ‘rebound’ malaria in study children who have reached the age of five years at the time 
of the last year of the trial and who are no longer eligible to receive either of the interventions. 
g. The acceptability to parents and health staff involved in delivery of the two interventions and the 
cost of adding RTS,S/AS01E to SMC.  
h. The presence of antibodies to a broad range of malaria antigens in children who have received 
RTS,S/AS01E alone, SMC alone and RTS,S/AS01E + SMC collected after three years of receipt of the 
interventions. 
 
4. STUDY DESIGN AND ENDPOINTS  
4.1 Study areas 
The trial will be conducted in Houndé health district, Burkina Faso and in Bougouni district, Mali.  The 
Houndé district is situated 300 km from Ouagadougou and 100 Km from Bobo-Dioulasso where the 
CHUSS, the 2nd National Reference Hospital (University Hospital), is located and which is the also a base 
of the Institut de Recherche en Sciences de la Santé, Direction Régionale (IRSS).   The study site in Mali is 
the district of Bougouni in the region of Sikasso, Mali, 150 km south of Bamako where the Malaria 
Research and Training Centre (MRTC) is based. 
 
Figure 1 : Map of Mali and Burkina Faso showing the two study sites Bougouni and Houndé. 
 
 
 

22 
 
 
The population of Houndé district belongs primarily to the Bwaba ethnic group and that of Bougouni 
primarily to the Bambara and Fula ethnic groups.  Farming is the main occupation in each area. Each 
district has a district hospital.  
 
Malaria, due predominantly to P. falciparum, is highly seasonal in both districts with over 80% of cases 
occurring during the rainy season (July – October) and during the following month. The prevalence of P. 
falciparum malaria in school age children in December 2018 was 24% in Bougouni and 61% in Houndé.  
The main malaria vector in each study area is Anopheles gambiae ss. A high proportion of children sleep 
under an LLIN.  The first line treatment for malaria in the public health system is artemether 
/lumefantrine in each district. Cases of uncomplicated malaria are treated at one of the health centres in 
the district and in Bougouni some cases are treated in the community by trained community health 
workers. Cases of severe malaria are managed in the district hospital.  
 
In Burkina Faso, drugs and vaccines are stored at IRSS, Bobo-Dioulasso where there is a dedicated 
storage room which has pharmaceutical refrigerators with dynamic cooling and an automatic defrosting 
system, power failure and open-door alarms.   The room is air conditioned 24 hours a day and the 
temperature maintained at 22°C with temperature and humidity controllers. In Mali, vaccines are stored 
at MRTC, Bamako, Mali. Vaccines are stored in a cold room with continuous monitoring of the 
temperature devices with alarm and telephone SMS and an email alert system. The system is also 
equipped with two back-up generators. Only authorized personnel have access to the cold room.  The 
cold room has a capacity of 14.28 m3 (2.45m long x 2.45m wide x 2.38m high).  Standard operating 
procedures are in place for vaccine reception, storage in the cold room and transfer to field sites on a 
daily basis.  Field laboratories, which are equipped to undertake parasitological and haematological 
investigations, have been established at each district hospital.  A trial in 16,000 children of adding 
azithromycin to the antimalarial drugs used for SMC was conducted successfully in these two study sites 
in 2014-2016 [16].  
4.2 Study endpoints 
4.2.1 Primary endpoint 
The primary end-point for the trial is the incidence of clinical episodes of malaria, defined as an episode 
of fever (temperature >= 37.5o C), or a history of fever within the past 48 hours, that is severe enough to 
require treatment at a health centre or by a community health worker and which is accompanied by a 
positive blood film with a parasite density of 5,000 parasites per µl or more.   
4.2.2 Secondary endpoints 
Secondary end-points for the trial include: 
a. Clinical episodes of an uncomplicated febrile illness (temperature >= 37.5o C), or a history of fever 
within the past 48 hours, with a positive blood film (any level of asexual parasitaemia) or a positive 
rapid diagnostic test (RDT) for malaria. 
b. Hospital admissions with malaria, including cases of severe malaria who meet WHO criteria for a 
diagnosis of severe malaria.  
c. The prevalence of malaria infection not severe enough to warrant a clinic visit detected in a subset 
of randomly selected children during home visits.  

23 
 
d. The prevalence of malaria parasitaemia, including gametocytaemia, moderate and severe anaemia 
and malnutrition at the end of the malaria transmission season.  
e. The safety of administering repeated doses of RTS,S/AS01E and tetanus toxoid as assessed  by the 
immediate response to vaccination and by the incidence of serious adverse events (SAEs), including 
any deaths, occurring at any time during the study with special reference to any cases of meningitis 
or cerebral malaria. 
4.2.3 Exploratory endpoints 
Exploratory end-points for the trial include: 
 
a. Anti-CSP antibody concentrations obtained before and after each booster dose, determined in a 
sub-sample of children. 
b. The prevalence of malaria parasitaemia at the end of the malaria transmission season in school-
age children resident in the study areas, as determined during the previous three years,  to 
provide information on whether there have been any changes in the overall level of malaria 
transmission in the study area during the five years of the study.   
c. The prevalence of mutations in the Th2 or Th3 locus of the Plasmodium falciparum csp gene in 
children who have received multiple doses of RTS,S/AS01E.  
d. The presence of molecular markers of resistance to SP and AQ in parasite positive samples 
collected during the final cross-sectional survey.  
e. The 28-day treatment outcome in children with asymptomatic malaria parasitaemia detected 
during the final cross-sectional survey and who are treated with SP+AQ.  
f. Evidence of ‘rebound’ malaria in study children who have reached the age of five years at the 
time of the last year of the trial and who are no longer eligible to receive either of the 
interventions.  
g. The presence of antibodies to a broad range of malaria antigens in children who have received 
RTS,S/AS01E alone, SMC alone and RTS,S/AS01E + SMC after three years of receipt of the 
interventions. 
 
5. STUDY RECRUITMENT, ENROLLMENT AND WITHDRAWAL  
5.1 Participant inclusion criteria 
In order to be eligible to participate in this study, a child must have been enrolled in the initial phase of 
the trial of seasonal vaccination with the RTS,S/AS01E vaccine and their parents or guardian must have 
provided consent for their inclusion in the extension study.   
 
A child who has reached the age of five years and who is currently enrolled in the trial but who is no 
longer eligible to receive either of the interventions will be followed until the end of the extension study 
on safety grounds and for evidence of “rebound” malaria. 
5.2 Participant exclusion criteria 
Any child who meets any of the following criteria will be excluded from participation in the extension 
study:  
a. The child has had an allergic reaction to the study drugs or vaccines. 

24 
 
b. The child had febrile convulsions on more than one occasion following vaccination. 
c. The child has developed a serious underlying illness such as severe malnutrition (weight for age or 
mid arm circumference Z scores < 3 SD) which in view of the investigators might impair the response 
to vaccination. 
d. The child has been enrolled in another malaria vaccine or other experimental malaria intervention 
study. 
5.3 National and community consent 
Approval of the trial by the national malaria control programme (NMCP) and the expanded programme 
of immunisation (EPI) of each country was obtained prior to the start of the first phase of the study. 
These organisations have been kept informed of its progress; directors of each NMCP have regularly 
attended project meetings. These organisations will be informed of the  results from the initial phase of 
the trial as soon as they are known and the nature of the extension study will be discussed with them. 
Results will also be presented to the local health authorities in the study areas.   
 
Community consent was obtained prior to the start of the first phase of the trial through community 
meetings. As soon as the final results of the trial are known, community meetings will be held to present 
these findings and their implications for the children who have participated in the trial.  However, 
parents will not be told the nature of the study group to which their child was allocated in order to 
retain blinding during the period of the extension period.  At these meetings, the parents or guardians of 
study children will be asked whether they found the number of contact points required for vaccination 
and drug administration acceptable to them and whether they were happy to continue with this 
approach to preventing malaria in their children. Community liaison groups will be established at each 
study site.  
 
5.4 Recruitment 
The first step for the extension study will be a visit to the families of all children enrolled in the initial 
phase of the trial to determine how many remain in the study area and are eligible for recruitment to 
the extension study. It is anticipated that approximately 4,800 will be eligible to join the trial. 
 
Once the families of study children have been identified (each child has an ID card with a unique 
number), project staff will explain the outcome of the first phase of the study and its implications for 
them in a local language that they understand. The family will then be asked if they are willing for their 
child to continue in the trial for a further two years and it will explained to them what this would 
involve. Families will not be told which group their child belonged to and this will not be known to the 
field or laboratory staff participating in the trial so as to obtain blinding.  Provided that the family 
provided their consent, and the child meets the inclusion criteria and does not fall within any of the 
exclusion criteria listed above, the child will be formally enrolled in the extension study and a new 
identify card with an updated photograph will be provided.  
5.5 Participant withdrawal or termination 
The following reasons may lead to withdrawal of individual subjects during the course of the extension 
study: 
• 
Withdrawal of informed consent by parent/guardian.  

25 
 
• 
Age >60 months on May 2020. 
• 
Any adverse event that, according to clinical judgment of the investigator, is considered as a 
definite contraindication to proceeding with the study procedures.  
• 
Complete loss to follow-up.  
• 
Any other protocol deviation that results in a significant risk to the subject’s safety. 
6. STUDY VACCINE 
6.1 Study vaccines and antimalarial medications 
 The Investigational Brochure (IB)  for RTS,S/AS01E  will be provided to the ethical and regulatory review 
committees. The candidate RTS,S/AS01 vaccine to be used has been developed and manufactured by 
GSK Biologicals. The vaccine is labelled and packed according to applicable regulatory requirements. 
 
RTS,S antigen is a lyophilized pellet containing 25 μg of RTS,S per vial. The pellet is reconstituted with 
adjuvant in liquid form and 0.5 mL of reconstituted vaccine contains 25 µg RTS,S. The AS01E adjuvant 
contains 25 µg of MPL®, 25 μg QS21 (QS21 is a triterpene glycoside purified from the bark of Quillaja 
saponaria) in a suspension of liposomes in phosphate buffered saline per 0.5 mL and is presented in 3 
mL monodose vials. The presentation of the reconstituted RTS,S/AS01E candidate malaria vaccine is an 
opalescent liquid. RTS,S/AS01E will be provided by GSK and sent directly to the study sites in Burkina 
Faso and Mali. A single dose consists of 0.5 ml of RTS,S/AS01E final preparation. After reconstitution, the 
vaccine is administered by slow intramuscular injection, using a fresh 25G needle with length of one inch 
(25 mm), into the left deltoid. Vaccine is injected within four hours of reconstitution (storage at +2°C to 
+8°C).  
 
Tetanus toxoid vaccine will be procured by LSHTM from the Serum Institute of India and sent directly to 
the study sites in Burkina Faso and Mali. Tetanus vaccine is provided in a 0.5ml single dose vial containing 
tetanus toxoid ≥ 40 IU adsorbed on Aluminium Phosphate ≤ 1.25 mg. Due to the impact of the COVID-19 
pandemic, it may not be possible to air freight the tetanus toxoid vaccine ordered from the Serum 
Institute of India to Burkina Faso and Mali in time for this to be administered in June 2020. In this 
situation, tetanus/diphtheria toxoids vaccine will be provided by the national infant immunization 
programmes of Burkina Faso and Mali from the stock utilized routinely in their national immunization 
programmes and obtained from recognized manufacturers through UNICEF. The vaccine contains an 
aluminum phosphate adjuvant.   Both vaccines are  administered by slow intramuscular injection into 
the deltoid muscle. 
6.1.1 Product storage and handling 
All vaccine vials/pre-filled syringes (RTS,S) and AS01E adjuvant and the tetanus or tetanus/diphtheria 
toxoids vaccine will be stored in a refrigerator (+2oC to +8oC) and  not frozen. All vaccine/adjuvant/water 
for injection vials will be stored in a safe place with no access for unauthorized personnel. The storage 
temperature will be monitored continuously with calibrated temperature monitoring device(s) and 
recorded according to SOPs at the investigator’s site. An alarm system and a back-up refrigerator will be 
available in case of power failure/breakdown. GSK and the study monitor will be contacted if the cold 
chain is broken, for example if vaccines become frozen or refrigeration fails.  
 

26 
 
GSK will manage temperature deviations during the primary shipment until the reception at the clinical 
trials site. Temperature excursions are the responsibility of the sponsor upon receipt of the RTS,S/AS01E 
study product. Any temperature excursion outside the range of 0.0 to +8.0°C (for +2 to +8°C/+36 to 
+46°F label storage condition) will be reported to the sponsor (LSTMH) and GSK. In case of temperature 
excursion below +2.0°C down to 0.0°C there will be  no need to report this deviation but adequate 
actions will be taken to restore the +2 to +8°C/+36 to +46°F label storage temperature conditions.  
 
Anti-malarial medications will be stored below 30oC in designated storage rooms. 
6.1.2 Preparation 
For this study, the commercial presentation of RTS,S/AS01E will be used, i.e. a two-dose glass vial of 
lyophilized RTS,S antigen (50 µg) to be reconstituted with a two-dose glass vial of AS01E Adjuvant System 
(1.0 ml). The final product for administration will be prepared by reconstitution of the lyophilized 
antigen with the liquid adjuvant. From the reconstituted vaccine vial, 0.5 mL will be administered for 
RTS,S/AS01E full doses. All vials of vaccine provided in this study are intended for single use only. Loading 
of syringes with RTS,S/AS01E or tetanus or tetanus/diphtheria toxoids, and masking syringes with tape to 
disguise their content, will be done by a person who takes  no further part in the trial.  Vaccines will be 
administered by a nurse or other category of health worker trained to give vaccines. 
6.1.3 Route of administration 
The route of administration for both RTS,S/AS01E and tetanus or tetanus/diphtheria toxoid will be by the 
intramuscular route to the deltoid muscle  
SP and AQ will be administered orally according to the dosages recommended by the National Malaria 
Control Programme.  
6.2 Study vaccine accountability procedures 
The study vaccines will be distributed to and maintained by the pharmacies in Burkina Faso and Mali and 
will include accounts of the amount of product shipped, documentation of adequate and safe handling 
and use, temperature logs for vaccine product and plans for returning or destroying unused product 
with the exception of any tetanus toxoid which, with permission of the sponsor, may be donated to the 
respective national EPI programmes.  
 
7. STUDY INTERVENTIONS 
7.1 Insecticide-treated bednets (LLIN) 
At the start of extension study, all child in the trial who can be identified will be issued with a new LLIN 
regardless  of whether or not their family consents for them to remain in the extension study. 
7.2 The RTS,S/AS01E vaccine 
As described previously, the RTS,S/AS01E vaccine is a virus like particle (VLP) composed of the CSP 
protein of P. falciparum and HBsAg which is given with the adjuvant, AS01E. The vaccine may cause local 
side effects such as pain and redness at the site of vaccination in approximately 20% of recipients and 
minor systemic effects such as drowsiness and irritability are sometimes observed following vaccination. 
Fever post vaccination occurs in about 10% of children and febrile convulsions occurred in about 1% of 

27 
 
5-17 month old recipients enrolled in the phase 3 trial [3] but no persistent neurological effects were 
recorded. The main safety issues related to administration of RTS,S/AS01E during the phase 3 trial were 
a statistically significant increase in meningitis  in children given the vaccine at the age of 5-17 months 
and an apparent increase in mortality in female vaccine recipients. However, during the first three years 
of the current trial, only five cases of febrile convulsion have been reported among the approximately 
4,000 children who have received three priming and two booster doses of RTS,AS01 or control vaccine 
and no proven case of meningitis has been detected.  Following a fifth dose of RTS,S/AS01E given in 
2019, no increase in reported local or systemic side effects compared to previous vaccine doses was 
noted. To ensure that minor local and systemic adverse events, such as a reaction at the vaccine site or 
fever, are not enhanced following repeated doses of RTS,S, adverse events during the first seven days 
after vaccination will be recorded in a sub-set of children. SAEs will be recorded  in all children 
throughout the period of the trial, as described below in Section 8 and Appendix 2.    
7.3 Comparator vaccines 
A single dose of tetanus or tetanus/diphtheria toxoid will be used as the comparator vaccine for children 
in SMC alone group in the first and second years of the extension study. A booster dose of tetanus or 
tetanus/diphtheria/ pertussis  vaccine at about the age of 18 months is recommended by WHO but is 
rarely given so that a majority of the children in the control group will receive some benefit that they 
would not have obtained had they not been enrolled in the trial. Tetanus or tetanus/diphtheria toxoid 
vaccine may produce some minor local and systemic effects but is very safe.  
7.4 Vaccine administration 
Vaccination will be undertaken during a mass campaign lasting one to two weeks during June 2021 and 
June 2022.  Children with a minor illness, such as mild diarrhea or a mild upper respiratory infection 
without fever, at the time they present for vaccination will be vaccinated. Those with recorded fever 
and/or other features of a more serious acute illness will not be vaccinated but will be treated for their 
illness and vaccinated when this has resolved. Children who miss the mass vaccination programme but 
who attend any of the subsequent rounds of SMC administration will be offered vaccination at that 
time. 
7.5 Seasonal Malaria Chemoprevention (SMC) 
Four or five courses of SMC with SP+AQ will be given at monthly intervals during the malaria 
transmission season to children in the SMC alone and SMC +RTS,S/AS01E groups.  A course of SMC for 
children aged over the age of one year comprises a single treatment of SP (500mg/25 mg) and AQ 
150mg on day 1 and AQ 150mg on days 2 and 3. SP and AQ and matching placebo will be obtained from 
Guilin Pharamceuticals, Shanghai, Co, a GMP-certified supplier. All treatments will be given under 
observation. SP can cause Stevens-Johnson syndrome, but this side effect has been seen very rarely 
when SP has been used for either intermittent preventive treatment of malaria in pregnancy or SMC.  
Amodiaquine is bitter and may cause vomiting but serious side effects, which include liver damage and 
neurological side effects, are very rare.  SMC with SP+AQ has proved to be very safe.  A study conducted 
in Senegal in which the consequence of administration of 800,000 doses of SMC with SP+AQ were 
followed carefully identified only three serious potentially drug related adverse effects (two cases of 
hepatic damage and one extra-pyramidal syndrome) [11]. Enhanced pharmaco- vigilance in 7 countries 
participating in the ACCESS-SMC project funded by UNITAID, through which approximately 15 and 30 

28 
 
million treatment courses were administered in 2015 and 2016, respectively, has confirmed that SMC 
with SP+AQ is very safe.   
 
SMC drugs will pre-packed by a pharmacist, who takes no further part in the trial, in re-sealable 
envelopes bearing the child’s unique number and containing tablets for the four cycles of treatment 
required for one malaria transmission season. Treatment with each dose of SMC is given by trained, paid 
volunteers at a central point in each study community under observation. Study children will be given  
an updated  identity card containing their photo, study identity number and date of birth. At the time of 
vaccination and/or SMC administration, a child’s photo ID card is scanned to ensure that the child is 
given the allocated intervention. Home visits are made to children who miss treatment on the 
designated day and their parents/guardians are asked if they would still like their child to receive SMC. If 
they agree, treatment is given at home.   
 
The interventions that children in each study group will receive during the extension period are 
summarised in Table 1. 
 
Table.1 Summary of the interventions that will be given to study children in each study group during the 
extension study 
 
 
Group1 (SMC) 
Group2 (RTSS) 
Group3 (RTSS+SMC) 
2020 
 
 
 
June 
Tetanus or 
Tetanus/diphtheria 
toxoids x 1 
RTSS/AS01 x 1 
RTSS/AS01 x 1 
July -Oct 
SMC x 4 
SMC placebo x 4 
SMC x 4 
 
2021 
 
 
 
June 
Tetanus or 
Tetatnus/diphtheria 
toxoids x 1 
RTSS/AS01 x 1 
RTSS/AS01 x 1 
July -Nov  
SMC x 4 (Mali) 
SMC x 5 (Burkina Faso) 
SMC placebo x 4 (Mali) 
SMC placebo x 5 (Burkina 
Faso) 
SMC x 4 (Mali) 
SMC x 5 (Burkina Faso) 
 
(SMC or placebo is given at monthly intervals on four or five (Burkina Faso 2021) occasions during the 
malaria transmission season.) 
 
7.6 Follow-up and measurement of endpoints 
The following follow-up procedures required to measure the study outcomes will be undertaken.  
 
7.6.1 Passive surveillance for uncomplicated and severe malaria. 
In each country, project staff are based in the district hospital and in the main dispensaries that serve 
the study communities and, working with health service staff, they will be responsible for identifying 
and documenting all cases of malaria who present to these  facilities. In Mali, cases of uncomplicated 
malaria may  be treated by community health workers who have been taught to diagnose malaria with a 
RDT and to treat RDT positive cases. Cases of suspected malaria (fever, history of fever within 48 hours 

29 
 
or any other symptom/sign suggestive of malaria) will be managed on the basis of their RDT result but 
blood films and filter paper strips will obtained from all these cases for subsequent confirmation of the 
malaria diagnosis.  
7.6.2 Active surveillance for malaria 
Each month throughout the study period, 96 randomly selected children (32 from each arm of the 
study) in each country will be visited at home, their temperature measured and a blood film collected 
for subsequent detection of asymptomatic parasitaemia. Any child who is febrile or who has other 
features suggestive of a diagnosis of malaria at the time of the home visit will have an RDT done and if 
this is positive s/he will be treated with artemether/lumefantrine.  This activity is suspended due to 
COVID-19. 
7.6.3 Prevalence of malaria parasitaemia and anaemia. 
A survey of all study children will be undertaken approximately one month after the last round of SMC 
administration at the end of each malaria transmission season. Temperature will be measured and any 
child who is febrile or who has other features suggestive of a diagnosis of malaria will have an RDT done; 
those who are positive will be treated with artemether/lumefantine. Finger prick blood samples will be 
collected for preparing blood slides and filter paper blood spots. The prevalence of parasitaemia, 
including the presence of gametocytes, will be detected by microscopy. 
7.6.4 Evaluation of mutations in the P.falciparum csp gene 
Because the efficacy of RTS,S/AS01E has been shown to be influenced by the occurrence of mutations in 
the csp gene [17] this will be measured in samples obtained from children with clinical episodes of 
malaria and in children with parasitaemia at the cross-sectional survey.   
7.6.5 Immune response to the RTS,S/AS01E vaccine 
Blood samples (4ml) will be collected from approximately 160 children in each of the groups (80 per 
country) who received RTS,S/AS01E and from approximately 20 children in the SMC alone group prior to 
and one month after administration of the third and fourth booster doses of RTS,SD/AS01 for 
measurement of anti-CSP antibodies. Children will be selected randomly for each blood draw.  
7.6.6 Malaria endemicity in the study area 
A malaria survey of schoolchildren will be conducted at approximately the same time as the cross-
sectional surveys are undertaken in study children (November 2020 and November 2021) to assess the 
malaria parasite prevalence at the end of the transmission in the study areas and to monitor the trend in 
the distribution of molecular SP resistance markers. Two hundred randomly selected school children per 
country (total 400) aged 6-12 years resident in the study area who are well and have not received SMC 
will be tested for malaria by microscopy to assess the prevalence of malaria parasitaemia at the end of 
each malaria transmissions season and to determine the overall level of malaria transmission and to 
assess the distribution of molecular markers of resistance to SP in the study area during the final period 
of the trial.   
7.6.7 Drug resistance 
Dried blood spots from children who have malaria parasitaemia detected by microscopy at the final 
cross-sectional survey in November 2021 will be used for analysis of molecular markers of resistance to 
SP and AQ at MRTC, Bamako.  

30 
 
7.6.8 In vivo measurement of drug sensitivity to SP+AQ   
At the end of the 2021 malaria transmission season, an in vivo study will be done to determine whether 
the local strains of P. falciparum still remain sensitive to SP+AQ.  At the time of the cross-sectional 
survey, a RDT for malaria will be done in addition to collection of blood slides and filter paper samples. 
Blood smears will be examined the same day, or as soon as possible afterwards, for any child who has a 
positive RDT test. Children who have microscopically-confirmed P. falciparum infection, but who are 
otherwise quite well, will receive a full treatment course of SP+AQ over three days, dosed according to 
age. Children will then be followed for 28 days according to a standard WHO treatment efficacy 
protocol, with parasitaemia and symptoms assessed at days 0, 1, 2, 4, 7, 14 and 28 after treatment. 
Children who have fever, a history of fever, or are unwell and who have a positive RDT will be treated 
with artemether-lumefantine. Infections occurring after day 7 (late parasitological failures) will be 
investigated to determine if the recurrent infection was due to a reinfection or a recrudescence using 
molecular markers.  
7.6.9 Investigation of possible rebound malaria 
Approximately 60% of trial children will, in line with national guidelines, not be eligible to receive SMC 
during the 2021 malaria transmission season as they will have reached the age of five years. These 
children may be at increased risk of malaria  due to the fact that they have been partially protected from 
malaria during the first few years of their life.  Employing the surveillance systems described in section 
7.1, these children will be identified if admitted to hospital with severe malaria or when presenting to a 
clinic with proven uncomplicated malaria. Details of how this case control study will be undertaken will 
be provided in another proposal that will be submitted to the ethics committee in due course.  
 
In order to investigate further the potential of four years of administration of SMC, seasonal vaccination 
with RTS,S/AS01E or both,  antibodies against a range of malaria antigens will be measured in a finger-
prick blood sample obtained from all study children at the start of the 2021 malaria transmission season 
and the patterns of response observed related to the  risk of malaria in the subsequent malaria 
transmission season. At the same time a questionnaire will be administered which asks about risk 
factors for malaria that might confound analysis of the association between antibody pattern and risk. A 
second blood spot for antibody determination will be obtained at the time of the cross-sectional survey 
described in section 7.6.3 above.   
 
Among children no longer eligible for SMC, the incidence rates of uncomplicated malaria and 
hospitalisation for malaria will be compared according to age, sex and prior adherence to study 
interventions (SMC history and vaccination history), to determine if children who have been well-
protected during the study period have been placed at increased risk during the period after SMC 
and/or vaccination is stopped, a possibility that has been little investigated.  To explore this issue 
further, we will develop case-control studies that will obtain information on additional relevant risk 
factors and potential confounders that are not routinely collected from all study children (e.g. measures 
of socio-economic status, housing construction, knowledge of malaria, use of LLIN, etc.) from cases of 
either uncomplicated or severe malaria and from controls sampled at random from the study cohort.     
 
8. ASSESSMENT OF SAFETY 
8.1  Safety assessments 

31 
 
Because there is no previous experience of the administration of sixth and seventh doses of 
RTS,S/AS01E, and to ensure there are no adverse outcomes following administration of a fifth dose of 
tetanus toxoid approximately 150  children in each country (50 in each intervention group) will be 
visited daily for seven days after their third and fourth booster doses and local and systemic side effects 
recorded. Additionally, the DSMB will undertake a safety review at the end of the 2020 malaria 
transmission season before progressing to the final year of the study. 
 
Surveillance will be maintained throughout the study period for any SAEs. Any SAEs that are (a) 
considered by the investigators to likely to be linked to the administration of a study vaccine or study 
drug or (b) are suspected cases of meningitis or cerebral malaria or (c) are fatal or life threatening will be 
thoroughly investigated and reported to GSK and to the DSMB within 72 hours of their detection. All 
SAEs, whether considered related to the study interventions or not will be tabulated in a blinded fashion 
and provided to the DSMB and to GSK at the times requested by the DSMB. SAEs will reported to the 
LSHTM ethics committee annually in a tabulated format. SUSARs will be reported to the sponsor and to 
LSHTM ethics committee within 72 hours of notification in a format similar to that outlined in LSHTM 
SOP 008. SAE’s will be reported to the ethics committees in Burkina Faso and in Mali in line with their 
instructions following review of the protocol. Details of definitions of adverse events and SAEs and of 
reporting procedures are described in Appendix 2.  
 
Experienced project staff based at the district hospitals are responsible for the identification of any child 
in the trial admitted to hospital and for ensuring their referral to a study physician. Hospital staff have 
been provided with additional training on the recognition of cases of meningitis, cerebral malaria or 
immune deficiency diseases and standard operating procedures have been developed for management 
of children suspected of having one of these conditions.  The aetiology of cases of meningitis is 
determined by microscopical examination of cerebrospinal fluid samples for bacteria and white blood 
cells and by subsequent PCR testing at a reference laboratory. The death of any study child is 
investigated and, if this occurred in the community, a verbal autopsy will be done.  
 
9. LABORATORY PROCEDURES 
9.1 Detection of malaria.  
A histidine rich protein (HRP2) based Rapid Diagnostic Test (RDT) will be used for the initial diagnosis of 
malaria and to guide treatment. All blood films will be read twice by two separate readers following the 
guidelines developed for the phase 3 RTS.S/AS01 trial [18].  Slides which are judged to be discordant for 
either positivity or parasite density will be read by a third reader.  For slides with high or medium 
density parasitaemia (> 400 parasites/μl) readings are considered discordant if the higher count divided 
by the lower count is > 2. In the case of slides with low density parasitaemia (< 400/μL), readings are 
considered discordant if the highest reading density is more than one log10 higher than the lowest 
reading. In cases when one reader gives a count > 400/μL and the other < 400/μL, the second criterion 
will apply. For cases of discrepancy in definition of positivity/negativity, the majority decision is adopted. 
If the majority decision is positive, the final result is the geometrical mean of the two positive readings. 
In the case of discrepancies in parasite density, the final result is the geometric mean of the two 
geometrically closest readings. 

32 
 
9.2 Detection of markers of resistance to SP and AQ.   
Parasite DNA will be extracted from dried blood spots and nested PCR reactions will used to detect the 
presence of mutations in the dhfr and dhps genes associated with resistance to pyrimethamine and 
sulphadoxine respectively, and the pfcrt and pfmdr mutations associated with resistance to 
amodiaquine [19-21]. PCR-RFLP will be used to detect the N511, C59R, S108N and I164L mutations in 
the dhfr gene, the A437G and K540E mutations in the dhps gene, the N86Y mutation in the pfmdr1 gene 
and the K76T mutation in the pfcrt gene.  
9.3 Differentiation of malaria re-infections and recrudescences  
DNA from dried blood spots will be used for molecular characterisation of the parasite using 
polymorphisms in the msp1, msp2 and CA1 polymerase genes [22]   
9.4 Detection of mutations in the csp gene of P. falciparum 
DNA will be extracted from selected blood spots collected from children with malaria and analysed for 
mutations at the Th2 or Th3 locus of the csp gene of P. falciparum associated with evidence of reduced 
efficacy following RTS,S/AS01E vaccination [17] These assays will be conducted at the Broad Institute, 
Boston.  
9.5 Measurement of haemoglobin concentration 
Haemoglobin concentration will measured colorimetrically using a Hemocue colorimeter (Hemocue AB, 
Angelholm, Sweden). 
9.6 Measurement of anti-CSP concentration 
Antibodies to CSP will be measured by a standardised ELISA used in many previous trials of RTS,S/AS01E 
at the University of Ghent [23] 
 
9.7   Measurement of a broad range of malaria antibodies.  
 
Antibodies to a broad range of malaria antigens will be measured using the Luminex assay at LSHTM. 
 
9.8  Measurement of malaria parasitaemia using  a novel  18S rRNA/rDNA biomarker 
 
Testing  for malaria parasite DNA will be undertaken in a sub-set of samples by  Prof Murphy at the 
University of Washington, Seattle, USA.  
 
10. STATISTICAL CONSIDERATIONS  
10.1 Statistical analysis plan 
All data will be collected and verified prior to analysis. Detailed statistical procedures, listings, table 
shells, and figures will be provided in a statistical analysis plan (SAP) prior to analysis. The SAP will be 
finalized before study close-out and database lock and will be submitted to the DSMB before the study 
code is broken.  
The following key statistical components and a detailed description will be documented in the SAP: 

33 
 
• 
Primary and secondary endpoints and how they will be measured. 
• 
Statistical methods and tests that will be used to analyze the endpoints. 
• 
Indication of whether the comparisons will be using one-tailed or two-tailed tests (with justification 
of the choice) and the level of significance to be used.  
• 
Identification of whether any adjustments to the significance level or the overall p value will be 
made to account for any planned or unplanned subgroup analyses or multiple testing  
• 
Specification of potential adjusted analyses and a statement with which covariates or factors will be 
included. 
• 
Planned exploratory analyses and justification of their importance. 
The number of subjects enrolled, vaccinated, completed, or withdrawn will be summarized. Reasons for 
withdrawal, when known, will be provided. Demographic data will be summarized by descriptive 
statistics and will include total number of observations (n), mean, standard deviation (SD) and range for 
continuous variables, and number and percentages for dichotomous variables.  
All the investigators involved in the collection of clinical or laboratory data will be blind as to which 
group the individual study children have been allocated. However, based on the likely final results from 
the  first phase of the trial the investigators are likely to know that none of the study groups was 
significantly inferior to another and it could be argued that this could influence how data are collected 
and analysed (anti-conservative bias). However, the investigators consider that this risk is small.  The 
trial statistician undertaking the analysis of the initial phase of the study will need to be able to link 
study findings to an individual child but measures will be undertaken to enable this to be done without 
the statistician being unblended for the period of the extension. This will be done by developing a 
second set of IDs for the trial participants with only the independent trial statistician knowing how the 
numbers link to the child’s initial number and study group. In this way, during the course of the 
extension study, the trial statistician dealing with data related to an individual child will not know the 
identity of the trial group to which that child belongs.  
10.2 Sample size and analysis of the primary efficacy endpoint  
At a survey conducted in April 2019, 5363 of the 5887 (91%) children enrolled in the first phase of the 
trial were contactable, an annual loss of about 5%. Assuming that this rate of attrition continues for a 
further year and that a similar proportion of families do not consent for their child to join the extension 
study, it is likely that approximately 4,800 children will enter the extension study. 
 
The primary analyses will cover the entire study period, ie. the initial three years of the study and the 
two year extension period. The entire study period is defined as the time from enrolment in June 2017 
until the end of the study year in which the child reaches five years of age and is no longer eligible for 
SMC, i.e. between 5 and 20 months of age (depending upon the exact age at first vaccination) and 60-71 
months of age (depending upon the timing of the child’s fifth birthday in relation to the SMC schedule). 
On the basis of date of enrolment, the exit dates above, and losses to follow-up as described above, we 
estimate that there will be approximately 20,625 person-years of follow-up during the entire study 
period: 6875 person-years per study group (i.e. an average of 3.5 person-years per child originally 
recruited). 
 

34 
 
The primary end-point for the trial is the incidence of clinical episodes of malaria. Based on data from 
the first two years of the ongoing study, we assume that the incidence of malaria will be 200 cases per 
1000 children per year over the entire study period. Based on this assumption, the study will have 90% 
power to detect a 12.0% or greater relative difference (absolute difference: 24 cases per 1000 child-
years) between the RTS,S/AS01E + SMC and the RTS,S/AS01E alone or SMC alone groups, through a 
standard superiority comparison. 
 
The study will also have 90% power using a two sided 95% confidence interval to exclude a 17.5%  
relative difference (absolute difference: 35 cases per 1000 child-years) in incidence of clinical malaria 
between children in the RTS,S/AS01E alone or SMC alone group, using a non-inferiority design, over the 
entire study period. To determine the level of confidence with which this margin can be excluded, we 
will also calculate two sided 90% and 99% confidence intervals.   
 
There will be approximately 4,100 child-years of observation in the year following the third booster; 
1367 child-years in each study group.   Assuming the incidence of clinical episodes of malaria remains at 
about 200 episodes per 1,000 children per year during this period, the study will have 90% power, 
through a standard superiority comparison, to detect a 25.9% or greater relative difference (absolute 
difference: 52 cases per 1000 child-years) between the RTS,S/AS01E + SMC and the RTS,S/AS01E alone or 
SMC alone group.  In the same period, the study will have 90% power, using a two sided 95% confidence 
interval, to exclude a 39.2% relative difference (absolute difference: 78 cases per 1000 child-years) in 
the incidence of clinical malaria between children in the RTS,S/AS01E alone or SMC alone group, using a 
non-inferiority design.   
 
A similar analysis will be undertaken for the final year of the study following administration of a fourth 
booster dose of the RTS,S/AS01E or comparator vaccine but study power will reduced because slightly 
more than half of the children enrolled (3860/5920) will not be eligible for SMC in 2021-22, and thus will 
not contribute further events nor person-time at risk to the study period.   
 
Both intention-to-treat and per-protocol analyses will be undertaken. Children who received any dose of 
SMC or vaccine will be included in the intention to treat analysis. Children who received all scheduled 
doses of SMC, or who were treatment for a clinical episode of malaria at a time when SMC would have 
been given, or all scheduled doses of vaccine will be included in the per protocol analysis for each year 
of the study. Results obtained in Burkina Faso and Mali will be analysed separately. Additional sub-
analyses will include analysis by age, gender, and bed net use during the transmission season, as 
determined by the history obtained at the cross-sectional surveys. 
10.3 Sample size and analysis of  secondary endpoint(s) 
10.3.1 Sample size for measurement of prevalence of parasitaemia in school children.  
Approximately 200 randomly selected children will be included in each school-survey. This sample size is 
based on the following assumptions: (1) the prevalence of malaria parasitaemia will be 50% and intra-
cluster correlation coefficient (ICC) will be 0.1 (this is based on the observations from the surveys 
undertaken previously in the same study areas;  (2) the design effect will be 1.9 if the cluster size is 10 
children; (3) refusal to take part in the survey will be <5%; (4) the acceptable 95% precision of the 
prevalence will be +/-10%.  

35 
 
10.3.2 Sample size for the serological assays 
For the analysis of the serological response to RTS,S/AS01E, comparisons will be made between mean 
anti-CSP antibody titres pre and post the sixth and seventh RTS,S/AS01E doses. Based on the standard 
deviations in antibody titres observed in children enrolled in the RTS,S/AS01E phase 2 and phase 3 trials, 
inclusion of around 160 individuals in each group (pre and post vaccination at each of the two time 
points) will give a study with approximately 80% power to detect a difference of 25% - 30% in mean titre 
between groups. 
10.3.3 Sample size for the in vivo study of anti-malarial drug resistance 
Assuming a 90% adequate clinical and parasitological response (ACPR) in asymptomatic children, for the 
study to be able to estimate this proportion with a precision of 5% at the 95% confidence level, 139 
children will need to be enrolled.  Anticipating up to 10% loss to follow-up, the number of children to be 
enrolled will be increased to a target of 155.  It should be possible to reach this target in Burkina Faso 
but this may not be possible in Mali where the prevalence of infection is now lower than in Burkina 
Faso. 
10.3.4 Sample size for the rebound study 
An important secondary analysis will be for potential ‘rebound’ malaria when children become too old 
to receive SMC and/or RTS,S/AS01E.  As slightly more than half of the children in the trial will no longer 
be eligible to receive SMC or RTS,S/AS01E in the last year of the trial,  the study provides an excellent 
opportunity to assess of the risk of ‘rebound’ malaria in children who have received the combination of 
RTS,S/AS01E and SMC during the previous four years with that of children who received just SMC 
(standard of care).  Here, the concern is that incidence will be higher among children who have received 
both SMC and RTS,S than among children who have received the single interventions. Consequently, the 
design will be a non-inferiority comparison (to rule out excess incidence in the RTS,S/AS01E + SMC 
group).  In our previous study in this area, incidence among children in the year they became too old to 
receive SMC was three-fold higher than in the last year they received SMC (2068 versus 677 cases per 
1000 person-years at risk).  Assuming that incidence among children who are no longer protected by 
SMC increases from 200 to 600 cases per 1000 person-years, and that there are 800 child-years at risk in 
each group during the rebound period, the study will have 90% power to exclude a 29.6% relative 
increase (absolute increase, 178 cases per 1000 child-years) in the incidence of clinical attacks of malaria 
among children who received RTS,S + SMC, compared with children who received SMC alone.  With the 
same assumptions, there will be 80% power to exclude a 25.6% relative increase (absolute increase, 153 
cases per 1000 child-years).  
 
11. SOCIO-ECONOMIC STUDIES  
The costs, both direct and indirect, of adding RTS,S/AS01E to SMC or substituting RTS,S for SMC will be 
undertaken and these studies will be described in a separate protocol.  
 
12. ASSESSMENT OF THE IMPACT OF THE COVID-19 PANDEMIC ON CONSULTATIONS AND 
HOSPITALISATIONS IN THE STUDY SITES 
The COVID-19 pandemic reached Mali and Burkina Faso in late March 2020 and, as in many countries, 
has impacted peoples’ lives and the use of health services. As of 17 December 2021, 19,062 cases 

36 
 
including 637 deaths were reported in Mali. The corresponding figures in Burkina Faso were 16,672 and 
296.  Although, based on published statistics, African countries including Mali and Burkina Faso are 
among the least affected by the virus the impact of the pandemic on the health services may still be 
large and vary from one area to another. In a study carried out by the Global Fund, a 23% drop in 
consultations was observed in services for children under 5 years of age in African countries [24]. The 
survey also found a 17% drop in malaria diagnoses in 2020, compared to 2019, in the 24 African 
countries surveyed.  To better describe the context of this trial we will examine the impact of the COVID-
19 pandemic on trends in health care seeking including pediatric consultations and admissions to 
hospitals in the areas where the RTS,S + SMC trial is being conducted. To achieve this, we will extract 
and examine all the data from heath registers for outpatients and hospital admission in the paediatric 
services of the district hospitals in the study areas over the period from January 2018 to December 
2021. Information to be collected will include the date of consultation or hospital admission, age, 
gender, place of residence (village/quartier), reason for consultation or admission, the diagnosis, the 
treatment, and outcomes of the hospitalisation. No personal identifiers will be collected. A descriptive 
analysis will be performed using the total number of consultations and hospitalisations and the number 
of consultations and admissions due to malaria over time by age categories before and during the 
COVID-19 pandemic, considering the malaria transmission season and various periods of the pandemic. 
The latter will be characterised as the beginning of the pandemic, the period of lifting of the restrictions 
by the government, the waves of the pandemic and the period between the waves as well as the 
introduction of the COVID-19 vaccine. Parametric and nonparametric tests will be used to compare the 
average number of consultations and hospitalisations. The average monthly changes in the number of 
consultations and hospitalisations including for malaria will be described graphically over time according 
to the characteristics of the patients (age, residence) and comparison between the trends before COVID-
19 and during various time periods during the pandemic as described above will be made. Comparisons 
will be made between the calendar months of 2018, 2019, 2020 and 2021 to explore variation in the 
seasonal pattern of care-seeking over different years. These data will be used to describe the context of 
the trial and facilitate interpretation of the trial results.   
 
13. DATA MANAGEMENT AND CLINICAL MONITORING 
Data collection and management will be undertaken using the Open Data Kit (ODK) 
(https://opendatakit.org) which was set up for the ongoing RTSS plus SMC trial and which is working 
well. This data collection and management system uses electronic case record forms (eCRF) loaded in 
tablet PCs. Project staff are well trained and competent in using ODK based eCRF and regularly upload 
data into a secured, central server.  Data collected using ODK will be uploaded to ODK Aggregate Server 
based at LSHTM. This is supported by the LSHTM IT department, with routine daily backups created and 
stored on separate servers. Data will be migrated from the ODK Aggregate server and loaded into MS 
Access databases in both Burkina Faso and Mali for generating queries and cleaning the databases. On a 
fortnightly basis, these separate country-based files will be merged into single tables (combining data 
from each country) for each of the eCRFs used in the study. These merged files will be exported from MS 
Access to CSV format and placed on a secure server at LSHTM where they will be accessible only to the 
trial statistician.  
 
An experienced independent GCP monitor (Raouf Osseni or one of his delegates) will continue to 
monitor the study to ensure the quality of the data collected and that GCP standards are met. The 

37 
 
monitor will conduct  two visit each year together including  a final close out visit. The monitor ensures 
that the trial is conducted according to the study protocol, that appropriate ethical procedures are in 
place and examines a random selection of clinical and laboratory records during each visit to confirm 
their validity. 
14. ETHICS/PROTECTION OF HUMAN SUBJECTS  
Inclusion in the trial of an RTS,S/AS01E alone group even though SMC is recommended policy is justified 
on the grounds that RTS,S/AS01E should provide some added protection against the malaria episodes 
that occur outside the main transmission season when SMC is given and that it may be easier to 
administer than SMC, thus creating a situation of equipoise. 
 
Individual, written, informed consent will be obtained from the family or legally recognised guardian of 
each child entered into the extensions study.  Ethical approval for the extension study will be obtained 
from the Ethics Committee of LSHTM, the Health Research Ethics Committee of Burkina Faso, the 
Institutional Ethics Committee of IRSS in Burkina Faso and the Ethics Committee of the Faculty of 
Medicine, Pharmacy and Dentistry, University of Bamako. Approval for the extension study will also be 
sought from the regulatory authorities in Burkina Faso and Mali.  Conduct of the trial will not impose any 
additional costs on the local health services. The project will contribute to the costs of routine clinical 
care of study subjects during the trial and to strengthening of  the district hospitals in the study areas. 
14.1 Future Use of Stored Specimens 
After the study is complete, sera, and dried blood spots will be stored at the MRTC in Mali, the IRSS in 
Burkina Faso or at the London School of Hygiene & Tropical Medicine in a biorepository. The samples 
will be kept for a maximum of 10 years. 
 
14.2 Compensation 
Participants will be insured against injury caused by the study according to legal requirements and 
compensation for research related injury (to include costs of long term and future medical care needs) is 
available, should it occur.   Appropriate treatment during the trial will be provided by the site personnel 
and paid for by the project.  
The participants will receive compensation for the time and travel of the protocol specified visits 
including the inclusion or vaccination visits, the end of season visits, the visits for serology and for the in 
vivo study. The compensation for the time and travel to the health center has been estimated to 1000 
FCFA (~ US $ 2) per visit. 
15. TRIAL MANAGEMENT AND ROLES OF THE INVESTIGATORS AND COLLABORATORS  
The London School of Hygiene & Tropical Medicine will be the main sponsor for the trial. Delegated 
responsibilities will be assigned locally.  The London School of Hygiene & Tropical Medicine holds Public 
Liability (‘’negligent harm’’) and Clinical Trial (non-negligent harm’’) insurance policies which apply to 
this trial.  The study may be subject to audit by the London School of Hygiene & Tropical Medicine under 
their remit as the sponsor, the Study Coordination Centre and other regulatory bodies to ensure 
adherence to GCP. 

38 
 
 
An independent trial steering committee, established at the start of the first phase on the trial provides 
scientific oversight and holds regular meetings either through teleconferencing or face-face meetings 
and will continue to do so during the extension study. Members of the steering committee are listed in 
appendix 3.  
 
A DSMB, established prior to the initiation of the first phase of the trial, oversees the safety of subjects 
in the trial and will continue to do so during the extension period.    Membership of the DSMB is shown 
in appendix 3. 
 
The trial adheres to the principles outlined in the International Conference on Harmonisation Good 
Clinical Practice (ICH GCP) guidelines, protocol and all applicable local regulations.  The extension study 
will be registered as a new trial on clinicaltrials.gov.  
 
The team from the IRSS, Burkina Faso, which includes Jean Bosco Ouédraogo, Halidou Tinto and Issaka 
Zongo, is responsible for conducting the part of the trial undertaken in Burkina Faso and they will 
participate in the analysis of the trial results.  The team from MRTC, which includes Alassane Dicko, and 
Issaka Sagara is responsible for conducting the part of the trial undertaken in Mali and will participate in 
the analysis of the trial results. The LSHTM team (Brian Greenwood, Daniel Chandramohan, Matthew 
Cairns, Paul Milligan, Jayne Webster and Karen Slater) provides epidemiological, statistical, 
administrative and financial management support.  
 
16. FUNDING  
Funding for the first phase of the trial has been provided by grants from the UK MRC/DFID/NIHR/WT 
Joint Global Health Trials scheme and PATH. Funding from the same donors is being sought to support 
the extension study. 
 
17. DISSEMINATION PLANS 
Results from the trial will be presented at national and international conferences, in peer reviewed 
journals, to the ethics committees and will be discussed with the study communities at the end of the 
study. Trial results will be shared with the WHO’s technical expert groups and Malaria Policy Advisory 
Group (MPAC). 
 
Strong links have been established already with the Ministries of Health, NMCPs and EPI programmes in 
Burkina Faso and Mali and these will facilitate the incorporation of RTS,S/AS01E vaccine into SMC/EPI 
programme  if this is found to be a useful intervention. The study team has also established good links 
with many other organisations involved in the delivery of SMC trials, including the ACCESS-SMC 
programme coordinated by the Malaria Consortium and with the WHO staff responsible for conducting 
the RTS,S/AS01E implementation studies Thus, if it is found that RTS,S/AS01E vaccine is a useful 
replacement for or addition to SMC regimens, routes have already been established through which this 
knowledge could be disseminated rapidly.  
 

39 
 
18.  REFERENCES  
[1] Cohen J, Nussenzweig V, Nussenzweig R et al.  From the circumsporozoite protein to the RTS,S/AS 
candidate vaccine. Human Vaccin 2010; 6: 90-96.  
[2] White MT, Verity R, Griffin JT et al. Immunogenicity of the RTS,S/AS01E malaria vaccine over time 
and implications for duration of vaccine efficacy: analysis of data from a phase 3 randomised 
controlled trial. Lancet Infect Dis 2015; 15: 1450-1458. 
[3] RTS,S Clinical Trials Partnership. Efficacy and safety of the RTS,S/AS01E 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: 31-45.  
[4] Tinto H,Otieno W, Gesase S et al. Long-term incidence of severe malaria following RTS,S/AS01E  
vaccination in children and infants in Africa: an open-label 3-year extension study of a phase 3  
randomised controlled trial. Lancet Infect Dis 2019; 19: 821-32. 
[5] Bojang K, Milligan PJ, Pinder M et al. Efficacy of RTS,S/AS02 malaria vaccine against Plasmodium 
falciparum infection in semi-immune adult men in The Gambia: a randomised trial. Lancet. 2001; 
358: 1927-1934.  
[6] http:www.who. int/mediacentre/news/release/2015/sage/en  
[7] https://www.afro.who.int/news/malaria-vaccine-launched-kenya-kenya-joins-ghana-and-malawi-
roll-out-landmark-vaccine-pilots. 
[8] Wilson AL. A systematic review and meta-analysis of the efficacy and safety of intermittent 
preventive treatment in children (IPTc). Plos One 2011; 6: e16976.  
[9] Dicko A, Diallo AI, Tembine I et al. Intermittent preventive treatment of malaria provides  
substantial protection against malaria in children already protected by an insecticide-treated 
bednet in Mali: a randomised, double-blind, placebo-controlled trial. PLoS Med 2011; 8: 1000407.  
[10] Konate AT, Yaro JB, Ouedraogo AZ et al. Intermittent preventive treatment of malaria provides 
substantial protection against malaria in children already protected by an insecticide-treated 
bednet in Burkina Faso : a randomised, double-blind, placebo-controlled trial. PLoS Med 2011; 8: 
e1000408. 
[11] Ndiaye JL, Cissé B, Ba EH et al. Safety of seasonal intermittent preventive treatment against malaria 
with sulfadoxine pyrimethamine + amodiaquine when delivered to children under 10 years of age 
by district health staff in Senegal. PLoS One 2016; 11: e1068421.   
[12] Cairns M, Roca-Feltrer A, Garske T et al. Estimating the public health impact of seasonal  malaria 
chemoprevention in African children. Nat Commun. 2012; 3: 881.  
[13] http://www.who.int/malaria/publications/atoz/who_smc_policy_recommendation/en/ index.html 
[14] Greenwood B, Dicko A, Sagara I, et al  Seasonal vaccination against malaria: a potential use for an 
imperfect malaria vaccine. Malar J. 2017; 16: 182. 
[15] Chandramohan D, Dicko A, Zongo I et al. Seasonal malaria vaccination: protocol of a Phase III trial of 
seasonal vaccination with the RTS,S/AS01E vaccine, seasonal malaria chemoprevention and the 
combination of vaccination and chemoprevention. BMJ Open, submitted. 
[16] Chandramohan, D  Dicko A, Zongo I et al. Effect of adding azithromycin to seasonal malaria 
chemoprevention. N Engl J Med 2019; 380: 2197-2206 
[17] Neafsey DE, Juraska M, Bedford T, et al. Genetic diversity and protective efficacy of RTS,S/AS01E 
malaria vaccine. N Engl J Med 2015; 373:2025-37. 

40 
 
[18] Swysen C,  Vekemans J, Bruls M et al. Development of standardized laboratory methods and quality 
processes for a phase III study of the RTS,S/AS01E candidate malaria vaccine. Mal J 2011; 10: e223. 
[19] Plowe CV, Djimde A, Bouare M et al. Pyrimethamine and proguanil resistance-conferring mutations 
in Plasmodium falciparum dihydrofolate reductase: polymerase chain reaction methods for 
surveillance in Africa. Am J Trop Med Hyg 1995; 52: 565-568.   
[20] Djimde A, Doumbo OK, Cortese JF et al. A molecular marker for chloroquine-resistant falciparum 
malaria. N Engl J Med 2001; 344: 257-63. 
[21] Djimde AA, Fofana B, Sagara I et al. Efficacy, safety and selection of molecular markers of drug 
resistance by two ACTs in Mali. Am J Trop Med Hyg 2008; 78: 455-461.   
[22] Felger I, Snounou G, Hastings I, Moehrle JJ, Beck HP.  PCR correction strategies for malaria drug 
trials: updates and clarifications. Lancet Infect Dis 2019 Sept 17th . pii: S1473-3099 (19) 30426-8. 
[23] Clement F, Dewar V, Van Braeckel E, et al. Validation of an enzyme-linked immunosorbent assay for 
the quantification of human IgG directed against the repeat region of the circumsporozoite protein 
of the parasite Plasmodium falciparum. Malar J. 2012 ;11:384. 
[24] The Global Fund. The impact of covid-19 on HIV, TB and malaria services and systems for health: a  
snapshot from 502 health facilities across Africa and Asia. April 2021. 
(https://www.theglobalfund.org/media/10776/covid-19_2020-disruption-impact_report_en.pdf ) 
Accessed December 18, 2021.      
 
 
 

41 
 
 
19. TIME AND EVENT TABLE 
 
 
Activities 
 
2020 
2021 
2022 
J F M A M J J A S O N D J F M A M J J A S O N D J F M A M J J A S O N D 
Protocol submission for ethics 
approval 
x  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Ethics and regulatory approval 
 
x  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Amend trial registration 
 
x  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Order study drugs 
x  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Vaccine shipment 
 
 
 
x  
 
 
 
 
 
 
 
 
 
 
x  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Steering committee meeting 
 
x  
 
 
 
 
 
x  
 
 
 
 
 
 
 
 
 
 
x  
 
 
 
 
 
 
 
 
 
 
x  
 
 
DSMB meeting 
x  
 
 
 
 
x  
 
 
 
 
 
 
 
 
 
 
X  
 
 
 
 
 
 
 
 
 
 
x  
 
 
 
 
Census of study children 
 
 
 
x  
 
 
 
 
 
 
 
 
 
 
x  
 
 
 
 
 
 
 
 
 
 
 
 
x  
 
 
 
 
 
Re-consenting children 
 
 
 
x  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Printing ID cards & labels  
 
 
 
 
x  
 
 
 
 
 
 
 
 
 
 
x  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Vaccination 
 
 
 
 
 
x  
 
 
 
 
 
 
 
 
 
 
x  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Administration of SMC 
 
 
 
 
 
 
 
x x x x 
 
 
 
 
 
 
 
 
X x x x 
 
 
 
 
 
 
 
 
 
 
 
 
 
Health facility based morbidity 
and mortality surveillance  
x x x x x x x x x x x 
 
x x x x x x X X x x x 
x x x 
x x x x  
 
 
 
 
 
Active surveillance of malaria 
infection 
x x x x x x x x x x x 
 
x X x x x x X X x x x 
x x x 
x x x x  
 
 
 
 
 
End of transmission surveys 
 
 
 
 
 
 
 
 
 
 
 
x  
 
 
 
 
 
 
 
 
 
 
x  
 
 
 
 
 
 
 
 
 
 
 
   Blood slide reading 
x x x x x x x x x x x 
x x x x x x x X X x x x 
x x x 
x x x x  
 
 
 
 
 
PCR assays 
 
 
 
 
 
 
 
 
 
 
 
 
x  
 
 
 
 
 
 
 
 
 
 
x  
 
 
 
 
 
 
 
 
 
 
Serology 
 
 
 
 
 
 
 
x  
 
 
 
 
 
 
 
 
 
 
X  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
GCP monitoring 
 
 
 
 
 
x  
 
 
 
 
 
 
 
 
 
 
 
X  
 
 
 
 
 
 
 
 
 
x  
 
 
 
 
 
Data management 
x x x x x x x x x x x 
 
x X x x x x X X x x x 
x x x 
x x x x x x x  
 
 
Blinded analysis & report for 
committees 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
x  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Locking of data 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
x  
 
 
Data analysis 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
x x  
 
Dissemination  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
x x 

Statistical Analysis Plan 
 
RTS,S-SMC Extension Study 
42 
 
20. APPENDICES 
Appendix 1 
 
A decision tree developed to aid in making the decision as to whether the trial should be extended.   
 
INTRODUCTION 
It is proposed that preliminary analysis should be conducted on data obtained up to the end of 2019, a 
few months before data collection has finished (March 2020), by an independent investigator in 
January or February 2020. This preliminary analysis will be undertaken to allow a decision to be taken 
early enough in 2020 for preparations to be made for continuation of the trial beyond June 2020 
without a break in continuity of the administration of the trial interventions or of surveillance. 
 
DATA AVAILABLE FOR THE PRELIMINARY ANALYSIS 
The data available for the preliminary analysis will include – 
a. Deaths.  
b. Deaths attributed to malaria. 
c. Hospital admissions. 
d. Hospital admissions due to malaria 
e. The incidence of clinical episodes of malaria (the primary trial end-point). 
f. The prevalence of malaria at the end of each transmission season.  
g. The presence of anaemia at the end of each transmission season.  
 
ANALYSES TO BE UNDERTAKEN FOR THE PRELIMINARY REVIEW 
Analyses of efficacy against clinical episodes of malaria, the primary trial end-point, will be undertaken 
for the overall period from the start of the trial in June 2017 until December 2019 and separately for 
the periods June 2017 to May 2018, June 208 to May 2019 and June 2019 to November 2019. Efficacy 
against severe malaria will be measured as a secondary analysis but the study is only powered to detect 
major differences between groups for this end-point. Safety evaluations will be based on the number 
and characteristics of serious adverse events recorded throughout the period of the trial.   Antibody 
titres to the CSP antigen measured before and after priming with the RTS.S/AS01 vaccine and before 
and after each of the two booster immunisations will be analysed in the final analysis but these results 
will not be available at the time of the preliminary review.   
The primary analysis will be based on data for both sites combined, with site as a stratification factor, 
but site-specific analyses will also be undertaken. A test of interaction will be used to determine if there 
is any evidence that efficacy of RTSS varies between the two trial sites. If a substantial difference is 
found, further analysis may be required.  
 
 
 

Statistical Analysis Plan 
 
RTS,S-SMC Extension Study 
43 
 
DECISIONS MADE ON THE GROUNDS OF EFFICACY 
Because of the inclusion of three study groups, a number of potential efficacy outcome are possible but 
analyses will focus on answering two main questions. 
a. Is efficacy against clinical episodes of malaria of the combination of RTS,S/AS01E plus SMC superior 
to that of SMC alone (the standard of care)?   
Deciding on the enhanced level of efficacy that would be sufficient to warrant a policy decision to 
implement the combination of RTS,S/AS01E and SMC,  and hence a justification for continuation of 
the trial, is difficult. However, based on the results of the RTS,S/AS01E phase 3 trial and the 
percentage  used frequently in setting a potential target, and hence sample size, for trials of other 
malaria control interventions a figure of 20% or more has been chosen  to define the superiority in 
efficacy against clinical episodes of malaria of the combination of RTS,S/AS01E and SMC over that 
achieved with SMC alone.   
Decision: If the trial data are consistent with an efficacy of the RTS,S +SMC combination over  SMC 
alone of at least 20% (the upper 95% confidence limit on the efficacy  is 20% or higher and the 
lower 95% confidence limit  exceeds zero),  the trial will continue with two or three groups 
depending on the outcome of the analysis described below. In the unlikely event that the 
combination of RTS,S/AS01E and SMC is not superior to either intervention used alone and 
RTS,S/AS01E given alone is not inferior to SMC given alone  consideration could be given to 
continuation of the trial with the RTS,S/AS01E and SMC alone arms. 
b. Is RTS,S/AS01E given alone at least as effective in preventing clinical episodes of malaria as SMC 
given alone (the standard of care)? 
SMC is the standard of care in both Burkina Faso and Mali.  Consequently, if it is  not possible  to 
establish that RTS,S/AS01E given alone is as effective as SMC in preventing clinical episodes of 
malaria,  it would  not be justifiable to continue with the RTS,S/AS01E alone group in the trial.  
The efficacy of SMC (against placebo or no SMC) has been well established in clinical trials and, 
more recently, through case control studies in Mali and Burkina Faso. Efficacy of SMC against no 
SMC was about 50% over the whole year, although higher during the four months of the malaria 
transmission season. The non-inferiority margin is the largest reduction in this efficacy that would 
be considered acceptable if RTSS/AS01 was to replace SMC. A reduction in efficacy from 50% to 
40%, which might be considered acceptable, translates to a 20% greater incidence in the 
RTSS/AS01-alone arm (the comparator) compared to the SMC-alone arm.  This reduction in efficacy 
might be considered acceptable taking into account the potential advantages of RTS,S/AS01E over 
SMC in terms of ease of delivery and hence the likelihood of being able to sustain high levels of 
coverage. We have, therefore, chosen a margin of 20% in clinical incidence between the 
RTS,S/AS01E alone and SMC alone groups as the margin on which a decision will be based (this is 
slightly higher than the 16.1%  margin used in the protocol to define the study’s power to measure 
this end-point) .    
If the incidence rate ratio RTS,S/AS01E alone: SMC alone can be shown to be less than 1.20 then we 
can conclude that RTS,S alone is non-inferior to SMC alone.  Two-sided 99%, 95% and 90% 
confidence intervals for the rate ratio will  indicate the degree of confidence with which the margin 
of 1.20 (relative increase of 20%) can be excluded. Examples of how the confidence intervals might 
be interpreted are shown in Figure 1.     
 
 
 
 

Statistical Analysis Plan 
 
RTS,S-SMC Extension Study 
44 
 
 
 
Figure 1. Interpretation of the possible findings in the comparison between RTS,S/AS01E and SMC.   
 
A separate consideration is the performance of RTS,S/AS01E alone, compared to the combined group.  
It could be argued that if the RTS,S/AS01E alone group is inferior to the RTS,S/AS01E + SMC group, 
children in the RTS,S/AS01E alone group should be given SMC. However, RTS,S/AS01E is not standard of 
care and it is uncertain whether the combination would be sufficiently more effective  to warrant its 
introduction. Therefore, it is justifiable to continue with the RTS,S/AS01E alone group, provided that it is 
not inferior to SMC, to investigate whether RTS,S/AS01E alone could be used as an alternative to SMC 
alone on grounds of increased feasibility or emergence of drug resistance.    
Decision: If RTS,S/AS01E is demonstrated to be non-inferior to SMC alone, using a relative margin of 
20%, it will be reasonable for this group to continue in the trial.  If non-inferiority cannot be 
demonstrated, the children in the RTS,S alone group will not continue; these children will receive SMC 
as recommended in the national malaria programme and be followed until the end of the study.  
The same questions will be considered in relation to the incidence of severe malaria (hospital 
admissions and deaths attributed to malaria) but the study is not powered to show differences 
between groups for these end-points Thus, although the findings from these analyses may help to 
support decisions based on the efficacy against clinical episodes of malaria, they will not be used alone 
to determine the decision on the future of the trial unless they raise issues of safety. 
 
DECISIONS MADE ON THE GROUNDS OF SAFETY 
. 
SMC alone is standard of care and so will act as the baseline for safety assessments (SMC has been 
shown in several large studies to be very safe).  Thus, the trial’s DSMB will be asked to consider the 
safety of children in the SMC plus RTS,S/AS01E and the RTS,S/AS01E alone groups in relation to findings 
in the SMC alone group based on an analysis of deaths overall, deaths attributed to malaria, SAEs 
overall and SAEs attributed to malaria. If there is a significant increase in the overall incidence of serious 
adverse events, primarily deaths or hospital admissions, not due to trauma or elective surgery or in the 
incidence of SAEs due to severe malaria in one of the RTS,S/AS01E groups compared with the SMC alone 

Statistical Analysis Plan 
 
RTS,S-SMC Extension Study 
45 
 
group, the DSMB may recommend that the affected group should not continue in the trial.  If there is a 
significant increase in both RTS,S/AS01E groups the DSMB may recommend that the trial should not be 
extended. Definition of what would considered to be sufficient evidence of risk in the groups receiving 
RTS,S/AS01E  will be a decision for the DSMB but, in such circumstances, a difference in the incidence of 
serious adverse events between groups that reaches a statistical significance of P = 0.01 or less is 
usually considered to be one potential reason for dropping a trial group. These data will also be made 
available to GSK. 
 
SEROLOGY 
Serological findings will not be available in time for the preliminary evaluation. 
 
CONCLUSIONS 
Deciding on whether to continue the RTS,S/AS01E + SMC trial and, if so, whether this should be with all 
three arms will require some subjective decisions based on balancing the potential risks and benefits of 
doing so. However, this document should help to provide a framework that will aid in making these 
decisions and a potential pathway is shown in Figure 2. 
 
 

Statistical Analysis Plan 
 
RTS,S-SMC Extension Study 
46 
 
Figure 2. Decision pathway  
 
 
 
 

Statistical Analysis Plan 
 
RTS,S-SMC Extension Study 
47 
 
Appendix 2 
Definitions of adverse and serious adverse events and reporting schedule 
A. Definition of an adverse event, adverse events of special interest, and serious adverse event 
A.1 Definition of adverse event (AE) 
An adverse event (AE) is defined as any clinical symptom or sign that occurs in a study child after 
administration of the study vaccine or drugs that may or may not have a causal relationship with the 
study drugs. Examples of an AE include -  
(1)   Occurrence of symptom such as fever, vomiting or diarrhoea in a child who did not have these 
symptoms prior to the administration of drugs;  (2)  Unexpected worsening of an existing condition.  
A.2 Definition of Adverse Events of Special Interest 
AEs of specific interest for safety monitoring include all seizures occurring within 30 days post-
vaccination, meningitis and pIMDs and will be recorded in the CRF. 
 
Meningitis cases: For the further evaluation of the safety signal of meningitis in the investigational 
vaccine groups all cases of meningitis occurring during the study will be reported as a SAE. 
 
pIMDs are a subset of AEs that include autoimmune diseases and other inflammatory and/or neurologic 
disorders of interest which may or may not have an autoimmune etiology. AEs that need to be recorded 
and reported as pIMDs include those listed in Table A. 
 
Table A. List of Potential Immune-mediated Diseases:  
Neuroinflammatory disorders 
Musculoskeletal disorders 
Skin disorders 
• 
Cranial nerve disorders, 
including paralyses/paresis (e.g. 
Bell’s palsy)  
• 
Optic neuritis 
• 
Multiple sclerosis 
• 
Transverse myelitis 
• 
Guillain-Barré syndrome, 
including Miller Fisher syndrome 
and other variants 
• 
Acute disseminated 
encephalomyelitis, including site 
specific variants: e.g. non-
infectious encephalitis, 
encephalomyelitis, myelitis, 
myeloradiculoneuritis 
• 
Myasthenia gravis, including 
Lambert-Eaton myasthenic 
syndrome  
• 
Immune-mediated peripheral 
neuropathies and plexopathies, 
• 
Systemic lupus 
erythematosus and 
associated conditions 
• 
Systemic scleroderma 
(Systemic sclerosis), 
including diffuse systemic 
form and CREST syndrome 
• 
Idiopathic inflammatory 
myopathies, including 
dermatomyositis  
• 
Polymyositis  
• 
Antisynthetase syndrome 
• 
Rheumatoid arthritis, and 
associated conditions 
including juvenile chronic 
arthritis and Still’s disease 
• 
Polymyalgia rheumatica 
• 
Spondyloarthritis, including 
ankylosing spondylitis, 
reactive arthritis (Reiter's 
• 
Psoriasis 
• 
Vitiligo 
• 
Erythema nodosum 
• 
Autoimmune bullous 
skin diseases (including 
pemphigus, 
pemphigoid and 
dermatitis 
herpetiformis) 
• 
Alopecia areata 
• 
Lichen planus 
• 
Sweet’s syndrome 
• 
Localised Scleroderma 
(Morphoea) 

Statistical Analysis Plan 
 
RTS,S-SMC Extension Study 
48 
 
(including chronic inflammatory 
demyelinating polyneuropathy, 
multifocal motor neuropathy 
and polyneuropathies associated 
with monoclonal gammopathy). 
• 
Narcolepsy 
Syndrome) and 
undifferentiated 
spondyloarthritis  
• 
Psoriatic arthropathy 
• 
Relapsing polychondritis 
• 
Mixed connective tissue 
disorder 
Vasculitides 
Blood disorders 
Others 
• 
Large vessels vasculitis 
including: giant cell arteritis 
such as Takayasu's arteritis 
and temporal arteritis.  
• 
Medium sized and/or small 
vessels vasculitis including: 
polyarteritis nodosa, 
Kawasaki's disease, 
microscopic polyangiitis, 
Wegener's granulomatosis, 
Churg–Strauss syndrome 
(allergic granulomatous 
angiitis), Buerger’s disease 
(thromboangiitis obliterans), 
necrotizing vasculitis and anti-
neutrophil cytoplasmic 
antibody (ANCA) positive 
vasculitis (type unspecified), 
Henoch-Schonlein purpura, 
Behcet's syndrome, 
leukocytoclastic vasculitis. 
• 
Autoimmune 
hemolytic anemia 
• 
Autoimmune 
thrombocytopenia 
• 
Antiphospholipid 
syndrome 
• 
Pernicious anemia 
• 
Autoimmune aplastic 
anaemia 
• 
Autoimmune 
neutropenia  
• 
Autoimmune 
pancytopenia 
• 
Autoimmune glomerulonephritis 
(including IgA nephropathy, 
glomerulonephritis rapidly 
progressive, membranous 
glomerulonephritis, 
membranoproliferative 
glomerulonephritis, and 
mesangioproliferative 
glomerulonephritis) 
• 
Ocular autoimmune diseases 
(including autoimmune uveitis 
and autoimmune retinopathy) 
• 
Autoimmune 
myocarditis/cardiomyopathySarc
oidosis 
• 
Stevens-Johnson syndrome 
• 
Sjögren’s syndrome 
• 
Idiopathic pulmonary fibrosis 
• 
Goodpasture syndrome 
• 
Raynaud’s phenomenon 
Liver disorders 
Gastrointestinal disorders 
Endocrine disorders 
• 
Autoimmune hepatitis 
• 
Primary biliary 
cirrhosis 
• 
Primary sclerosing 
cholangitis 
• 
Autoimmune 
cholangitis 
• 
Inflammatory Bowel 
disease, including Crohn’s 
disease, ulcerative colitis, 
microscopic colitis, 
ulcerative proctitis  
• 
Celiac disease 
• 
Autoimmune pancreatitis 
• 
Autoimmune thyroiditis (including 
Hashimoto thyroiditis) 
• 
Grave's or Basedow’s disease 
• 
Diabetes mellitus type I 
• 
Addison’s disease 
• 
Polyglandular autoimmune 
syndrome 
• 
Autoimmune hypophysitis 
 
 
A3. Definition of a serious adverse event (SAE) 

Statistical Analysis Plan 
 
RTS,S-SMC Extension Study 
49 
 
A serious adverse event is any clinical condition that fulfils at least one of the following criteria: 
(1)      Results in death. 
(2)      Results in admission to hospital. 
(3)      Is life-threatening (the child was at risk of death at the time of the adverse event). 
(4)      Results in disability/incapacity.  
 
B. Severity, relationship of event to study drug or vaccine and outcome 
The severity of a clinical adverse event is to be scored according to the following scale: 
(1)     Mild: Awareness of sign or symptom, but easily tolerated.                                                                                                   
(2)     Moderate:  Discomfort enough to cause interference with usual activity                                                                           
(3)    Severe:  Incapacitating with inability to work or perform usual activity.                                                                               
(4)    Life-threatening: Patient at risk of death at the time of the event.                                                                                       
(5)    Death. 
C. Assessment of Causality   
The relationship between the study vaccines and drugs and the occurrence of each SAE will be 
determined by the project physician in consultation with the site PIs based on their clinical judgment. 
Alternative causes, such as the natural history of the underlying diseases, concomitant therapy, other 
risk factors, and the temporal relationship of the event to the study drug will be considered and 
investigated.  The site PIs will consult the lead PIs and the DSMB if this is deemed to be necessary. 
There may be situations where the PIs have very minimal information about a SAE to include in the 
initial report.  However, every attempt will be made to make an assessment of causality for every SAE 
prior for reporting to the IDMC.  The PIs may change their opinion of causality in light of follow-up 
information, and may amend the SAE case report form accordingly.   
The relationship of an adverse event to study vaccine or drug will be assessed according to the 
following definitions: 
(1) Definitely unrelated: events that had occurred prior to administration of the study drugs or events 
that are obviously unrelated to the study (e.g. accidental injury). 
(2) Unlikely: There is no reasonable temporal association between the study drug and the suspected 
event and the event could have been produced by the child's clinical state or other concomitant 
medications. 
(3) Possible: The suspected adverse event may or may not have a reasonable temporal association 
with the administration of study drug but the nature of the event is such that an association with the 
study drug cannot be ruled out. The event could be related to the child's clinical state or by 
concomitant medications. 
(4) Probable: The suspected adverse event follows a reasonable temporal sequence after 
administration of study drugs, abates upon discontinuation of the drug, and cannot be reasonably 
explained by the known clinical state of the child. 
(5) Definitely related:  events that have no uncertainty in their association to the administration of 
study drugs. 
The outcome of each AE must be assessed according to the following classification: 

Statistical Analysis Plan 
 
RTS,S-SMC Extension Study 
50 
 
1) Completely recovered : 
The child has fully recovered with no observable 
residual effects. 
2) Not yet completely recovered : 
The child’s condition has improved, but still has some 
residual effects. 
3) Deterioration : 
The child’s overall condition has worsened 
4) Permanent damage : 
The AE has resulted in a permanent impairment 
5) Death : 
The child died due to the AE. 
6) Ongoing : 
The AE remains the same as at onset. 
7) Unknown : 
The outcome of the AE is not known because  of lost 
to follow-up 
D. Reporting of adverse events and SAEs 
All serious adverse events will be reported using a SAE report form which will have a detailed narrative 
of the events including information on the date the event started, severity, possible relationship to 
study drugs, concomitant medications, action taken, and outcome of the event.   
 
A list of all SAEs will be compiled monthly or three monthly as requested by the DSMB and provided to 
the DSMB and GSK. Any SAE potentially related to the vaccine or drug administration will be reported to 
the DSMB and institutional Ethics Committees within 72 hour. 
SAEs will be reported promptly to GSK within the timeframes described in Table A, once the 
investigator determines that the event meets the protocol definition of a SAE. 
 
Table B. Timeframes for submitting serious adverse event and other events reports to GSK Biologicals 
Type of Event 
Initial Reports 
Follow-up of Relevant Information on a 
Previous Report 
Timeframe 
Documents 
Timeframe 
Documents 
SAEs 
72 hours*‡ 
electronic Expedited 
Adverse Events Report 
72 hours*** 
electronic Expedited Adverse 
Events Report 
AEs of specific 
interest** 
72 hours*‡ 
electronic Expedited 
Adverse Events Report 
72 hours*** 
electronic Expedited Adverse 
Events Report 
 
* Timeframe allowed after receipt or awareness of the information. 
** AEs of specific interest include meningitis and pIMDs. 
*** Within receipt of information on the outcome of the SAE 
‡ The investigator will be required to confirm review of the SAE/AEs of specific interest causality by 
ticking the ‘reviewed’ box in the GSK electronic Expedited Adverse Events Report within 72 hours of 
submission of the SAE/AEs of specific interest. 
 
Reporting events of special interest 
AEs of specific interest (pIMDs, and meningitis occurring within 30 or 42 days of vaccination) will be 
reported promptly to GSK within the timeframes described in Table B, once the investigator determines 
that the event meets the protocol definition of an AEs of specific interest (pIMDs and meningitis within 
30 or 42 days of vaccination). 
 
 
Contact information for reporting serious adverse events and AEs of specific interest 

Statistical Analysis Plan 
 
RTS,S-SMC Extension Study 
51 
 
Study Contact for Reporting SAEs and AEs of specific interest* 
Refer to the local study contact information document. 
Back-up Study Contact for Reporting SAEs and AEs of specific interest* 
24/24 hour and 7/7 day availability: 
 
GSK Biologicals Clinical Safety & Pharmacovigilance 
Outside US & Canada sites: 
Fax: +32 2 656 51 16 or +32 2 656 80 09 
Email address: Rix.CT-safety-vac@gsk.com 
 
 
 

Statistical Analysis Plan 
 
RTS,S-SMC Extension Study 
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Appendix 3 
Membership of the trial committees. 
 
Trial Steering Committee 
Members of the trial steering committee are – 
 
Daniel Chandramohan, LSHTM, London, UK (investigator). 
Alassane Dicko, MRTC, Bamako, Mali (investigator). 
Brian Greenwood, LSHTM, UK (investigator). 
Jean Bosco Ouedraogo, IRSS Bobo-Dioulasso, Burkina Faso (investigator). 
Opokua Ofori-Anyinam, GSK, Brussels, Belgium (independent member). 
Kwadwo Koram, Noguchi Memorial Research Institute, Accra, Ghana (independent member). 
Joaniter Nankabirwa, Makerere University, Kampala, Uganda (independent member). 
Chris Ockenhouse, MVI Path, Washington, USA (independent member). 
Caroline Harris (replacing Morven Roberts) MRC Head Office, London (donor representative). 
Feiko ter Kuile, LSTM, Liverpool, UK (independent member) (chair). 
Mahamdou Thera (MRTC, Bamako, Mali (independent member). 
 
Data, Safety and Monitoring Board 
Members of the Data Safety and Monitoring Board are -        
Umberto D’Alessandro, MRC Unit, The Gambia. 
Blaise Genton, Swiss Tropical and Public Health Institute, Basle, Switzerland (chair). 
Mainga Hamaluba, KEMRI Wellcome Trust, Kilifi, Kenya 
Francesca Little, University of Capetown, Capetown, South Africa. 
Malcolm Molyneux, LSTM, Liverpool, UK 
Jean-Louis Ndiaye, University Cheikh Anta Diop, Dakar, Senegal 
 
 
 
 
 
 

Statistical Analysis Plan 
 
RTS,S-SMC Extension Study 
53 
 
 
 
23.06.22 
 
Seasonal vaccination with the RTS,S/AS01E malaria vaccine given with or without 
seasonal malaria chemoprevention:  extension of a randomised, double-blind 
Phase 3 trial until children reach the age of five years.  
 
 
Brief Title: A trial of RTS,S/AS01E and SMC: extension study 
 
(Protocol Identifying Number - PATH: CVIA 083) 
(Protocol Identifying Number - GSK: MALARIA-106 EXT:099 Y4-5 SUPP) 
 
 
Revisions to the Statistical Analysis Plan: 
  
Revision 
Number 
Date of 
Revision 
Page 
Number(s) 
Details 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Statistical Analysis Plan 
 
RTS,S-SMC Extension Study 
54 
 
 
Summary Study Information 
 
Study title 
Seasonal vaccination with the RTS,S/AS01E malaria vaccine given with or without seasonal 
malaria chemoprevention:  extension of a randomised, double-blind Phase 3 trial until children 
reach the age of five years.  
 
Clinical trial registration 
Clinicaltrials.gov Identifier: NCT04319380  
 
This study is an extension of an earlier clinical trial, NCT03143218:  ‘A comparative trial of 
seasonal vaccination with the malaria vaccine RTS,S/AS01E, seasonal malaria chemoprevention, 
and of the two interventions combined.’ 
 
Study duration 
 
Original trial:    
April 2017 – March 2020;  
Extension Study: 
April 2020 – March 2022. 
 
Study site 
The study is being conducted in Houndé district, Burkina Faso and in Bougouni district, Mali.   
 
 
Study objectives 
 
Original Trial 
The original trial sought to determine  
 
1. Whether seasonal vaccination with RTS,S/AS01E was non-inferior to four monthly 
courses of seasonal malaria chemoprevention (SMC) with sulphadoxine-pyrimethamine 
plus amodiaquine (SP+AQ) in preventing clinical malaria and other adverse outcomes.  
 
2. Whether the combination of these two interventions (i.e. seasonal vaccination with 
RTS,S/AS01 E and SMC with SP+AQ) was superior to RTS,S/AS01 E alone or SMC alone in 
preventing clinical malaria and other adverse outcomes. 
 
These aspects were considered to be of equal priority. 
 
 
Extension Study  
 
The extension study, undertaken in children aged three or four years old who had previously 
received five doses of the RTS,S/AS01 E malaria vaccine or comparator vaccines, together with 
seasonal malaria chemoprevention (SMC) with sulphadoxine-pyrimethamine (SP) plus  
amodiaquine (AQ) or placebo set out to determine whether : 
 
1. Vaccination with a booster dose of the RTS,S/AS01 E malaria vaccine at the beginning of 
the malaria transmission until children reach the age of five years is non-inferior to  SMC 
in preventing clinical attacks of malaria and would be easier to deliver than SMC. 

Statistical Analysis Plan 
 
RTS,S-SMC Extension Study 
55 
 
 
2. Administration of further doses of RTS,S/AS01 E at the beginning of the malaria 
transmission until children reach the age of five years  provides additional  protection 
against clinical episodes of malaria when given together with SMC.    
 
 
A key question for policy makers and malaria control programme managers is the impact of the 
two interventions and their combination in the first five years of life (the age range for which 
seasonal malaria chemoprevention is the current standard of care in most countries where it is 
policy). The primary focus of the overall study is, therefore, determination of the impact of the 
two interventions and their combination over the whole five-year period (the three-year initial 
study, plus the two-year extension), and its interpretation will, therefore, be based on the 
cumulative incidence of malaria until children reach the age of five years, and are no longer 
eligible for SMC.  
 
The primary objective of the extension study is determination of potential added value of 
extending administration of the two interventions and their combination beyond the age of 
three years. This will be based on the results obtained in the final two years of the study, based 
on the objectives described above. 
 
 
Important note on age groups within the study cohort, and eligibility for interventions 
 
In most SMC programmes, children are eligible to receive the intervention if they are aged at 
least 3 months, and were under 60 months at the time of the first SMC administration each year 
(Senegal is an exception, with children aged 3-119 months eligible for SMC).   
 
The original trial cohort was recruited in April 2017, when children were aged between 5-17 
months.  This means that some of the study cohort reached the age of five years prior to the 
2021 rainy season and were, therefore, not eligible to receive the trial interventions in the final 
year of the extension study. 
 
In the two-year extension phase of the trial, all children enrolled in the initial phase of the study 
received study interventions in 2020.  In 2021, children enrolled in the initial phase of the study 
were eligible to receive study interventions if they were aged up to, and including, exactly 5 
years (60 months) on the first of June 2021.  Children above this age (i.e. with dates of birth on 
31/05/2016, or earlier) remained under observation using identical surveillance measures as 
part of longer-term follow-up of the study cohort (to be reported separately) aimed at 
determining whether children in the trial are at any increased risk of malaria in the period after 
the interventions are no longer given.,  Other eligibility criteria are described below.  
 
In the text below, the terms ‘older’ and ‘younger’ children are used as follows:  
 
‘Older children’: children who were above the age of five years on 01 June 2021 (DOB 
31/05/2016, or earlier), and who were not eligible for interventions in 2021.  
 
‘Younger children’:  children who were exactly five years of age, or below on 01 June 2021 
(DOB 01/06/2016, or later), and who were eligible to receive interventions in 2021. 

Statistical Analysis Plan 
 
RTS,S-SMC Extension Study 
56 
 
 
Study groups 
This is a double-blind, individually-randomised trial with three study arms.  For reference, the 
interventions given during the initial three-year trial are shown first, with the interventions given 
during the extension study shown separately. 
 
Group 1 – SMC arm - ‘SMC alone’   
Original Trial:  
In 2017, children in the SMC group were given three doses of rabies vaccine at monthly 
intervals and four rounds of SMC with SP+AQ at monthly intervals.   In 2018 and 2019, these 
children received one dose of a control vaccine (Hepatitis A) and four rounds of SMC at 
monthly intervals.   
 
Extension study:  
All children in the SMC group received a single dose of a Tetanus Toxoid (in Burkina Faso) or 
Tetanus/Diphtheria toxoids (in Mali) vaccine in June 2020, and four rounds of SMC at monthly 
intervals between July and October 2020.   
Children in the SMC group exactly five years of age, or below, on 1st June 2021 received an 
additional dose of Tetanus toxoid vaccine in June 2021 (in both Countries), and either four 
rounds of SMC (in Mali) or five rounds of SMC (in Burkina Faso) at monthly intervals between 
July and October 2021.  This change in Burkina Faso for 2021 resulted from the National Malaria 
Control Programme choosing to deploy 5 courses of SMC in Houndé district. 
  
Group 2 – RTSS/ AS01E arm – ‘RTS,S alone’ 
 
Original Trial:  
In 2017, children in the RTS,S/AS01E group received three doses of RTSS/AS01E vaccine and four 
rounds of placebo-SMC at monthly intervals.   In 2018 and 2019, children received one dose of 
RTS,S/AS01E vaccine in June (prior to the malaria transmission season) and four rounds of 
placebo-SMC at monthly intervals.   
 
Extension study:  
All children in the RTS,S alone group received a single dose of RTS,S/AS01E in June 2020 (prior 
to the malaria transmission season), and four courses of placebo SMC at monthly intervals 
between July and October 2020.    
Children in the RTS,S alone group exactly five years of age, or below, on 1st June 2021 received 
an additional dose of RTS,S/AS01E vaccine in June 2021 (prior to the malaria transmission 
season), and four courses of placebo SMC (in Mali) or five courses of placebo SMC (in Burkina 
Faso) at monthly intervals between July and October 2021.   
  
Group 3 – RTS,S/AS01+SMC arm – ‘Combined arm’  
 
Original trial:   
In 2017, children in the combined group were given three doses of RTS,S/AS01E vaccine and 
four rounds of SMC with SP+AQ at monthly intervals.   In 2018 and 2019, children received one 
dose of RTS,S/AS01E vaccine in June (prior to the malaria transmission season) and four rounds 
of SMC with SP+AQ at monthly intervals. 
 
Extension study:  

Statistical Analysis Plan 
 
RTS,S-SMC Extension Study 
57 
 
All children in the Combined group received a single dose of RTS,S/AS01E in June 2020 (prior to 
the malaria transmission season), and four courses of SMC at monthly intervals between July 
and October 2020.    
Children in the Combined group exactly five years of age, or below, on 1st June 2021 received 
an additional dose of RTS,S/AS01E vaccine in June 2021 (prior to the malaria transmission 
season), and either four rounds of SMC (in Mali) or five rounds of SMC (in Burkina Faso) at 
monthly intervals between July and October 2021.   
   
The study groups, and abbreviated names used for ease of reference, are shown in the table 
below. The interventions given during the initial three-year trial, and the two-year extension are 
shown in separate sections. 
 
Table 1.  Study Interventions  
  
 
Group 
 
 
1) SMC  
‘SMC alone’ 
 
2) RTS,S/AS01E  
‘RTS,S alone’ 
 
3) RTS,S/AS01E +SMC 
‘Combined group’ 
Initial three-year trial 
April/May – June/July 
2017 
Rabies vaccine x 3 
RTS,S/AS01E x 3 
RTS,S/AS01E x 3 
July/Aug – Oct/Nov 
2017 
SMC x 4 
SMC placebo x 4 
SMC x 4 
 
 
 
 
June 2018 
Hep A vaccine x 1       
RTS,S/AS01E x 1 
RTS,S/AS01E x 1 
July-Oct 2018 
SMC x 4 
SMC placebo x 4 
SMC x 4 
 
 
 
 
June 2019 
Hep A vaccine x 1       
RTS,S/AS01E x 1 
RTS,S/AS01E x 1 
July-Oct 2019 
SMC x 4 
SMC placebo x 4 
SMC x 4 
 
 
 
 
 
Two-year Extension Study 
June 2020 
Tetanus (BF) or 
Tetanus/Diphtheria 
toxoids (Mali) x 1# 
RTS,S/AS01E x 1 
RTS,S/AS01E x 1 
July-Oct 2020 
SMC x 4 
SMC placebo x 4 
SMC x 4 
 
 
 
 
 
 
 
 
June 2021* 
Tetanus toxoid x 1# 
RTS,S/AS01E x 1 
RTS,S/AS01E x 1 
July-Nov 2021*$ 
SMC x 4  
(Mali) 
 
SMC x 5  
(Burkina Faso) 
SMC placebo x 4 
(Mali) 
 
SMC placebo x 5  
(Burkina Faso) 
SMC x 4  
(Mali) 
 
SMC x 5  
(Burkina Faso) 
 
* Only children aged 5 years or below on June 1st 2021 were eligible to receive study intervention in 2021. 
 
# In 2020, due to difficulties shipping tetanus toxoid vaccine from India as a result of the COVID-19 outbreak, children in group 1 in 
Burkina Faso received tetanus toxoid vaccine from stocks held by Burkina Faso’s national infant immunization programme. Children 
in group 1 in Mali received tetanus/diphtheria toxoids vaccine from stock held by Mali’s national infant immunization programme.  
In 2021, children in both countries received tetanus toxoid vaccine supplied by the Serum Institute of India. 
 
$ In Burkina Faso, in 2021, the National Malaria Control Programme began to deliver 5 monthly courses of SMC in the study area.  
To ensure that our intervention reflected the current standard of care, the study team also administered 5 SMC courses in 2021.

Statistical Analysis Plan 
 
RTS,S-SMC Extension Study 
58 
 
 
Figure 1.  Schematic of Key Study Activities in the Initial Trial Period, and in the Two-Year Extension 
 
 
 
Only children aged below 5 years on June 1st 2021 were eligible to receive study intervention in 2021.

Statistical Analysis Plan 
 
RTS,S-SMC Extension Study 
59 
 
 
Precise timing of interventions during the original trial, and the extension study 
 
Administration of RTS,S/AS01E or control vaccine: 
Original trial:  
Administration of the first dose of study vaccines began in late April 2017 in all three study 
groups, and was completed by mid-May.  The third dose was given between late June and early 
July 2017.  In 2018 and 2019, single doses of RTS,S/AS01E or control vaccine were given in the 
first two weeks of June.   
 
Extension study:  
In June 2020, vaccinations were given to all children in the study. In June 2021, vaccinations 
were given to all children exactly five years of age, or below, on 1st June 2021. 
 
 
Administration of SMC or placebo SMC:   
Original trial: 
In 2017, administration of the first SMC course began in late July and was completed by the end 
of the first week of August, with subsequent cycles on a monthly basis thereafter.  In 2018 and 
2019, administration of the first SMC course began in the second week of July, and was 
completed by mid-July. Subsequent courses were delivered monthly thereafter.  Earlier delivery 
in 2018 and 2019 was necessary to ensure that drug administration was completed before SMC 
delivery through the national malaria control programme took place. 
 
Extension study:  
In 2020, SMC was administered to all children, as four monthly courses, starting in July.  In 2021, 
all children exactly five years of age, or below, on 1st June 2021 received SMC, starting in July 
2021.  In Mali, children received four courses of SMC in 2021.  In Burkina Faso, children received 
5 cycles in 2021, because the national programme decided to deliver five cycles of SMC in 
Houndé district. 
 
 
 
 
 

Statistical Analysis Plan 
 
RTS,S-SMC Extension Study 
60 
 
Study population in the original trial 
 
Children of either sex were eligible for inclusion in the trial, provided that they were 5-17 
months of age on the scheduled date of first vaccination in April 2017, they were living 
permanently in the study area, and the consent of a parent or legally acceptable representative 
was obtained. 
 
Children with a history of an adverse reaction to SP or AQ, known to have a serious underlying 
illness including known HIV infection not well controlled by treatment, having severe 
malnutrition (z scores < 3 SD) or known to have previously received a malaria vaccine were 
excluded from the trial.   
 
Children known to have received SMC during the year prior to enrolment (either through the 
previous study in these districts (AZ-SMC, NCT02211729), or from the national programme) 
were not excluded from this study. 
 
The list of eligible children in each country was sorted by location (village), age in months, 
gender and prior receipt of SMC, before assigning randomisation codes in permuted blocks of 
9, to give an implicit stratification on these factors. 
 
Children who were randomized but who did not receive the first dose of study vaccines in 2017 
were deemed to have withdrawn consent and were not followed up further.  Consequently, 
analyses from baseline of all the children who remained under observation constitute a 
modified ITT population, rather than the full ITT population.  Analyses during specific years of 
the study, or during the extension study period only, reflect the children remaining in follow-up 
at that point, rather than analysis by intention to treat. 
 
Inclusion criteria for the Extension study  
 
In order to be eligible to participate in the extension study, a child must have been enrolled in 
the initial phase of the trial of seasonal vaccination with the RTS,S/AS01E vaccine and their 
parents or guardian must have provided consent for their inclusion in the extension study.   
 
As described above, children above the age of 5 years on 1st June 2021 were no longer eligible 
to receive either of the interventions, but were followed until the end of the extension study on 
safety grounds and to begin longer-term monitoring for any increased risk of malaria in the 
period after the interventions were no longer given. 
 
Exclusion criteria for the extension study 
 
Any child who met any of the following criteria was excluded from participation in the extension 
study:  
a. 
The child had had an allergic reaction to the study drugs or vaccines. 
b. 
The child had febrile convulsions on more than one occasion following vaccination. 
c. 
The child had a serious underlying illness, such as severe malnutrition (weight for age or 
mid arm circumference Z scores < 3 SD), which in view of the investigators might impair the 
response to vaccination. 
d. 
The child had been enrolled in another malaria vaccine or other experimental malaria 
intervention study. 

Statistical Analysis Plan 
 
RTS,S-SMC Extension Study 
61 
 
 
Figure 2.  CONSORT chart for the initial phase of the trial and the extension study 
 
 
 
 
 
* Some of the numbers in the figure above are provisional, and may change for the final analysis once 
all data cleaning is complete.   

Statistical Analysis Plan 
 
RTS,S-SMC Extension Study 
62 
 
 
Illustrative sample size calculations for the extension study period  
 
In Burkina Faso and Mali, 2777 and 3143 children respectively, received the first dose of study 
vaccine and were enrolled in the initial trial.  In Burkina Faso 2410 out of 2510 children 
remaining in follow-up at the end of the three-year trial consented to join the extension study. 
In Mali, 2624 out of 2680 children remaining in follow-up after three years consented to join the 
extension phase of the trial (Figure 2). 
 
Non-inferiority Comparison: SMC for four months of the year has an efficacy, assuming 
receipt of all four monthly cycles, of about 85% during this 4-month period. If, without 
intervention, the peak four months of malaria incidence would account for 60% of annual cases 
(with the other 40% falling during the other months) this equates to an efficacy over 12 months 
of at least 50%. The non-inferiority margin is the largest reduction in this efficacy that policy 
makers would be likely to accept if RTS,S/AS01 was to replace SMC, taking into account of the 
potential advantages of RTS,S over SMC in terms of ease of delivery and the potential to sustain 
a high level of coverage. A reduction in annual efficacy from 50% to 40% was considered the 
largest difference that would be likely to be acceptable. This lower efficacy would translate to a 
20% greater incidence in the RTS,S/AS01-alone arm compared to the SMC-alone arm.  
 
The study has greater than 90% power to exclude, at the 2.5% significance level, a relative 
difference in the incidence of clinical malaria of 20% over the five year study period between 
the RTS,S/AS01 and the SMC alone groups if these two interventions were equally effective.  To 
determine the level of confidence with which this margin can be excluded, we will also calculate 
two sided 90% and 99% confidence intervals.  With the anticipated incidence rates, there is 
adequate power to reject a smaller margin. However, 20% was considered the largest difference 
that would be considered unimportant.   
 
Superiority Comparison: The primary end-point for the trial is the incidence of clinical 
episodes of malaria. Based on incidence during the first three years of the trial (305 cases per 
1000 child-years in the SMC alone group), and taking account of the fact that the incidence of 
clinical episodes of malaria in children still protected by study interventions may decline with 
age, we assume that the incidence of malaria will be at least 200 cases per 1000 child-years at 
risk over the entire study period. Based on this assumption, the study will have 90% power to 
detect a 12.0% or greater relative difference (absolute difference: 24 cases per 1000 child-years) 
between the RTS,S/AS01E + SMC combined group and the RTS,S/AS01E alone or SMC alone 
groups, through a standard superiority comparison during the five years of the trial.  In the first 
three years of the trial, the incidence was about 60% lower in the combined group (rather than 
12%), so the extension study should have very high power for the superiority comparisons. 
 
 
 
Analyses in individual years 
 
There will be approximately 4,100 child-years of observation in the year following the third 
booster; 1367 child-years in each study group.   Assuming the incidence of clinical episodes of 
malaria remains at about 200 episodes per 1,000 children per year during this period, the study 
will have 90% power, through a standard superiority comparison, to detect a 25.9% or greater 

Statistical Analysis Plan 
 
RTS,S-SMC Extension Study 
63 
 
relative difference (absolute difference: 52 cases per 1000 child-years) between the Combined 
RTS,S/AS01E + SMC group and the RTS,S/AS01E alone or SMC alone group.   
 
A similar analysis will be undertaken for the final year of the study following administration of a 
fourth booster dose of the RTS,S/AS01E or comparator vaccine but study power will reduced 
because slightly more than half of the children enrolled (3860/5920) will not be eligible for SMC 
in 2021-22, and thus will not contribute further events nor person-time at risk to the study 
period.   
 
 
 
Study outcomes  
 
Primary Endpoint 
The primary end-point is the incidence of episodes of clinical malaria, as defined below, treated 
at a study health centre or hospital. 
 
1.1 
Clinical malaria is defined as an episode of fever (either measured axillary temperature ≥ 37.5°C, 
or a history of fever within the past 48 hours), and a positive blood film, with a Plasmodium falciparum 
parasite density of 5,000 per µl or more.  This cut-off was used in the original three-year trial, as well as in 
previous studies of SMC in Burkina Faso and Mali, and in the phase 3 studies of the RTS,S/AS01 vaccine 
(1, 2, 5-7).  
All passively-detected episodes of clinical malaria will be included in the analysis.  Specifically, 
this includes visits to outpatient clinics and hospitals, as well as morbidity detected at the time 
of SMC or at the end of transmission season survey. These contacts can be considered ‘passive’ 
because the caregiver had to bring the child to the contact and because the SMC or survey was 
conducted at the health facility in many cases. As the focus is on passively detected cases, 
morbidity detected during home visits for serological sampling, and for the weekly parasitaemia 
survey (which was undertaken only in the initial phase of the study) are excluded.  Morbidity 
detected at the time of vaccination is also excluded, because some children were visited at 
home and brought to the clinic in order to be vaccinated, so cases found at the time of 
vaccination cannot be regarded as passively detected.  
 
To avoid double counting of disease episodes which result in more than one healthcare contact, 
episodes of the primary outcome documented within 7 days of a previous episode will not be 
counted. No adjustment is necessary to the person-time at risk (11). 
 
 
Secondary Endpoints 
 
Secondary end-points - not listed in order of priority - include the following: 
 
2.1 
Morbidity events detected passively at study health centres and hospitals 
As for the primary outcome, episodes of the outcomes listed below which occur within 7 days 
of a previous event of the same type will be discounted. No adjustment is necessary to the 
person-time at risk (11). 
 

Statistical Analysis Plan 
 
RTS,S-SMC Extension Study 
64 
 
2.1.1 
Clinical malaria with P. falciparum parasitaemia of any density.  Defined as an 
episode of fever (either measured temperature ≥ 37.5°C, or a history of fever within the past 48 
hours), and a positive blood film for P. falciparum parasites.  This includes hospitalisations for 
malaria meeting the above criteria (i.e. fever or history of fever, plus slide confirmed P. 
falciparum malaria of any density).   
 
2.1.2 
Clinical malaria confirmed by rapid diagnostic test.  Defined as an episode of fever 
(either measured temperature ≥ 37.5°C, or a history of fever within the past 48 hours), and a 
positive rapid diagnostic test (RDT).  
  
2.1.3    Clinical malaria with non-falciparum parasitaemia of any density.  Defined as an 
episode of fever (either measured temperature ≥ 37.5°C, or a history of fever within the past 48 
hours), and a positive blood film for non-falciparum Plasmodium parasites.   
 
2.1.4    Clinical malaria with Plasmodium spp. infection of any density.  Defined as an 
episode of fever (either measured temperature ≥ 37.5°C, or a history of fever within the past 48 
hours), and a positive blood film for any Plasmodium spp. parasites (i.e. including P. falciparum).   
 
2.2 
Severe outcomes detected passively at study health centres and hospitals, and 
through verbal autopsies 
For all hospital admissions and deaths, the primary diagnosis by a study physician was reviewed 
by a second independent clinician.  A third clinician reviewed cases of disagreement to reach a 
consensus primary diagnosis.  All verbal autopsies were also reviewed by the same process to 
obtain a consensus cause of death. 
 
2.2.1 
Hospital admissions due to any cause   
 
2.2.2. Hospital admissions excluding those due to external causes or surgical conditions 
 
2.2.3  Hospital admissions due to malaria.  Defined as hospital admissions where malaria 
was the primary diagnosis, supported by a positive blood smear. Children who were admitted 
with a diagnosis of severe anaemia or neurological signs, with documented antimalarial 
treatment in the past 4 weeks, were also deemed to be due to malaria if their malaria RDT was 
positive (even if the blood smear result was negative or the blood smear result was not 
available).  Additional analyses of children who meet the WHO criteria for a diagnosis of severe 
malaria including those with a) cerebral malaria, b) severe anaemia and c) other forms of severe 
malaria will be undertaken. 
  
2.2.4 
The incidence of blood transfusions in study hospitals 
 
2.2.4 
Deaths due to any cause  
 
2.2.6 
Deaths due to any cause excluding external causes and surgical conditions 
 
2.2.7 
Deaths due to malaria.  Defined as hospital admissions resulting in death, where 
malaria was recorded as the primary cause of death supported by a positive blood smear.  
Children who were admitted with a diagnosis of severe anaemia or neurological signs, with 
documented antimalarial treatment in the past 4 weeks, were also deemed to be due to malaria 

Statistical Analysis Plan 
 
RTS,S-SMC Extension Study 
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if their malaria RDT was positive (even if the blood smear result was negative or the blood 
smear result was not available).  Deaths in the community will also be included when malaria is 
assigned by a panel of three independent physicians as the primary cause of death from verbal 
autopsy . 
 
2.3 
Outcomes measured at cross-sectional surveys at the end of the malaria 
transmission season 
For each of the outcomes below, results will be analysed and presented separately for the two 
surveys conducted during the extension study (conducted in November/December 2020 and 
November/December 2021). 
  
2.3.1 
The prevalence of asexual stage P. falciparum infection of any density 
2.3.2 
The prevalence of asexual stage P. falciparum infection with density ≥ 5000 per ul  
2.3.3  The prevalence of sexual stage P. falciparum infection (i.e. gametocytes) 
2.3.4 
The arithmetic mean P. falciparum parasite density, including samples which are parasite 
negatives as having a density of zero.  
2.3.5 
The prevalence of asexual stage infection of non-falciparum Plasmodium species. 
2.3.6 
The prevalence of sexual stage infection (i.e. gametocytes) of non-falciparum 
Plasmodium species. 
2.3.7 
The mean haemoglobin concentration in g/dL. 
2.3.8  The prevalence of anaemia, defined as measured Hb < 10 g/dL.   
2.3.9 
The prevalence of moderate anaemia, defined as measured Hb < 7 g/dL.   
2.3.10 The prevalence of severe anaemia, defined as measured Hb < 5 g/dL.  
 
2.4 
The antibody response to sixth and seventh doses of the RTS,S/AS01E vaccine 
2.4.1 
The Plasmodium falciparum anti-CSP antibody titre before and after the sixth and 
seventh doses (third and fourth ‘booster’ doses) of the RTS,S/AS01E malaria vaccine. 
2.4.2    The association between the magnitude of the anti-CSP antibody response to the sixth 
and seventh doses of the RTS,S/AS01E vaccine and the risk of malaria during the subsequent 
malaria transmission season.    
  
2.5 
The treatment efficacy of the SP+AQ combination used for SMC 
2.5.1 
The percentage of children with asymptomatic P. falciparum parasitaemia at the cross-
sectional survey in November/December 2021, who, when treated with SP+AQ, have an 
adequate clinical and parasitological response (ACPR) after 28 days.  
 
Serious Adverse Events 
In addition to the comparison of incidence rates described above, serious adverse events (SAEs) 
defined as hospitalisations or death, occurring at any time during the study will be tabulated by 
study group according to their cause.   
 
Outcomes measured among school-age children 
To help interpret results obtained in study children., end of season surveys have also been 
conducted among school-age children in the study areas at the same time as the end of 
transmission season surveys.  The following outcomes will be calculated for school-age children. 
 
3.1. 
The prevalence of asexual stage P. falciparum infection of any density 
3.2 
The prevalence of asexual stage P. falciparum infection with a density ≥ 5000 per ul 

Statistical Analysis Plan 
 
RTS,S-SMC Extension Study 
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3.3 
The arithmetic mean P. falciparum parasite density, including samples which are parasite 
negatives as having a density of zero. 
 
Analysis periods and person-years at risk 
 
Several analysis periods are of interest for the primary outcome of clinical malaria.  These are 
detailed in the figures and text that follow.   
 
Hypothesis testing and p-values will be presented for the whole intervention period (Period A 
characterised below) and for the two periods of interest in the extension study (periods B and C 
in the list below).  Numbers of events and hazard ratios or other measures of effect (with 
confidence intervals) will be tabulated for other analysis populations. 
 
 
A) Whole intervention period (all children).     
The primary analysis population will be the whole intervention period.  This is the period 
starting from enrolment in April 2017 until the end of the intervention year in which children 
reached 5 years of age (31 March 2021 for older children, 31 March 2022 for younger children). 
B) Study Year 4, to assess effectiveness of the sixth dose of RTS,S/AS01E 
All study children still in follow-up at the start of the fourth year of the study will be included, 
starting from 1st April 2020 until 31 March 2021 (i.e., covering the 2020 rainy season and 
subsequent dry season). 
C) Study Year 5, to assess effectiveness of the seventh dose of RTS,S/AS01E (younger 
children only) 
Older children were not eligible for interventions in 2021. Consequently, this analysis will 
include only younger children, starting from 1st April 2021 until 31 March 2022 (i.e., covering the 
2021 rainy season and subsequent dry season). 
D) From study start until the end of the study year 4 (to assess the overall effectiveness of 
up to six doses) 
All children originally enrolled in the trial were scheduled to receive study interventions during 
the first four years of the trial. This period will be analysed, starting from enrolment in April 
2017 until 31 March 2021, after the end of the 2020 rainy season. 
E) Whole intervention period, including only younger children (those eligible for 
interventions in 2021) 
Starting from enrolment in April 2017 until 31 March 2022 (i.e. after the end of the 2021 rainy 
season, in which interventions were given to younger children for the last time).  This period 
reflects the ‘full’ intervention over five years, with up to 7 doses of RTS,S and/or five complete 
seasons of SMC prior to 5 years of age. 
 

Statistical Analysis Plan 
 
RTS,S-SMC Extension Study 
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Figure 3.  Schematic of the analysis periods referred to in the text 
 
 
         
Dark blue boxes show the periods that will be included in each analysis.  *Older children are those aged 5 years or above on 1st June 2021, who were not eligible for 
interventions in Study Year 5 (2021).  $Younger children were aged below 5 years on 1st June 2021, so were eligible for interventions in 2021.  

Statistical Analysis Plan 
 
RTS,S-SMC Extension Study 
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Analysis strategy 
 
Modified Intention to Treat 
The primary analysis will be by modified intention to treat (mITT).  The mITT population will 
include all children who were screened and who received the first dose of RTS,S/AS01E or 
control vaccine, irrespective of the number of doses of subsequent vaccines or SMC/SMC 
placebo received.   
 
Children will contribute time at risk from the date of the first vaccination contact in 2017, until 
the end of the intervention period, which is either  
 
i) 31st March 2021, if born prior to 1st June 2016 (and thus over the age of five years in June 
2021), or 
ii) 31st March, 2022, if born on or after 1st June 2016, and thus still under the age of five year in 
June 2021.  
 
 
Prior to the end of the intervention period, children may be censored;  
1) on the date last seen if lost-to-follow-up (LTFU),  
2) on the date of permanent exit from the study area, or  
3) on the date of death,  
 
Children who temporarily left the study area with known exit and re-entry dates will have the 
corresponding person-time excluded from the analysis by intention to treat (and per protocol, if 
leaving the study area does not result in missed treatments).   
 
Per Protocol Analysis 
As a secondary analysis, the primary outcome of clinical malaria will also be analysed per 
protocol (PP).  The PP population will be defined separately for each year of the study.  Children 
who were vaccinated at all scheduled vaccination contacts in a particular year (3 in 2017, 1 in 
each subsequent year) and who, in the same year, were also seen at the first SMC/SMC placebo 
contact each month (4 per year) will be considered as ‘per protocol’ for that year.   
 
Children who attended for SMC administration but who did not receive SMC because they had 
malaria and were referred for treatment will be included in the per protocol analysis.  
 
‘Per protocol’ is defined differently for the two interventions (vaccination and SMC).  For 
vaccination, a child must have received all vaccination doses that year; for SMC a child must 
only have attended all SMC contacts that year. This difference is necessary because the primary 
outcome of the trial (clinical malaria) can result in a specific SMC dose being missed 
permanently, whereas if a child had malaria at the time of vaccination, catch-up was attempted 
later in the season.  The per protocol conditions will be applied equally to all three groups, i.e. 
to be considered as per protocol, a child must have received all doses of vaccine AND attended 
all SMC contacts, irrespective of which of these were active and placebo.  
 
 
All secondary outcomes will be analysed by modified intention to treat, as described above.   
 

Statistical Analysis Plan 
 
RTS,S-SMC Extension Study 
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Strategy to address multiplicity 
 
Hypothesis testing for analyses of the primary outcome will follow the closed testing procedure, 
whereby there is initially a test of the null hypothesis that the incidence in the three groups is 
the same. If this is rejected at the 5% level, pairwise comparisons will be done also using a 5% 
significance level.  Pairwise comparisons can be considered statistically significant only if the 
overall null hypothesis is rejected.   P-values and 95% confidence intervals will be presented. 
 
For the non-inferiority comparison of the primary outcome described above, we will calculate 
two-sided 95% confidence intervals, equivalent to the use of a one-sided significance level of 
2.5%, as is recommended (4).  To illustrate the level of confidence with which non-inferiority can 
be declared, we will plot the point estimate for the hazard ratio with two-sided 90, 95 and 99% 
confidence intervals, and the non-inferiority margin (a hazard ratio of 1.2, which would reflect 
20% higher incidence of clinical malaria in children in the RTS,S/AS01E alone group, relative to 
the SMC alone group). 
 
For secondary outcomes, we will present 95% confidence intervals, without adjustment for 
multiplicity, stating that no adjustment was made.   
 
For safety outcomes, we will present p-values and 95% confidence intervals, without adjustment 
for multiplicity. 
 
 

Statistical Analysis Plan 
 
RTS,S-SMC Extension Study 
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Trial profile – Original Study 
 
The original trial profile showed the number of individual children enumerated at the initial 
census, the number of children eligible, and the number of children for whom consent was 
obtained.  The profile also showed, for all the eligible children seen at the census who were 
randomised, the number of children seen at the first study contact who received the first dose 
of vaccine and who joined the study.   
 
The number of children who exited the study population by the end of the first, second and 
third year of the original trial were shown, with reasons (where known) tabulated.  
 
Uptake of the study interventions will be summarised, including: 
- 
the number that received different combinations of vaccine doses;  
- 
the distribution of the interval between doses;  
- 
the number that received 0,1,2,3,4 SMC treatments each year;  
- 
the actual timing of SMC cycle 1 in relation to the malaria transmission season;  
- 
the mean and range of the intervals between the monthly SMC courses;  
- 
the adherence to daily doses of SMC each month 
 
Separate profiles were produced for each of the two trial centres. 
 
 
Trial Profile - Extension Study 
The number of individuals eligible to join the two-year extension study, and the number who 
gave consent to participate will be shown. 
 
The number of children who exited the study population by the end of the fourth (2020 
intervention year) will be tabulated with reasons (where known) indicated.   
 
Children who reached the age of 5 years prior to the 2021 intervention year will be shown as a 
separate category (this does not count as ‘loss to follow-up’ as these children were excluded 
from further intervention by design, and continued to be monitored for longer-term for 
morbidity post-intervention).   
 
Among children who remained below the age of five years in June 2021, the number of children 
who exited the study population by the end of the fifth (2021 intervention year) will be 
tabulated, with reasons (where known) indicated.  
 
 
Uptake of the study interventions during the intervention period will be summarised, including: 
- 
the number that received different combinations of vaccine doses;  
- 
the distribution of the interval between doses;  
- 
the number that received 0,1,2,3,4 (and 5 in Burkina Faso, in 2021) SMC treatments each 
year;  
- 
the actual timing of SMC cycle 1 in relation to the malaria transmission season;  
- 
the mean and range of the intervals between the monthly SMC courses;  
- 
the adherence to daily doses of SMC each month 
 

Statistical Analysis Plan 
 
RTS,S-SMC Extension Study 
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Statistical methods 
 
Reference group 
As SMC is the current standard of care, the SMC alone group will be considered as the 
reference group for comparisons with RTS,S/AS01E alone, and the combined group.  
Comparisons will also be made between the RTS,S/AS01E alone and the combined group. 
 
Primary endpoint 
 
Hazard Ratios  
The hazard ratio for the primary outcome will be estimated using Cox regression models, with a 
robust standard error (i.e. the Andersen-Gill extension of the Cox model) to account for 
potential clustering of episodes within children.  The Efron method will be used for tied event 
times. 
 
The timescale will be calendar time.  For analyses of the whole study period, calendar time will 
start from 1st April 2017, i.e. allowing delayed entry according to the precise timing of the first 
vaccination contact.  This ensures that risk sets in the Cox models are comparable with respect 
to the timing of onset of transmission each year, and the timing of SMC.  Due to variable timing 
in vaccine dose 1 in 2017, this would not be the case if the data were analysed on the time in 
study timescale.  For the same reasons, analyses of the effectiveness of the sixth and seventh 
doses of RTS,S, calendar time will start from 1st April 2020 and 1st April 2021, respectively (i.e. 
the beginning of the study year in which the sixth and seventh doses were administered).   
 
Nelson-Aalen Cumulative hazards will be plotted for each group to show the mean number of 
events per child during the study and the timing of events, and Kaplan Meier failure estimates 
will be plotted to show the risks during the study. 
 
 
Incidence rate differences 
In addition to the hazard ratios from Cox regression, as described above, the incidence rate 
differences (IRD) will also be calculated, i.e. the cases averted per 1000 child-years at risk.  This 
is recommended in the updated CONSORT guidelines (8), as this gives an indication of the 
reduction in incidence attributable to the interventions, i.e. the absolute public health impact in 
similar contexts.  This is an important question for the overall effect of the two interventions 
under study in this trial, which is a question of key interest for policy makers.  This is also 
relevant to the interpretation of efficacy over successive years of the study.  For example, if the 
hazard ratio between the intervention groups moves towards unity as children become older, 
but malaria incidence increases in older children, the absolute impact of the interventions could 
remain as important (or even more important), in older children. Alternatively, if the hazard ratio 
remains similar to that seen in the initial intervention period but incidence falls in older children, 
extension of vaccination into older children may not be warranted.    
 
The IRD will be calculated using ordinary least squares regression of transformed variables for 
the intervention group, country and person-time at risk, as described by Xu et al. (9). This 
method uses a robust standard error and controls for unequal follow-up time, as well as 

Statistical Analysis Plan 
 
RTS,S-SMC Extension Study 
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quantitative or multiple covariates. To aid interpretation, the risk of the primary outcome will 
also be estimated from the Kaplan-Meier estimate of the risk.   
 
Proportion of children remaining free of malaria in individual years of the study 
 
The proportion of children who remained free of malaria (1-risk), in individual study years will 
be calculated from the Kaplan-Meier estimates in each intervention group.  The difference in 
this proportion between arms, with a 95% CI, and the relative difference (% increase in fraction 
remaining free of malaria) will also be calculated and presented.  
 
Secondary endpoints 
Secondary outcomes which are passively detected events, will be analysed in a similar way as 
for the primary outcome, i.e. estimating the hazard ratio using Cox regression with a robust 
standard error.   
 
The prevalence ratio of secondary endpoints measured at the weekly survey (aggregated into 
three-month periods), and at end of season surveys (including P. falciparum parasitaemia, 
anaemia, etc) will be estimated using Poisson regression, with a robust standard error for the 
individual, as described in Zou, 2004 (10).  
 
Linear regression models will be used to compare mean haemoglobin concentration between 
the groups.  
 
Arithmetic mean parasite densities (including in the calculation samples which are parasite 
negatives, as having density of zero), will be compared between arms using Poisson regression 
with a robust standard error.   
 
Covariates 
All analyses (primary outcomes and secondary outcomes) will stratify on study country only 
(Burkina Faso or Mali). 
 
For the primary outcome, we will also build a model adjusting for the following potential 
confounders:   
- 
Age at enrolment  
- 
Child’s Sex 
- 
Bednet use at baseline in 2017 
 
 
 
Pre-specified subgroup analyses, interactions and exploratory analyses  
 
As described above, the primary analysis will be pooled across the two study centres, stratified 
by (i.e. adjusted for) country.  Efficacy (ratio and difference) measures will be presented for both 
sites combined.  Investigation of any differences in intervention effects between the centres 
(formally, evidence for an interaction between intervention group and study centre) is pre-
specified due to possible differences in performance of these interventions under different 
malaria transmission intensity.  All outcomes will, therefore, also be tabulated by centre, and 
site-specific efficacy estimates will be presented.   

Statistical Analysis Plan 
 
RTS,S-SMC Extension Study 
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Investigation of differences between study groups in each study year is also pre-specified, as 
described above, because it is possible that the efficacy of RTS,S/AS01 booster doses changes 
over time, or following successive doses.  This will be assessed by exploring evidence for an 
interaction between intervention group and study year. 
  
Evidence for effect modification by age at enrolment will be explored.  It is possible that 
vaccination will perform differently according to the extent of prior exposure to malaria.  As 
described above, analyses of the first four years of the study (analysis population D) will be 
conducted splitting children according to their age in June 2021 (children aged 5 years or more, 
vs. children who were still under 5 years; younger children subsequently received an extra year 
of intervention). 
 
Finally, evidence for effect modification by child’s sex (observed in the RTS,S Phase III trial) will 
be explored by inclusion of an interaction term in the Cox models for the primary outcome over 
the whole analysis period (period A). Severe malaria meeting the WHO definition and malaria 
deaths will also be included as exploratory analyses. 
 
 
Pre-specified Secondary analyses  
   
 
Safety signals from the RTS,S/AS01 phase 3 studies  
The incidence of meningitis has been very low in the study cohort, with no events in the first 
three years of the trial.  A 95% confidence interval for the incidence rate of meningitis among 
children vaccinated with RTS,S/AS01, over the whole intervention period, up to the age of five 
years, will be calculated. 
The incidence of cerebral malaria among children vaccinated with RTS,S/AS01E will be 
investigated by comparing the RTS,S/AS01 groups with the SMC alone group, controlling for 
SMC status using an indicator variable.  Cox regression will be used to obtain the hazard ratio 
and its 95% confidence interval over the whole intervention period, up to the age of five years. 
The incidence of febrile convulsions not related to malaria or another obvious cause among 
children vaccinated with RTS,S/AS01 will be investigated as for cerebral malaria, i.e. by 
comparing the RTS,S groups with the SMC alone group, controlling for SMC status using an 
indicator variable.  Cox regression will be used to obtain the hazard ratio and its 95% 
confidence interval over the whole intervention period, up to the age of five years.  The excess 
cases per 1000 child-years at risk, based on the incidence rate differences will also be 
presented.  The subset of febrile convulsions that occurred within 7 days of vaccination will also 
be analysed.  
An exploratory analysis for data from the first three years of the trial found no evidence that 
RTS,S/AS01 increases mortality in girls.  This will be repeated over the whole intervention 
period, up to the age of five years.    
This was done by comparing the incidence of deaths using Cox regression, with an interaction 
between a dummy variable indicating receipt of RTS,S/AS01E and gender.  The Wald test p-
value for the interaction term will be used to assess evidence for effect modification. This model 
will also include a dummy variable for SMC to adjust for SMC receipt.  This will enable the 

Statistical Analysis Plan 
 
RTS,S-SMC Extension Study 
74 
 
female: male mortality ratio and its 95% confidence interval to be calculated separately for 
RTS,S/AS01E recipients, and non-recipients.  We will use indicator variables to obtain the ratio of 
these ratios, with the 95% confidence interval.   We will also present the mortality ratio for RTS,S 
recipients versus non-recipients separately for males and females. Since it is hypothesised that 
this effect may be age-dependent, we will also carry out these analyses restricted to the period 
after the first booster dose.  
 
Ancillary analyses to be reported separately 
1.  Analysis of the prevalence and frequency of molecular markers of resistance to SP and AQ in 
children with P. falciparum infection, among samples collected at the end of season cross-
sectional survey in 2019, and at the final cross-sectional survey in December 2021.  The 
molecular markers of interest include the dhfr 51-59-108 triple mutation, dhps-A437G, and 
dhps-K540E mutations for resistance to SP, and the pfcrt K76T and pfmdr1 N86Y mutations for 
resistance to AQ.     
2.  Secondary analyses are planned to investigate the association between malaria event history, 
and completeness of adherence to study interventions, with malaria incidence over time.  
 
3. Regression approaches applied to data from the first three years of the trial will be applied to 
data from the final two years of the study, to obtain estimate the duration and profile of 
protection over time from the sixth and seventh doses of RTS,S/AS01E and SMC.  Further 
secondary analyses will explore the changing relative benefits of SMC and RTS,S/AS01E with age 
and transmission intensity (by comparing efficacy profiles with age between Burkina Faso, which 
has higher incidence rates, with Mali). 
 
4. Analyses of the primary vaccine effect, through stratification on event number, to account for 
potential differences in the acquisition of immunity in the different study groups over the 
course of the trial, and to determine whether any appearance of diminishing effectiveness of 
the interventions over time, could be explained by differences in acquired immunity in the 
single intervention groups compared to the combined group. 
 
5.  Analyses of rebound morbidity.  The study cohort is being followed up beyond the 
observation period of the extension study, to determine whether prior randomisation group, 
adherence to the allocated interventions, and disease history is associated with morbidity post-
intervention.  While older children in the study cohort have been observed during their first 
year post-interventions (during the 2021 rainy season), it would be premature to draw 
conclusions about delayed morbidity post-intervention, as these older children who received 
their final (sixth) dose of RTS,S vaccine in June 2020 may continue to be partially protected from 
vaccination in 2021.  For this reason, analyses to investigate morbidity in the post-intervention 
period will be reported separately after a longer period of follow-up.  
 
 
 

Statistical Analysis Plan 
 
RTS,S-SMC Extension Study 
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Database and randomisation codes  
 
Data have been collected using electronic case record forms (eCRF) developed using Open Data 
Kit (ODK) software. Tablet PCs are used to document all intervention contacts and all active 
surveillance contacts such as the end of season cross-sectional surveys. For passive case 
detection, tablet PCs loaded with eCRFs are available at all study health centres that provide 
treatment. Data are transferred from the eCRFs held in the research sites to a central data 
management team. Automatic checks are performed on clinical and laboratory forms to ensure 
that they are complete and contain valid responses prior to transferring data.  Further checking 
and cleaning of the data (including checks for duplicate entries, consistency and range checks 
of variables) is then carried out by the data management teams in Burkina Faso and Mali using 
MS Access. 
 
The consistency of merges between different database tables will be undertaken blind to 
randomisation group.  The analysis databases and analysis programmes (written as Stata do 
files) will also be prepared for the primary and key secondary analyses before the 
randomisation code is broken.  
 
A final version of the database for analysis, following approval by the Data and Safety 
Monitoring Board (DSMB) will be archived on the LSHTM MyFiles system, and a copy sent to 
the chair of the DSMB for their records. 
 
The study treatment group will only be released by the independent statistician when the final 
database is ready and authorisation is given by the DSMB.  
 
All data used for analysis of the main trial report will be annotated and archived. Stata code 
used for the analyses will also be archived. 
 
 
 
 

Statistical Analysis Plan 
 
RTS,S-SMC Extension Study 
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Planned main tables for published report  
 
Dummy Table 1: Incidence of the primary outcome until the end of the intervention period: number of cases of clinical malaria; person-years at risk 
(PYAR); rates per 1000 person-years; and P-values from tests of homogeneity among all study children.  Results will also be shown by country with 
results of the test of interaction by country. 
 
 
No. 
children 
PYAR 
No. 
events 
Rate/1000 
(95% CI) 
Rate Ratio  
(95% CI) 
 
Test of 
homogeneity1 
Interaction 
by Country 
All children 
 
 
 
 
 
 
 
 
SMC 
2000 
6000.0 
1333 
180.0 (120.0, 240.0) 
(Reference) 
 
P=0.0 
 
RTS,S 
2000 
6000.0 
1333 
180.0 (120.0, 240.0) 
1.00 (0.90, 1.10) 
Reference 
 
 
Combined 
2000 
6000.0 
1333 
180.0 (120.0, 240.0) 
1.00 (0.90, 1.10) 
1.00 (0.90, 1.10) 
 
 
Total 
6000 
18000.0 
4000 
180.0 (120.0, 240.0) 
- 
- 
- 
 
 
 
 
 
 
 
 
 
 
Burkina Faso 
 
 
 
 
 
 
 
P=0.0 
SMC 
2000 
6000.0 
1333 
180.0 (120.0, 240.0) 
(Reference) 
 
P=0.0 
 
RTS,S 
2000 
6000.0 
1333 
180.0 (120.0, 240.0) 
1.00 (0.90, 1.10) 
Reference 
 
 
Combined 
2000 
6000.0 
1333 
180.0 (120.0, 240.0) 
1.00 (0.90, 1.10) 
1.00 (0.90, 1.10) 
 
 
Total 
6000 
18000.0 
4000 
180.0 (120.0, 240.0) 
- 
- 
- 
 
 
 
 
 
 
 
 
 
 
Mali 
 
 
 
 
 
 
 
 
SMC 
2000 
6000.0 
1333 
180.0 (120.0, 240.0) 
(Reference) 
 
P=0.0 
 
RTS,S 
2000 
6000.0 
1333 
180.0 (120.0, 240.0) 
1.00 (0.90, 1.10) 
Reference 
 
 
Combined 
2000 
6000.0 
1333 
180.0 (120.0, 240.0) 
1.00 (0.90, 1.10) 
1.00 (0.90, 1.10) 
 
 
Total 
6000 
18000.0 
4000 
180.0 (120.0, 240.0) 
- 
- 
- 
 
 
Numbers are included only to give an idea of layout / spacing.  
 

Statistical Analysis Plan 
 
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Dummy Table 2:  Incidence of the primary outcome by study year: number of cases of clinical malaria; person-years at risk (PYAR); rates per 1000 
person-years; and P-values from tests of homogeneity among all study children.  The results of the test of interaction by study year will also be shown. 
 
 
No. 
children 
PYAR 
No. 
events 
Rate/1000 
(95% CI) 
Rate Ratio  
(95% CI) 
 
Test of 
homogeneity1 
Interaction 
by Year 
Study Year 4 
 
 
 
 
 
 
 
 
SMC 
2000 
2000.0 
1333 
180.0 (120.0, 240.0) 
(Reference) 
 
P=0.0 
 
RTS,S 
2000 
2000.0 
1333 
180.0 (120.0, 240.0) 
1.00 (0.90, 1.10) 
Reference 
 
 
Combined 
2000 
2000.0 
1333 
180.0 (120.0, 240.0) 
1.00 (0.90, 1.10) 
1.00 (0.90, 1.10) 
 
 
Total 
6000 
6000.0 
4000 
180.0 (120.0, 240.0) 
- 
- 
- 
 
 
 
 
 
 
 
 
 
 
Study Year 5 
 
 
 
 
 
 
 
P=0.0 
SMC 
1000 
1000.0 
1333 
180.0 (120.0, 240.0) 
(Reference) 
 
P=0.0 
 
RTS,S 
1000 
1000.0 
1333 
180.0 (120.0, 240.0) 
1.00 (0.90, 1.10) 
Reference 
 
 
Combined 
1000 
1000.0 
1333 
180.0 (120.0, 240.0) 
1.00 (0.90, 1.10) 
1.00 (0.90, 1.10) 
 
 
Total 
3000 
3000.0 
4000 
180.0 (120.0, 240.0) 
- 
- 
- 
 
 
 
Numbers are included only to give an idea of layout / spacing.   
Similar tables will be used to report the incidence of passively detected secondary outcomes. 
 

Statistical Analysis Plan 
 
RTS,S-SMC Extension Study 
78 
 
Dummy Table 3:  Prevalence of P. falciparum infection at the end of malaria transmission season surveys: number of children tested; number with the 
outcome of interest; prevalence (95% CI); prevalence ratio (95% CI) and P-values from tests of homogeneity will be shown.   
 
 
No. children 
with result 
No. with  
outcome 
Prevalence 
(95% CI) 
Prevalence Ratios  
(95% CI) 
Test of 
homogeneity1 
P. falciparum infection 
 
 
 
 
 
 
 
 
 
 
 
 
 
All study children – 2020 
 
 
 
 
 
 
SMC 
2000 
200 
10.0 (5.00, 15.0) 
(Reference) 
 
P=0.0 
RTS,S 
2000 
200 
10.0 (5.00, 15.0) 
1.00 (0.90, 1.10) 
Reference 
 
Combined 
2000 
200 
10.0 (5.00, 15.0) 
1.00 (0.90, 1.10) 
1.00 (0.90, 1.10) 
 
Total 
6000 
600 
10.0 (5.00, 15.0) 
- 
- 
- 
 
 
 
 
 
 
 
All study children – 2021 
 
 
 
 
 
 
SMC 
2000 
200 
10.0 (5.00, 15.0) 
(Reference) 
 
P=0.0 
RTS,S 
2000 
200 
10.0 (5.00, 15.0) 
1.00 (0.90, 1.10) 
Reference 
 
Combined 
2000 
200 
10.0 (5.00, 15.0) 
1.00 (0.90, 1.10) 
1.00 (0.90, 1.10) 
 
Total 
6000 
600 
10.0 (5.00, 15.0) 
- 
- 
- 
 
 
Numbers are included only to give an idea of layout / spacing.   
Similar tables will be used to report the prevalence of other secondary outcomes measured at the end of transmission season surveys, and weekly 
surveys. 
 
 

Statistical Analysis Plan 
 
RTS,S-SMC Extension Study 
79 
 
Dummy Figure 1.  This will show i) cumulative hazards of malaria, by treatment group and ii) the risk of malaria, by treatment group.  As an example, 
the plot from the first 3 years of the trial is shown. 
 
 

Statistical Analysis Plan 
 
RTS,S-SMC Extension Study 
80 
 
Dummy Figure 2.    This will show the hazard ratios for the primary outcome, including 90, 95 and 99% confidence intervals, and the non-inferiority 
margin for the comparison of RTS,S alone and SMC alone.  As an example, the plot from the first 3 years of the trial is shown. 
 
 
 

Statistical Analysis Plan 
 
RTS,S-SMC Extension Study 
81 
 
 
References 
 
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Dicko A, Diallo AI, Tembine I, Dicko Y, Dara N, Sidibe Y, et al. Intermittent Preventive Treatment of Malaria Provides 
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