A Phase IIIB Comparative Trial of Seasonal Vaccination With the Malaria Vaccine RTS,S/AS01, Seasonal Malaria Chemoprevention and of the Two Interventions Combined
Phase: PHASE3
Conditions: Malaria,Falciparum, Children, Only
Sponsor: London School of Hygiene and Tropical Medicine

This supplement contains the following items. 
 
1. Original protocol, final protocol, summary of changes. 
2. Original statistical analysis plan, final statistical analysis plan, 
summary of changes. 
 
Clinical trials registration no. NCT03143218 

 
 
 
1 
Original protocol approved by LSHTM Ethics Committee 5.1.2017   
 
 
STUDY PROTOCOL 
 
A PHASE IIIB COMPARATIVE TRIAL OF SEASONAL VACCINATION WITH THE 
MALARIA VACCINE RTS,S/AS01, SEASONAL MALARIA CHEMOPREVENTION AND OF 
THE TWO INTERVENTIONS COMBINED. 
 
Study Identification number  
207284 (MALARIA-099) 
Brief Title: 
 
 
 
RTS,S/AS01 and SMC Trial 
Protocol Version & date: 
Original protocol approved by LSHTM Ethics Committee 
5.1.2017 (December 2016) 
Sponsor: 
 
 
 
London School of Hygiene & Tropical Medicine, 
 
 
 
 
 
Keppel Street, WC1E 7HT, 
 
 
 
 
 
London 
 
Lead Principal Investigators:  
Daniel Chandramohan & Brian Greenwood 
Site Study Directors:  
 
Jean Bosco Ouedraogo & Ogobara Doumbo  
 

 
 
 
2 
Contact details of the principal Investigators 
Brian Greenwood 
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 
London School of Hygiene & 
Tropical Medicine, Keppel St.,  
London WC1E 7HT, UK. 
 
Tel:  + 44 207 927 2322 
E-mail: 
daniel.chandramohan@lshtm.ac.uk   
 
Contact details of site study directors 
 
Jean Bosco Ouedraogo 
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. 
or 
Institut des Sciences et 
Techniques (INSTech), 540 
Avenue de Sya, 01 BP 2779, 
Bobo-Dioulasso, Burkina Faso. 
Tel (226) 20981880 or 20970811 
Tél/Fax (226) 20974868 
E-mail: 
jbouedraogo.irssbobo@fasonet.bf    
or  jbouedrago@gmail.com 
 
Ogobara Doumbo 
Malaria Research & Training 
Center; University of  Sciences 
Techniques and Technologies of 
Bamako, P.O. Box 1805, Bamako, 
Mali. 
 
Tel: +223 2022 8109 
E-mail: okd@icermali.org 
 
 
Contact details of site principal investigators in Burkina Faso 
 
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. tintohalidou@yahoo.fr or 
tintoh@fasonet.bf    
 
Issaka Zongo 
Institut de Recherche en Sciences 
de la Santé, Direction Régionale 
de l’Ouest (IRSS-DRO)  
399 Avenue de la Liberté 
Tel : (00226) 20 981 880 
Email: issaka.zongo@lshtm.ac.uk or 
zongo_issaka@yahoo.fr  
 

 
 
 
3 
 01 BP 545 Bobo-Dioulasso 01, 
Burkina Faso. 
 
 
Contact details of site principal investigators in Mali 
 
Alassane Dicko 
 
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: adicko@icermali.org 
 
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 
 
 
Contact details of other investigators and collaborators 
 
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 
 
Matthew Cairns 
Faculty of Epidemiology and 
Public Health, London School of 
Hygiene & Tropical  Medicine, 
Keppel St.,  
London WC1E 7HT, UK. 
 
Tel:  + 44 207 958 8104  
E-mail:  matthew.cairns@lshtm.ac.uk 
Irene Kuepfer 
Faculty of Infectious and Tropical 
Diseases, London School of 
Hygiene & Tropical Medicine, 
Keppel St., London WC1E 7HT, 
UK. 
 
Tel + 44 207 927 2322 
Email: irene.kuepfer@lshtm.ac.uk 
 

 
 
 
4 
Silke Fernandes 
Faculty of Public Health and 
Policy, London School of Hygiene 
& Tropical Medicine, Keppel St.,  
London WC1E 7HT, UK. 
Tel: +44 (0)20 7 
E-mail: silke.fernandes@lshtm.ac.uk 
 
 
 
Kara Hanson 
Faculty of Public Health and 
Policy, London School of Hygiene 
& Tropical  Medicine, Keppel St.,  
London WC1E 7HT, UK. 
Tel: +44 (0)20 7 
Email: kara.hanson@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 
Email: 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 
 
 
The signatures below confirm agreement by the individuals authorised by the sponsor and principal 
participating institution at the clinical site that the study will be conducted in compliance with 
protocol version 2 dated September 2016. 
 
Study Director in Mali 
NAME (PRINTED) 
DATE 
 
……………………………………… 
……………………………… 
……… 
Study Director in Burkina Faso 
NAME (PRINTED) 
DATE 
 
……………………………………… 
……………………………… 
……… 
Study Director in the UK 
NAME (PRINTED) 
DATE 
 

 
 
 
5 
 
                                                     LIST OF ABBREVIATIONS 
 
ACT 
Artemisinin Combination Therapy 
AQ 
Amodiaquine 
CSP 
Circumsporozoite protein. 
DSMB 
Data Safety and Monitoring Board 
EMA 
European Medicines Agency 
GCP 
Good Clinical Practice 
IRSS 
Institut de Recherche en Sciences de la Santé 
ITN 
Insecticide-treated bednet 
LDH 
Lactic dehydrogenase 
LSHTM 
London School of Hygiene & Tropical Medicine 
MMV 
Medicines for Malaria Venture 
MPAC 
Malaria Policy Advisory Committee 
MRTC 
Malaria Research and Training Centre 
NMCP 
National Malaria Control Programme 
SMC 
Seasonal Malaria Chemoprevention 
RDT 
Rapid Diagnostic Test 
SP  
Sulphadoxine/pyrimethamine 
WHO 
World Health Organization 
 
 

 
 
 
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                                                         PROTOCOL SUMMARY 
 
Title 
A comparative trial of seasonal vaccination with the malaria vaccine 
RTS,S/AS01, seasonal malaria chemoprevention and of the two 
interventions combined. 
Study objective 
This trial seeks to determine whether –  
 
1. Seasonal vaccination following priming with the RTS,S/AS01 malaria 
vaccine would be non-inferior to  Seasonal Malaria Chemoprevention 
(SMC) with sulphadoxine/pyrimethamine (SP) + amodiaquine (AQ) in 
preventing malaria in children in the areas of the Sahel and sub-Sahel 
of Africa where malaria is still a major public health challenge and 
whether RTS,S/AS01  would be easier to deliver than SMC.  
2. RTS,S/AS01 would provide additional, useful and cost effective 
protection against malaria if given together with SMC in areas with 
highly seasonal malaria transmission and reduce the risk of the 
emergence of resistance to the antimalarials used for SMC. 
 
Study design 
This will be a double-blind, individually randomised trial with three study 
arms.  The study groups are as follow: 
                             Group1 (SMC)         Group2 (RTSS)      Group3 (RTSS+SMC) 
Year1: Feb-April  Rabies vaccine x 3     RTSS/AS01 x 3           RTSS/AS01 x 3 
Year1: Aug-Nov        SMC x 4                SMC placebo x 4           SMC x 4 
Year2: Aug-Nov   HepA vaccine x 1      RTSS/AS01 x 1           RTSS/AS01 x 1 
                                   SMC x 4                 SMC placebo x 4          SMC x 4 
Year3: Aug-Nov  HepA vaccine  x 1      RTSS/AS01 x 1           RTSS/AS01 x 1 
                                 SMC x 4                   SMC placebo x 4          SMC x 4 
A decision will be made by the steering committee in year 2 on whether 
all children in Groups 2 and 3 will receive a fractional dose of RTS,S/AS01 
for their booster doses in years 2 and 3 or a full dose or whether children 
will be randomised to receive either a full or a fractional dose. This 
decision will be guided by the results of ongoing RTS,S/AS01 studies.  
Seasonal vaccination with either RTS,S/AS01 or control vaccine will be 
undertaken approximately one month before the start of the malaria 
transmission season and the first administration of SMC. 
Study site 
The trial will be conducted in Hounde district, Burkina Faso and in 
Bougouni district, Mali, sites of an on-going trial of the impact on 

 
 
 
7 
mortality and hospital admissions of adding azithromycin to the SP + AQ 
used for SMC.   
 
Study population 
Children of either sex, 5-17 months of age on the scheduled date of 
administration of the first dose of RTS,S/AS01 vaccine (February 2017) 
who are living permanently in the study area will be eligible for inclusion 
in the trial provided  the  consent of a parent or legally acceptable 
representative is 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 received a malaria vaccine will be 
excluded from the trial. Children known to have received SMC during the 
year prior to enrolment will not be excluded from the trial but will be 
distributed equally between study groups at the time of randomisation. 
  
Group1 - SMC arm 
Children in the control group will be given three doses of rabies vaccine 
(February-April) and four rounds of SMC (SP+AQ) (August to November) 
at monthly intervals in year 1. In years 2 and 3 they will receive one dose 
of a control vaccine (Hepatitis A) in June and four rounds of SMC (August-
November) at monthly intervals.  
  
Group 2 – RTSS/ 
AS01 arm 
Children in this intervention group one will be given three doses of 
RTS,S/AS01 vaccine (February-April) and four rounds of SMC placebo 
(August to November) at monthly intervals in year 1. In years 2 and 3 they 
will be given one dose of RTS,S/AS01 vaccine (June) and four rounds of 
SMC placebo (August-November) at monthly intervals. 
   
Group 3 – SMC + 
RTS,S/AS01 arm  
Children in the intervention group two will be given three doses of 
RTS,S/AS01 vaccine (February-April) and four rounds of SMC in year 1 
(August to November) at monthly intervals. In years 2 and 3 they will be 
given one dose of RTS/AS01 vaccine (June) and four rounds of SMC 
(August-November) at monthly intervals. 
   
Primary endpoint 
The primary end-point for the trial will be 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 and which is accompanied by a 
positive blood film with a parasite density of 5,000 per µl or more.   
Secondary 
endpoints 
Secondary end-points for the trial will 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 
which meet WHO criteria for a diagnosis of severe malaria.  

 
 
 
8 
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 the 
malaria transmission season.  
e. Serious adverse events (SAEs), including any deaths, occurring at any 
time during the study with special reference to any cases of meningitis 
and cerebral malaria (WHO case definition). 
f. Anti-CSP antibody concentrations obtained after priming and after 
each booster dose, determined in a sub-sample of children.   
g. The presence of molecular markers of resistance to SP and AQ in 
parasite positive samples collected at each annual cross-sectional 
survey.   
h. The presence of polymorphisms in the csp gene of Plasmodium 
falciparum isolates from children who have received RTS,S/AS01 that 
differ from those of the isolate used in the preparation of the vaccine.  
 
Secondary end-point (b) will be important for the economic evaluation of 
the interventions. Particular attention will be paid to the occurrence of 
any cases of meningitis and cerebral malaria [end-point (e)] as an increase 
in the incidence of these conditions was identified as a potential safety 
signal in the phase 3 RTS,S/AS01 trial. The trial will not be large enough to 
measure an impact on mortality but all deaths will be recorded and 
investigated. Evaluation of the clinical and immunological response to a 
fifth dose of RTS,S/AS01 is highlighted as an important research objective 
by the WHO.  
Sample size 
 Three thousand children will be recruited in Burkina Faso and in Mali 
(total 6,000) and these children will be followed for three years.  
Study duration 
October 2016 – June 2020 
 
 
 

 
 
 
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TABLE OF CONTENTS 
1 
BACKGROUND AND RATIONALE ............................................................................................... 10 
2 
STUDY OBJECTIVES .................................................................................................................. 12 
3 
STUDY AREA............................................................................................................................. 12 
4 
COMMUNITY SENSITISATION ................................................................................................... 14 
5 
TRIAL POPULATION .................................................................................................................. 15 
6 
SAMPLE SIZE ............................................................................................................................ 16 
7 
THE INTERVENTIONS ................................................................................................................ 17 
8 
IMPLEMENTATION OF THE INTERVENTIONS ............................................................................ 20 
9       CONTRAINDICATIONS TO VACCINATION   ……………………………………………………………………………………………20 
10 FOLLOW-UP AND MEASUREMENT OF OUTCOMES ...................... Error! Bookmark not defined.2 
11   LABORATORY PROCEDURES.................................................................................................... 244 
12   SOCIO-ECONOMIC STUDIES .................................................................................................... 254 
13   DATA MANAGEMENT ............................................................................................................... 26 
14   STUDY OUTCOMES ................................................................................................................. 277 
15   ANALYSIS ................................................................................................................................ 288 
16  ETHICS ..................................................................................................................................... 299 
17  TRIAL MANAGEMENT ................................................................................................................ 29 
18  ROLES OF THE INVESTIGATORS AND COLLABORATORS ............................................................. 30 
19  DISSEMINATION PLANS   .…………………………………………………………………………………………….…………..30 
20   REFERENCES..……………………………………………………………………..…………………………………………………..32 
21   ACTIVITIES SCHEDULE 
..…………………………………………………………………………………………………………..344 
22   APPENDICES ..……………………………………………………………………………………………………………………….. 
366 
 

 
 
 
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1     BACKGROUND AND RATIONALE 
The RTS,S/AS01 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/AS01 induces a strong 
antibody response to the P. falciparum CSP and high titres of anti-CSP antibody are associated with 
protection [2]. Following a long process of development, a phase 3 study of RTS,S/AS01 conducted 
in 15,439 children in 7 countries in Africa showed that three doses of RTS,S/AS01 given with a one 
month interval between doses, followed by a fourth dose 18 months post 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 given to infants at the age of 6-
12 weeks [3]. RTS,S/AS01 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 [4].  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, 4]. In July 2015, the 
European Medicines Agency reviewed efficacy and safety data on RTS.S/AS01 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 [5]. These 
included the pilot implementation of RTS,S/AS01 in children aged 5-17 months in 3-5 settings with 
moderate-to-high malaria transmission intensity, with a preference for areas where SMC is not 
being delivered, and evaluation of alternative approaches to deployment of the vaccine.  Recent 
evidence [6] from challenge studies conducted in American adult volunteers suggests that a higher 
level of protection can be obtained when the third dose of the priming schedule is reduced to one 
fifth of the usual amount and delayed until approximately 6 months post dose 2, and when a 
reduced dose is used for boosting. In these studies, a vaccine efficacy of 86% was achieved three 
weeks following priming and 90% efficacy following boosting with a fractional dose. This 
encouraging result is now being followed in further studies. 
 

 
 
 
11 
SMC involves monthly administration of an antimalarial drug or drug combination in a full 
therapeutic course to children  on three of four occasions during the period of highest risk of 
malaria infection. Studies undertaken in several countries in West Africa, including Burkina Faso 
and Mali, have shown 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% [7-9]. 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 [10]. Recent studies have defined the areas where SMC would be 
an appropriate intervention based on the seasonality and incidence of malaria [11]. These include 
most of the Sahel and sub-Sahel, population approximately 200 million, and possibly other areas in 
southern and eastern Africa. 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. 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 implementation of SMC at scale is in 
progress in many countries in this region through the UNITAID supported SMC ACCESS programme 
and the support of other major donor organisations. 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).  
 
SMC is effective but its delivery is demanding on the recipient and provider, requiring four contacts 
each malaria transmission season if anti-malarials are given to mothers to administer at home and 
12 contacts if directly observed treatment is employed. In addition, SMC is threatened by the 
emergence of resistance to SP and AQ and there are currently no other combinations of licensed 
antimalarials that could be used to replace them. It is likely to be 5-10 years before novel 
antimalarials under development could be deployed for SMC.  In contrast to SMC, seasonal 
vaccination with RTS,S/AS01  would require only one visit each transmission season after priming. 
RTS,S/AS01 may be a little less effective than  SMC during the malaria transmission season but this 
may be balanced by provision of protection during the dry season, when some malaria transmission 
still occurs and when SMC would provide no benefit. There is, therefore, a need for a comparative 

 
 
 
12 
study of these two interventions. In some areas where SMC is currently being deployed, and other 
malaria control interventions such as long-lasting insecticide treated nets used widely, the 
incidence of malaria in young children remains high (0.4 episodes per year in children under the age 
of five years in SMC recipients in Burkina Faso).  Thus, determining whether RTS,S/AS01 would 
provide added, useful protection to SMC in such situations is also important. It might also be able to 
protect some children who, because of side effects, are unable or unwilling to take SMC.    
 
Although the EMA has given a positive opinion on RTS,S/AS01, it is not yet certain how this partially 
effective malaria vaccine can be used most effectively [12]. Three, large-scale pilot implementation 
studies are being planned by WHO but it is unlikely that, following WHO recommendations, any of 
these will be conducted in a country where SMC is being delivered. The WHO recommendations on 
RTS,S/AS01 indicate the need for research on  alternative approaches to the delivery of this vaccine 
[13]. Exploration of the potential of the vaccine to prevent seasonal malaria, taking advantage of its 
high but rapidly waning efficacy, meets this recommendation and is, therefore, timely. 
 
2     STUDY OBJECTIVES  
The trial seeks to determine whether –  
a. Seasonal vaccination following priming with the RTS,S/AS01 malaria vaccine would be non-
inferior to  SMC SP + AQ in preventing malaria in children in the areas of the Sahel and sub-
Sahel of Africa where malaria is still a major public health challenge and whether it would be 
easier to deliver.  
b. RTS,S/AS01 would provide additional, useful and cost effective protection against malaria if 
given together with SMC in areas with highly seasonal malaria transmission and reduce the 
risk of the emergence of resistance to the antimalarials used for SMC. 
 
3     STUDY AREA 
The trial will be conducted in Hounde health district, Burkina Faso and in Bougouni Koulikoro 
district, Mali.  The Hounde 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 

 
 
 
13 
and which is the also a base of the IRSS.   The study site in Mali is the district of Bougouni in the 
region of Sikasso, Mali, 150 km south of Bamako where the MRTC is based.  
 
Figure 1 : Map of Mali and Burkina Faso showing the two sites. 
 
 
 
 
 
 
 
 
 
 
 
 
 
The population of Hounde 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 Plasmodium 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 2015 was 53% in 
Bugouni and 62% in Hounde.  The main malaria vector in each study area is Anopheles gambiae ss. 
A high proportion of children sleep under an ITN in Bougouni (96%) but the percentage is less in 
Hounde. 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. During the 
two years of the SMC + AZ the incidence of clinically suspected meningitis in children aged 3 - 59 
Hounde 
Bougouni 

 
 
 
14 
months in Bougouni was approximately 0.11 per 1,000 per year and that of cerebral malaria 0.88 
per 1,000 per year. 
 
During the past three years, Hounde and Bougouni districts have been the site of a trial, involving 
approximately 20,000 children aged 3-59 months, which has been evaluating the impact of adding 
azithromycin to the SP + AQ used for SMC in preventing overall mortality and hospital admissions 
with non-traumatic illnesses. This trial finishes at the end of 2016 which would allow a smooth 
transition to the new trial, incorporating many of the well trained staff who have conducted the 
previous study. Field laboratories, which are equipped to undertake parasitological and 
haematological investigations, have been established at each district hospital and efficient data 
management systems set up.   
 
In Mali vaccines will be stored at MRTC, Bamako, Mali. The MRTC has a long history (> 12 years) of 
testing vaccines including in collaboration with GSK, the WRAIR, the NIAID/NIH and Sanaria. 
Vaccines will be stored in a cold room with continuous monitoring of the temperature devices with 
alarm and telephone SMS and 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.  In Burkina 
Faso, vaccines will be stored at IRSS, in Bobo-Dioulasso where there is a dedicated storage room for 
drugs that 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 average with temperature and humidity controllers.  
 
4     COMMUNITY SENSITISATION 
The objectives of the study and the way in which it will be conducted have already been discussed 
with staff of the national malaria control progamme (NMCP) and expanded infant immunisation 
programme (EPI) in both Burkina Faso and Mali and their support for the trial obtained in principle.  
These discussions will be continued in the coming months. Community approval will be sought 
through meetings with leaders of the study communities and through open meetings held in the 

 
 
 
15 
study communities. Community leaders will be consulted prior to the start of the intervention on 
the best ways of achieving high compliance with vaccination, drug delivery and follow-up. 
 
5      TRIAL POPULATION 
After obtaining permission from the community leaders for the trial, a household census will be 
conducted in January 2017 and all households within the study areas with children 5-17 months of 
age on February 1st 2017 will be enumerated. At the census, a preliminary screening of potentially 
eligible children will be undertaken. Potentially eligible children and their caretakers will be visited 
again and written informed consent will be obtained from their caretakers for their inclusion in the 
trial before the administration of the first dose of study vaccines. Children entered into the trial will 
be assigned a unique ID number and their demographic data (date of birth and/or age, and gender), 
use of insecticide treated nets (ITN) and history of receiving SMC during the last transmission 
season will be collected. The census data will be updated in April/May 2018 and 2019 prior to the 
administration of the booster doses of vaccine.  Eligible children will be allocated randomly to one 
of the three study arms in permuted blocks of 12 using standard randomisation procedures (table). 
Children who have a history of receiving SMC in the previous years will be distributed equally 
between the three study groups 
 
A child will be eligible for inclusion in the trial if -   
a. 
The child is a permanent resident of the study area and likely to remain a resident for the 
duration of the trial. 
b. 
The child is 5 - 17 months of age at the time of first vaccination.   
c. 
A parent or legally recognised guardian provides informed consent for the child to join the 
trial.   
A child will be ineligible for inclusion in the trial if -  
a. 
The child is a transient resident in the study area. 
b. 
The child is in care. 
c. 
The age of the child is outside the stipulated range.  
d. 
The child has a history of an adverse reaction to SP or AQ. 

 
 
 
16 
e. 
The child has a serious underlying illness, including known HIV infection, unless this is well 
controlled by treatment, or severe malnutrition (weight for age or mid arm circumference Z 
scores < 3 SD). 
f. 
The child is known to have an immune deficiency disease or is receiving an 
immunosuppressive drug. 
g. 
The child has previously received a malaria vaccine. 
h. 
The child is enrolled in another malaria intervention trial. 
i. 
The parents or guardians do not provide informed consent.  
 
Table. Study groups                                          
                                         Group1 (SMC alone)      Group2 (RTS,S alone)      Group3 (RTS,S+SMC) 
Year1: Feb-April                  Rabies vaccine x 3              RTSS/AS01 x 3                 RTSS/AS01 x 3 
            Aug-Nov                         SMC x 4                      SMC placebo x 4                    SMC x 4 
Year2: Aug-Nov                     HepA vaccine x 1            RTSS/AS01 x 1                   RTSS/AS01 x 1 
                                                      SMC x 4                 SMC placebo x 4                      SMC x 4 
Year3: Aug-Nov                   HepA vaccine x 1            RTSS/AS01 x 1                      RTSS/AS01 x 1 
                                                    SMC x 4                   SMC placebo x 4                      SMC x 4 
SMC or placebo will be given at monthly intervals on four occasions during the malaria transmission 
season. 
 
6    SAMPLE SIZE 
Three thousand children will be recruited in Burkina Faso and a similar number in Mali (total 6,000) 
and these children will be followed for three years. A low dropout rate of around 5 % per year (15% 
overall) is anticipated based on findings from the current SMC+AZ study, although it is possible that 
the dropout rate may be higher during a vaccine trial as a vaccine trial has not been conducted 
previously in the study districts.  Since the relative efficacy of SMC and RTS,S/AS01 is uncertain, the 
conservative approach of recruiting equal numbers of children into each arm of the study (2,000 
per arm) will be followed. Results obtained over a period of three years of observation in the two 
study sites will be combined for the primary analysis of the incidence of clinical malaria. An analysis 

 
 
 
17 
will also be undertaken of efficacy in years 2 and 3 combined ie. to the efficacy of administration of 
a booster dose.  It is not anticipated that an unblinded interim analysis will be undertaken.  
 
During the current SMC study in the proposed trial sites, the incidence of clinical episodes of 
malaria during the first year of the study (2014) was 442 and 382 per 1000 children in Mali and in 
Burkina Faso respectively. Complete morbidity data are not yet available for the second year of the 
study (2016) but malaria incidence was higher than in 2015. For the sample size calculations below, 
we have, therefore, assumed a conservative incidence rate of 300 cases per 1000 children over a 
calendar year. Using a two sided 95% confidence interval, a three-arm study which enrolled 2000 
children in each arm (1000 per arm per centre) would have, for the non-inferiority comparisons, 
90% power to exclude a relative difference in incidence between RTS,S/AS01 and SMC given alone 
of 16.1% over the three-year study period and of 28.6% or more for each individual year. The study 
would also have 80% power to exclude a relative difference in incidence of 13.9% over the whole 
study period and of 24.7% in each year. The differences that could be detected will be smaller if, as 
suspected, the incidence of clinical malaria is greater than 300 cases per 1,000 children per year. 
For the superiority comparisons of the combined interventions with SMC alone, using a two-sided 
95% confidence interval, the study would have 90% power to detect a difference greater than 
11.1% over the three years of the study and of 19.2% in each year.  
 
For the analysis of the serological response to RTS,S/AS01, comparisons will be made between 
mean anti-CSP antibody titres pre and post the primary series of vaccination and before and after 
the two subsequent booster doses. Based on the standard deviations in antibody titres observed in 
children enrolled in the RTS,S/AS01 phase 2 and phase 3 trials, inclusion of around 160 individuals 
in each group (pre and post vaccination at each of the three time points) will give a study with 
approximately 80% power to detect a difference of 25% - 30% in mean titre between children who 
receive RTS,S/AS01 with or without co-administration of SMC.  
 
7     THE INTERVENTIONS 
RTS,S/AS01 vaccine: Three doses of RTS,S/AS01 will be given to children allocated to the RTSS or 
RTSS+SMC groups at monthly intervals during the 2017 dry season (February to April) followed by a 
fourth and fifth dose at the beginning of the 2018 and 2019 malaria transmission seasons.  A 

 
 
 
18 
decision will be made by the steering committee in the first quarter of 2018, based on the 
information available from on-going studies at that time, whether or not to use a fractional dose for 
the booster immunisation.   
 
The efficacy of RTS,S/AS01 against both severe and uncomplicated malaria in children vaccinated at 
the ages of 5 – 17 months and subsequently given a booster dose has been assessed by both the 
EMA and WHO to outweigh the vaccines side effects. The vaccine causes 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 common following vaccination. Fever post vaccination 
occurs in about 10% of children and febrile convulsions occurred in about 1% of 5-17 month old 
recipients enrolled in the phase 3 trial [3] but no persistent neurological effects were recorded. The 
main safety issue related to administration of RTS,S/AS01 is the unexplained, statistically significant 
increase in meningitis observed in children given the vaccine at the age of 5-17 months during the 
phase 3 trial (about 4 per 1,000 during a four-year period of follow up);  this was not observed 
when the  vaccine was given at the age of 6-12 weeks.  
 
 Meningitis was caused by a variety of organisms and did not show any temporal relationship to 
vaccination and more than 40% of cases occurred at one centre in Malawi.   The EMA concluded 
that the increase in cases of meningitis was probably a chance finding and that the benefits of the 
vaccine exceeded any safety issues. However, the agency recommended further evaluation of the 
incidence of meningitis in vaccine recipients when RTS,S/AS01 was deployed and this will be done 
in this study. Any study children admitted to hospital with suspected meningitis will be investigated 
as fully as possible and CSF samples obtained for microbiological diagnosis by PCR.  In addition, 
there was a suggestion that the proportion of cases of severe malaria which were classified as 
cerebral malaria was increased in the RTS,S/AS01 recipients, although the incidence of severe 
malaria overall was reduced.  Therefore, the incidence of cases of severe malaria meeting the WHO 
definition [ref] will be monitored carefully. 
 
RTS,S/AS01 malaria vaccine should not be administered to subjects with known hypersensitivity to 
any component of the vaccine, to a previous dose of RTS,S/AS01 E malaria vaccine or who is known 
to be hypersensitive to hepatitis B vaccine. 

 
 
 
19 
 
Control vaccines: Rabies vaccine will be used as the control vaccine for the primary series of 
vaccinations during the dry season in year 1 (2017) for those allocated to the SMC group. The rabies 
vaccine used will be a licensed, WHO approved vaccine purchased through UNICEF. This is likely to 
be RabipurR, previously produced by Novartis but now by GSK. This vaccine is produced in chick 
embryo cells and contains polygeline and residues of chicken proteins, and it may contain traces of 
neomycin, chlortetracycline and amphotericin and it is, therefore, contraindicated in subjects with 
an history of a severe hypersensitivity to any of the ingredients in the vaccine. Minor local and 
systemic reactions are common (>1:100, <1.10)   after vaccination with rabies vaccine and 
neurological complications including Guillain Barré syndrome have been described but are very rare 
(<1:10.000). 
 
 Hepatitis A vaccine (HAVRIXR), a licensed inactivated hepatitis A vaccine produced by GSK, will be 
used for the booster dose in years 2 and 3. HAVRIXR is contraindicated in subjects who have had an 
allergic reaction to prior administration of the vaccine or who are sensitive to neomycin. This 
vaccine may also cause minor local and systemic reactions ((>1:100, <1.10). Severe reactions, 
including anaphylaxis have been described but are very rare (<1.10,000).   
 
Seasonal malaria chemoprevention: Four courses of SMC with SP+AQ will be given at monthly 
intervals during the malaria transmission season in line with WHO’s recommendation and national 
policy [8]. A course of SMC for children aged over the age of one year will comprise a single 
treatment of SP (500mg/25 mg) and AQ 150mg on day 1 and AQ 150mg on days 2 and 3. Infants 
will receive half of these doses. SP and AQ and matching placebo will be obtained from a GMP 
certified supplier, if possible in a dispersible form.  All treatments will be given under observation.  
Children who do not receive SMC will receive a matching placebo. Discussions are taking place with 
the manufacturer of the SP+AQ combination used for SMC (Guilin Pharamceuticals, Shanghai, Co) 
and the Medicines for Malaria Venture as to whether it will be possible for the company to prepare 
a dispersible, matching placebo. If this is not possible, tablets of SP and AQ and of matching placebo 
tablets produced by the same company will be used.  
 

 
 
 
20 
Approximately 5 million children have received SMC with SP + AQ during the 2015 rainy season 
across the Sahel and sub-Sahel (http://www.malariaconsortium.org/pages/access-smc.htm)  
 and this drug combination has been shown to be remarkably safe [10]. 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. The possibility that SMC with SP+AQ might induce resistance to these drugs in P. 
falciparum has been investigated in a number of trials of SMC. Selection of parasites carrying 
mutations which confer resistance to pyrimethamine or sulphadoxine has been demonstrated in 
some but not all studies [7]. However, because the prevalence of parasitaemia in children who 
received SMC was substantially less than in the control group, the total number of parasites 
carrying resistance markers was less in children who had received SMC than in control children. 
Extensive use of SP for intermittent preventive treatment in pregnant women has not accelerated 
resistance to SP in West Africa.   Preliminary analysis of samples obtained from children in the 
ongoing SMC trial in the study sites at the end of 2014 did not show levels of mutations in dhfr and 
dhps genes likely to be associated with in vivo resistance to SP.   The potential risk of inducing 
resistance to SP or AQ by SMC was reviewed carefully by a WHO Technical Expert Group and WHO’s 
Malaria Policy Advisory Group and considered to be an acceptable risk in light of the major benefits 
conveyed by the intervention. Malaria parasites isolated at the end of the transmissions season will 
be tested for resistance markers to SP. 
 
8     IMPLEMENTATION OF THE INTERVENTIONS 
Vaccination.  RTS,S/AS01 will be provided in  a two-doses glass vial of lyophilized RTS,S antigen to 
be reconstituted with a two-dose glass vial of AS01 Adjuvant System. The final product for 
administration will be prepared by reconstitution of the lyophilized antigen with the liquid adjuvant 
to deliver two doses (1.0 ml). A single dose consists of 0.5 ml of RTS,S/AS01 final preparation. All 
vials of vaccine provided in this study are intended for single use only. After reconstitution the 
vaccine will be administered by slow IM injection, using a fresh 25G needle with length of one inch 
(25 mm), in the left deltoid. Vaccine will be injected within four hours of reconstitution (storage at 
+2°C to +8°C). Syringes containing RTS,S/AS01 or the control vaccine will be prepared by a 
pharmacist who takes no other part in the trial. Loading of syringes with vaccines and masking with 

 
 
 
21 
tape to blind the person administering the vaccine as to its nature 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. 
Rabies vaccine, 1.0 ml in volume is given by intramuscular injection. The paediatric dose of     
HAVRIX R is 0.5 ml and it is also administered by intramuscular injection. 
SMC. SMC drugs will be 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 four cycles of 
treatment required for one full malaria transmission season appropriate for the child’s age. 
Treatment with each dose of SMC will be given by trained, paid volunteers at a central point in each 
study community under observation. Study children will be given an 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 will be scanned to ensure that the child is given the allocated 
intervention. Home visits will be made to children who miss treatment on the designated day and 
their parents/guardians will be asked if they would still like their child to receive SMC. If they agree, 
treatment will be given at home.   
                                                                                                                                                                                
All children will be given an ITN at the commencement of the 2017 rainy season.  
 
9     CONTRAINDICATIONS TO SUBSEQUENT VACCINATION 
Contraindications to administration of a further dose of vaccine in any child include –  
a. Anaphylaxis following administration of the first dose of the vaccine. 
b.  Any condition that in the judgment of the investigator would make intramuscular injection 
unsafe.  
c. The occurrence of a new adverse event (AE) or the exacerbation of an existing AE that, in 
the opinion of the investigator, exposes the subject to unacceptable risk from subsequent 
vaccination.    
d. An acute disease and/or fever at the time of vaccination.  Fever is defined as temperature 
 37.5°C/99.5°F for oral, axillary or tympanic route, or  38.0°C/100.4°F for rectal route. 

 
 
 
22 
Subjects whose fever resolves with treatment may be vaccinated provided that 
revaccination falls within the stipulated time period. 
Subjects with a minor illness (such as mild diarrhea, mild upper respiratory infection) without fever 
can be administered all vaccines. 
10      FOLLOW-UP AND MEASUREMENT OF OUTCOMES 
The following follow-up procedures required to measure the study outcomes will be undertaken – 
a.    Passive surveillance for cases of uncomplicated and severe malaria. In each country, 
project staff will be 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 health facilities. In 
Mali, cases of uncomplicated malaria may also be treated by community health workers 
who have been taught to diagnose malaria with a RDT and to treat RDT positive cases and 
these cases will also be recorded if they meet the inclusion criteria. Cases of suspected 
malaria (fever, history of fever within 48 hours or any other symptom/sign suggestive of 
malaria) will be tested with a RDT and managed on the basis of their RDT result and blood 
films will be obtained from all these cases for subsequent confirmation of the diagnosis.  
b.  Active surveillance for malaria. Each month during the malaria transmission season, 90 
randomly selected children (30 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 parasitaemias. Any child who is febrile or who has other features 
suggestive of a diagnosis of malaria will have an RDT done and those who are positive will 
be treated with a full course of an ACT.  
c.    Prevalence of malaria parasitaemia and anaemia. A survey of all study children will be 
done one month after the last round of SMC administration at the end 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 and those 
who are positive will be treated with a full course of an ACT. Finger prick blood samples 
will be collected for preparing blood slides and blood spots on filter paper from all 

 
 
 
23 
children. The prevalence of parasitaemia, including the presence of gametocytes, will be 
detected by microscopy. 
d.    Serious Adverse Events. Project staff based at the district hospitals will be responsible for 
the identification of any child in the trial admitted to hospital and ensuring their referral to 
a study physician. Hospital staff will be provided with additional training on the recognition 
of cases of meningitis, cerebral malaria or immune deficiency diseases and standard 
operating procedures will be developed for management of children suspected of having 
one of these conditions.  Definitions for meningitis and cerebral malaria, currently being 
developed by WHO for use in the pilot RTS,S/AS01 implementation trials will be used. The 
aetiology of cases of meningitis will be 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 will be investigated and, if 
this occurred in the community, a verbal autopsy will be done.  
 
Surveillance of all children will be maintained throughout the study period for any Serious 
Adverse Events (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 a time decided by the DSMB, 
perhaps three monthly.    Details of definitions of adverse events and SAEs and of reporting 
procedures are described in appendix 1. 
e.    Immune response to the vaccine.  Blood samples (2ml) will be collected from 160 children 
in each of the groups who receive RTS,S/AS01  prior to administering the first dose of 
vaccine and one month after third dose of the primary series of  vaccination, and then in 
year 2 and 3 before giving the booster dose and one month after administration of the 
fourth  and fifth doses of vaccine for measurement of anti-CSP antibodies.  
f.   Drug resistance: Dried blood spots from children who have malaria parasitaemia detected 
by microscopy at each annual cross-sectional survey will be used for analysis of molecular 
markers of resistance to SP and AQ  at MRTC, Bamako and at IRSS, Bobo-Dioulasso. A 

 
 
 
24 
subset of samples will also be analysed at LSHTM as a test of quality control. A sub-set of 
positive samples will be tested  for the presence of HRP2 deletions which may give rise to a 
false negative RDT.  
g.   Polymorphisms in the P. falciparum CSP.  DNA will be obtained from a randomly selected 
group of children with clinical episodes of malaria to measure polymorphisms in the P. 
falciparum csp gene to determine how closely these match  the genetic structure of the 
isolate used in the production of the CSP vaccine as analysis of parasites collected during the 
phase 3 RTS,S trial showed that vaccine efficacy was higher against parasites with a CSP 
protein that was homologous with the strain used to produce the RTS,S/AS01 vaccine than 
against less well matched parasite strains [14].  
 
11     LABORATORY PROCEDURES 
a. 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. Blood films collected at 
the same time will be read subsequently by two microscopists. All slides will be read twice 
by two separate readers following the guidelines developed for the phase 3 RTS.S/AS01 trial 
[15]. 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/μl) 
readings will be 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 will be 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 will be adopted. If the majority decision is positive, the final result will be 
the geometrical mean of the two positive readings. In the case of discrepancies in parasite 
density, the final result will be the geometric mean of the two geometrically closest 
readings. 
b. Detection of markers of resistance to SP.  Parasite DNA will be extracted from dried blood 
spots and nested PCR reactions will be 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  [16-18]. PCR-

 
 
 
25 
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. 
c. Measurement of haemoglobin concentration. Haemoglobin concentration will be 
measured colorimetrically using a Hemocue colorimeter (Hemocue AB, Angelholm, 
Sweden). 
d. Measurement of anti-CSP concentration. Antibodies to CSP will be measured by a 
standardised ELISA at the University of Ghent in the laboratory of Professor Leroux-Roels as 
used in many previous trials of RTS,S/AS01. 
e. Detection of polymorphisms in the csp gene. Sequencing of the C terminal region of the CSP 
protein will be undertaken using methods described previously for detecting polymorphisms 
in this region of the csp gene [14].  
 
12     SOCIO-ECONOMIC STUDIES 
Objectives. The objectives of the socio-economic component of the trial are to determine: 
a. The cost-effectiveness of a) RTS, S vaccine versus SMC and b) the combination of both 
versus SMC alone. 
b. The acceptability of the two interventions (separately and combined) to the health care 
deliverers and to the study communities. 
c. The feasibility of introducing two malaria control strategies simultaneously from the health 
system perspective. 
Economic Evaluation. Data on the costs of a clinical case of malaria and of a hospital admission with 
malaria to both the health care deliverers and recipients have already been collected in the two 
study areas during the course of the SMC+AZ trial and this information will be updated. The costs of 
adverse events, in particular of meningitis, will be estimated using published literature.  
Information on the costs of delivery of SMC outside an intervention study has also been gathered 
by the SMC ACCESS team and this will facilitate determination of the costs of this intervention.  The 
costs of adding RTS,S/AS01 to the routine vaccination programme will require specific study 
through observations, key stakeholder interviews and review of relevant documents  and files. The 
delivery costs will be estimated through a combination of a step-down and ingredients-approach 
costing methodology. A cost effectiveness analysis of the two interventions and their use in 

 
 
 
26 
combination will incorporate the results from the trial, the costs collected during this study as well 
as during previous studies and then model the cost effectiveness of the interventions from a 
societal perspective in a decision tree model. The final outcomes used in the model will be 
costs/DALYs averted. The costs of scaling up the optimum intervention at regional/ national level 
will be investigated 
Acceptability. The acceptability of the two interventions to the health care deliverers and the 
preference of study communities for each of the interventions will be investigated through 
socioeconomic surveys and focus group discussions involving both the families of trial participants 
and those involved in administration of the trial interventions.  
Feasibility. Investigating the feasibility of delivery of RTS,S/AS01 outside the routine EPI programme 
will be a major objective of the large pilot studies being planned by WHO. Experience gained on the 
logistic challenges posed to the national immunisation programme of delivering RTS,S/AS01 as a 
seasonal vaccine will be shared with both the national immunisation programmes in Burkina Faso 
and Mali and also with WHO.   
 
13     DATA MANAGEMENT  
To ensure data is fit for purpose, questionnaires will be tested prior to use and staff will receive 
training prior to and at regular intervals during data collection. Data will be managed using the 
DataFax system which has been set up for the SMC + AZ trial and which is working well. This system 
is based on electronic transfer of the CRFs from the research sites where the data are automatically 
captured and validated. The MRTC data management team are responsible for the training and 
support of the IRSS data management team, with overall support from the NIAID/NIH central data 
management team.  Data will be uploaded to the DataFax system at the earliest opportunity to 
enable queries to be validated and issues resolved as soon as possible after data collection. 
Automatic checks will be performed on clinical and laboratory forms to ensure they are complete 
and contain valid responses prior to uploading by the local data managers at both sites. 
 
An experienced independent GCP monitor (Raouf Osseni) currently monitoring the SMC + AZ trial will 
be contracted to ensure the quality of the data collected and that GCP standards are met. The 
monitor will conduct a trial initiation visit, a close out visit and at least one additional visit each year. 
The monitor will ensure that the trial is  conducted according to the study protocol, that appropriate 

 
 
 
27 
ethical procedures are in place and s/he will examine a random selection of clinical and laboratory 
records during each visit to confirm their validity. 
 
14     STUDY OUTCOMES 
Primary outcome: The primary outcome measure of the trial is the incidence of clinical 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 outcomes: Secondary outcomes include: - 
a. Blood slide or rapid diagnostic test (RDT) positive malaria defined as a clinical episode 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 parasitemia) or a positive RDT. 
b. Hospital admissions with malaria, including cases of severe malaria which meet WHO 
criteria for a diagnosis of severe malaria.  
c. The prevalence of malaria infection (symptomatic or asymptomatic parasitaemia) 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 the malaria transmission season.  
e. Serious adverse events (SAEs), including any deaths, occurring at any time during the study 
with special reference to any cases of meningitis, cerebral malaria or immune deficiency 
illness. 
f. Anti-CSP concentrations obtained after priming and after each booster dose, determined in 
a sub-sample of children. 
g. Comparison of the anti-CSP concentrations among children who received co-administration 
of SMC and RTS,S/AS01 versus those who received RTS,S/AS01 alone.   
h. The presence of molecular markers of resistance to SP and AQ in parasite positive samples 
collected at each annual cross-sectional survey. 

 
 
 
28 
i. The match of polymorphisms in the P. falciparum csp gene of parasite isolates obtained 
from children with clinical episodes of malaria to the genetic structure of the strain of 
parasite sued to develop the RTS.S/AS01 vaccine.   
 
Particular attention will be paid to the occurrence of any cases of meningitis  as this was identified 
as a potential safety signal in the phase 3 RTS,S/AS01 trial. The trial will not be large enough to 
measure an impact on mortality but all deaths will be recorded and investigated including by verbal 
autopsy if these occur at home. Safety data will be provided to GSK in the format requested by the 
company. Evaluation of the clinical and immunological response to a fifth dose of RTS,S/AS01 is 
highlighted as an important research objective by the WHO.  
 
15     ANALYSIS  
The primary endpoint of incidence of all episodes of clinical malaria over the study period will be 
analysed using Cox regression models with a robust standard error to account for clustering of 
episodes within individuals (i.e. the Andersen-Gill extension of the Cox model). This will estimate 
the total effect of the interventions. The proportion of children in each group who experience one 
episode of malaria will be compared as a secondary outcome using Kaplan-Meier estimates: 
evidence for provision of complete protection through the combination of SMC and vaccination 
each year will be explored using recently published methods [19, 20]. For the comparison of 
RTS,S/AS01 plus SMC to the other interventions, a standard superiority comparison will be 
performed, calculating two-sided 95% confidence intervals for the hazard ratio. For the non-
inferiority comparison of RTS,S/AS01 to SMC, the two-sided 95% confidence interval for the hazard 
ratio will be compared to a pre-specified non-inferiority margin. The trial is powered to have more 
than 80% power to exclude a difference of 15% in the incidence of clinical malaria over the study 
period, a difference considered to be clinically important. Demonstration of a smaller difference 
would require a substantially larger trial and is considered to be not clinically relevant.  If the lower 
limit of the 95% does not overlap zero (i.e. it is clear that RTS,S is superior to SMC) then a 
superiority comparison will be performed as for the combined intervention. 
 
A formal analysis plan will be prepared and approved by the  Data Safety Monitoring Board (DSMB) 
appointed for the trial before the study code is broken. Both intention to treat and per protocol 

 
 
 
29 
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 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 but the study is only powered to meet its primary 
and major secondary end-points if results from both countries are combined. Additional sub-
analyses will include analysis by age, gender, bed net use during the transmission season, as 
determined by the history obtained at the cross-sectional surveys, and socio-economic status as 
determined by the educational level and occupation of the child’s family. 
16     ETHICS    
 Individual, written, informed consent will be obtained from the family or legally recognised 
guardian of each child entered into the trial.  Ethical approval 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.   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 the district hospitals in the study areas. 
 
17    TRIAL MANAGEMENT 
The London School of Hygiene & Tropical Medicine will act as the main sponsor for the trial. 
Delegated responsibilities may 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. 
 
An independent trial steering committee, which will provide scientific oversight, has been 
established and their approval of the protocol will be obtained. The steering committee will hold 
teleconferencing or face-face meeting annually to monitor progress and advise on the scientific 
content of the study. In addition, a DSMB has been established to oversee the safety of the trial and 

 
 
 
30 
a clinical trial monitor will be appointed to ensure that the trial is conducted to GCP standards. 
Membership of the trial steering committee and the DSMB is shown in appendix 2. 
The trial management committee will include the LSHTM PIs, site PIs and the trial administrator.  
The trial management committee is responsible for overseeing the trial and its members will 
communicate regularly by teleconferences.  
 
The trial will adhere to the principles outlined in the International Conference on Harmonisation 
Good Clinical Practice (ICH GCP) guidelines, protocol and all applicable local regulations.   The trial 
will be registered on clinicaltrials.gov. 
 
18  ROLES OF THE INVESTIGATORS AND COLLABORATORS 
The team from the IRSS, Burkina Faso which includes Jean Bosco Ouédraogo, Halidou Tinto and 
Issaka Zongo will be responsible for conducting the part of the trial undertaken in Burkina Faso and 
will participate in the analysis of the trial results.  The team from MRTC which includes Alassane 
Dicko, Ogobara Doumbo, and Issaka Sagara will be 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, Irene Kuepfer, Matthew Cairns, Paul Milligan, Layla Yiannikaris, 
Karen Slater and Amit Bhasin) will provide epidemiological, statistical, administrative and financial 
management support. Silke Fernandes and Kara Hanson from the LSHTM will be responsible for the 
economic aspects of the study. A consultant will be recruited to assist the design of the 
acceptability and feasibility studies. 
 
19    DISSEMINATION PLANS 
Results from the trial will be presented at national and international conferences and in peer 
reviewed journals 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 in connection with the implementation of SMC and these 

 
 
 
31 
links will facilitate the incorporation of RTS,S/AS01 vaccine into SMC/EPI programme  if this is found 
to be a useful intervention. The study team has established good links with many other 
organisations involved in the delivery of SMC trials including the SMC ACCESS programme 
coordinated by the Malaria Consortium and with the WHO staff responsible for conducting the 
RTS,S/AS01 implementation studies Thus, if it is found that RTS,S/AS01 vaccine is a useful 
replacement or addition to SMC regimens, routes have already been established through which this 
knowledge could be disseminated rapidly.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
32 
20  REFERENCES 
[1]   Cohen J, Nussenzweig V, Nussenzweig R et al.  From the circumsporozoite protein to the 
RTS,S/AS candidate vaccine. Human Vaccin 2010; 6:90-6.  
[2]   White MT, Verity R, Griffin JT  et al. Immunogenicity of the RTS,S/AS01 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/AS01 malaria vaccine with or 
without a booster dose in infants and children in Africa: final results of a phase 3, individually 
randomised, controlled trial. Lancet 2015; 386: 31-45.  
 [4]  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;358:1927-34.  
[5]   http:www.who. int/mediacentre/news/release/2015/sage/en  
[6]   Regules JA, Cicatelli SB, Bennett JW et al. Fractional third and fourth dose of RTS,S/AS01 
malaria candidate vaccine: a phase 2a controlled human malaria parasite infection and 
immunogenicity study.  J Infect Dis 2016; 214:762-771. 
[7]  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.  
[8]  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.  
[9]  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. 
[10] 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, in press.   
[11] 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.  
[12] Penny MA, Verity R, Bever CA et al. The public health impact and   cost-effectiveness of malaria 
vaccine candidate RTS,S/AS01: a systematic comparison of predictions from four mathematical 
models. Lancet 2016; 387: 367-375. 
[13] World Health Organization. Weekly Epidemiol Rec 2015; 90:681-700.   
[14] Neafsey DE, Juraska M, Bedford T,et al   Genetic diversity and protective efficacy of the  
RTS,S/AS01 malaria vaccine. N Engl J Med 2015; 373: 2025-2037.  

 
 
 
33 
 
[15] 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/AS01 candidate malaria vaccine. Mal J 2011; 
10: e223.    
                                                                                           
[16] Plowe CV, Djimde A, Bouare M et al. Pyrimethamine and proguanil reistance-conferring 
mutations in plasmodium falciparum dihydrofolate reductase: polymerase chain reaction 
methods for surveillance in Africa. Am J Trop Med Hyg 1995; 52:565-568.   
[17] Djimde A, Doumbo OK, Cortese JF et al. A molecular marker for chloroquine-resistant 
falciparum malaria   N Engl J Med 2001; 344: 257-63. 
[18] 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.                                                                 
[19] Cairns M, Cheung YB, Xu Y et al.  Analysis of preventive interventions for malaria: exploring 
partial and complete protection, and total and direct intervention effects.  AmerJ Epidemiol 
2015; 181:1008-17. 
[20] Xu Y, Cheung YB, Lam KF et al.  Estimation of summary protective efficacy using a frailty 
mixture model for recurrent event time data. Stat Med 2012; 31: 4023-39.  
 

 
 
 
34 
21      ACTIVITIES SCHEDULE 
 
Activities 
2016 
2017 
2018 
2019 
2020 
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 J 
F 
M A M J 
J 
A S 
O N D 
J-
M 
A-
J 
Protocol submission for 
ethics approval 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Ethics and regulatory 
approval 
 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Staff recruitment  
 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Trial registration 
 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Order study drugs 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Vaccine shipment 
 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Steering committee meeting 
x 
 
 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
x 
 
 
 
 
 
 
 
 
 
 
x 
 
IDMC meeting 
 
 
 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
x 
 
 
 
 
 
 
 
 
 
 
x 
 
Census and consenting of 
children 
 
 
 
x 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Randomisation  
 
 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Printing IDcards, labels 
(vaccine & drugs) 
 
 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Vaccination 
 
 
 
 
 
x 
x 
x 
 
 
 
 
 
 
 
 
 
 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
x 
 
 
 
 
 
 
 
 
 
Administration of SMC 
 
 
 
 
 
 
 
 
 
 
 
x 
x 
x 
x 
 
 
 
 
 
 
 
 
x 
x 
x 
x 
 
 
 
 
 
 
 
 
x 
x 
x 
x 
 
 
 
Health facility based 
surveillance of  malaria 
morbidity and mortality 
 
 
 
 
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 
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 
x 
x 
x 
x 
x 
x 
 
 
End of transmission surveys 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
x 
 
 
Repeat household census 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
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 
x 
x 
x 
x 
x 
x 
 
PCR assays 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
x 
 
 
 
 
 
 
 
 
 
x 
x 
 
 
 
 
 
 
 
 
 
x 
x 
 

 
 
 
35 
 
 
Serology 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
GCP monitoring 
 
 
 
 
x 
 
 
 
 
 
 
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 
x 
x 
x 
x 
 
Blinded analysis & report for 
committees 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
Locking of data 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
x 
 
Data analysis 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
xx 
xx 
Dissemination  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  x 

 
 
 
36 
22    APPENDICES 
 
Appendix 1: Definitions of adverse and serious adverse events and reporting schedule 
a. Definition of an adverse event and serious adverse event 
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 serious adverse event (SAE) is any clinical condition that fulfils at least one of the following 
criteria: 
a. 
results in death, 
b. 
results in admission to hospital, 
c. 
is life-threatening (the child was at risk of death at the time of the adverse event), 
d. 
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: Patients 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 

 
 
 
37 
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: 
 
Completely recovered : 
The child has fully recovered with no observable 
residual effects 
 
Not yet completely 
recovered : 
The child’s condition has improved, but still has 
some residual effects 
 
Deterioration : 
The child’s overall condition has worsened 
 
Permanent damage : 
The AE has resulted in a permanent impairment 
 
Death : 
The child died due to the AE 
 
Ongoing : 
The AE remains the same as at onset 
 
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.   
 

 
 
 
38 
 A list of all SAEs will be compiled monthly or three monthly and provided to the DSMB and GSK as 
requested. Any SAE potentially related to the vaccine or drug administration will be reported to the 
DSMB and institutional Ethics Committees within 72 hours and GSK within 96 hours.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
39 
 Appendix 2.  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)                                                                        
Morven Roberts  MRC Head Office, London, (donor representative)                                                   
Feiko ter Kuile, LSTM, Liverpool, UK  (independent member)                                                                                            
Mahamdou Thera (MRTC, Bamako, Mali (independent member)                                                  
 Data, Safety and Monitoring Board 
Members of the Data Safety and Monitoring Board are -        
Sheick Oumar Coulibaly, University de Ougadougou, Burkina Faso and WHO, Brazzaville. 
Umberto D’Alessandro, MRC Unit, The Gambia. 
Blaise Genton, Swiss Tropical and Public Health Institute, Basle, Switzerland (chair). 
Francesca Little, University of Capetown, Capetown, South Africa. 
Malcolm Molyneux, MLW Research Programme, College of Medicine, Blantyre, Malawi. 
 
 
 
 

 
 
1 
FINAL 
April 2020 
 
STUDY PROTOCOL 
 
A PHASE IIIB COMPARATIVE TRIAL OF SEASONAL VACCINATION WITH THE MALARIA 
VACCINE RTS,S/AS01, SEASONAL MALARIA CHEMOPREVENTION AND OF THE TWO 
INTERVENTIONS COMBINED. 
 
Study Identification number  
207284 (MALARIA-099) 
Brief Title: 
 
 
 
RTS,S/AS01 and SMC Trial 
Protocol Version & date: 
 
Final April 2020 
Sponsor: 
 
 
 
London School of Hygiene & Tropical Medicine, 
 
 
 
 
 
Keppel Street, WC1E 7HT, 
 
 
 
 
 
London 
 
Lead Principal Investigators:  
Daniel Chandramohan & Brian Greenwood 
Site Study Directors:  
 
Jean Bosco Ouedraogo & Alassane Dicko  
 

 
 
2 
Contact details of the principal Investigators 
Brian Greenwood 
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 
London School of Hygiene & 
Tropical Medicine, Keppel St.,  
London WC1E 7HT, UK. 
 
Tel:  + 44 207 927 2322 
E-mail: 
daniel.chandramohan@lshtm.ac.uk   
 
Contact details of site study directors 
 
Jean Bosco Ouedraogo 
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. 
or 
Institut des Sciences et 
Techniques (INSTech), 540 
Avenue de Sya, 01 BP 2779, 
Bobo-Dioulasso, Burkina Faso. 
Tel (226) 20981880 or 20970811 
Tél/Fax (226) 20974868 
E-mail: 
jbouedraogo.irssbobo@fasonet.bf    
or  jbouedrago@gmail.com 
 
Alassan 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 
 
 
Contact details of site principal investigators in Burkina Faso 
 
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. tintohalidou@yahoo.fr or 
tintoh@fasonet.bf    
 
Issaka Zongo 
Institut de Recherche en Sciences 
de la Santé, Direction Régionale 
de l’Ouest (IRSS-DRO)  
399 Avenue de la Liberté 
Tel : (00226) 20 981 880 
Email: issaka.zongo@lshtm.ac.uk or 
zongo_issaka@yahoo.fr  
 

 
 
3 
 01 BP 545 Bobo-Dioulasso 01, 
Burkina Faso. 
 
 
Contact details of site principal investigators in Mali 
 
Alassane Dicko 
 
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: adicko@icermali.org 
 
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 
 
 
Contact details of other investigators and collaborators 
 
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 
 
Matthew Cairns 
Faculty of Epidemiology and 
Public Health, London School of 
Hygiene & Tropical  Medicine, 
Keppel St.,  
London WC1E 7HT, UK. 
 
Tel:  + 44 207 958 8104  
E-mail:  matthew.cairns@lshtm.ac.uk 
Irene Kuepfer 
Faculty of Infectious and Tropical 
Diseases, London School of 
Hygiene & Tropical Medicine, 
Keppel St., London WC1E 7HT, 
UK. 
 
Tel + 44 207 927 2322 
Email: irene.kuepfer@lshtm.ac.uk 
 

 
 
4 
Silke Fernandes 
Faculty of Public Health and 
Policy, London School of Hygiene 
& Tropical Medicine, Keppel St.,  
London WC1E 7HT, UK. 
Tel: +44 (0)20 7 
E-mail: silke.fernandes@lshtm.ac.uk 
 
 
 
Kara Hanson 
Faculty of Public Health and 
Policy, London School of Hygiene 
& Tropical  Medicine, Keppel St.,  
London WC1E 7HT, UK. 
Tel: +44 (0)20 7 
Email: kara.hanson@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 
Email: 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 
 
 
The signatures below confirm agreement by the individuals authorised by the sponsor and principal 
participating institution at the clinical site that the study will be conducted in compliance with 
protocol version 4 dated 7 December 2017. 
 
Study Director in Mali 
NAME (PRINTED) 
DATE 
 
……………………………………… 
……………………………… 
……… 
Study Director in Burkina Faso 
NAME (PRINTED) 
DATE 
 
……………………………………… 
……………………………… 
……… 
Study Director in the UK 
NAME (PRINTED) 
DATE 
 

 
 
5 
 
                                                    
 
  LIST OF ABBREVIATIONS 
 
ACT 
Artemisinin Combination Therapy 
AQ 
Amodiaquine 
CSP 
Circumsporozoite protein. 
DSMB 
Data Safety and Monitoring Board 
EMA 
European Medicines Agency 
GCP 
Good Clinical Practice 
IRSS 
Institut de Recherche en Sciences de la Santé 
ITN 
Insecticide-treated bednet 
LDH 
Lactic dehydrogenase 
LSHTM 
London School of Hygiene & Tropical Medicine 
MMV 
Medicines for Malaria Venture 
MPAC 
Malaria Policy Advisory Committee 
MRTC 
Malaria Research and Training Centre 
NMCP 
National Malaria Control Programme 
SMC 
Seasonal Malaria Chemoprevention 
RDT 
Rapid Diagnostic Test 
SP  
Sulphadoxine/pyrimethamine 
WHO 
World Health Organization 
 
 

 
 
6 
                                                         PROTOCOL SUMMARY 
 
Title 
A comparative trial of seasonal vaccination with the malaria vaccine 
RTS,S/AS01, seasonal malaria chemoprevention and of the two 
interventions combined. 
Study objective 
This trial seeks to determine whether –  
 
1. Seasonal vaccination following priming with the RTS,S/AS01 malaria 
vaccine would be non-inferior to  Seasonal Malaria Chemoprevention 
(SMC) with sulphadoxine/pyrimethamine (SP) + amodiaquine (AQ) in 
preventing malaria in children in the areas of the Sahel and sub-Sahel 
of Africa where malaria is still a major public health challenge and 
whether RTS,S/AS01  would be easier to deliver than SMC.  
2. RTS,S/AS01 would provide additional, useful and cost effective 
protection against malaria if given together with SMC in areas with 
highly seasonal malaria transmission and reduce the risk of the 
emergence of resistance to the antimalarials used for SMC. 
 
Study design 
This is a double-blind, individually randomised trial with three study arms.  
The study groups are as follow: 
                             Group1 (SMC)         Group2 (RTSS)      Group3 (RTSS+SMC) 
Year1: April-June  Rabies vaccine x 3     RTSS/AS01 x 3           RTSS/AS01 x 3 
Year1: Aug-Nov        SMC x 4                SMC placebo x 4           SMC x 4 
Year2: Aug-Nov   HepA vaccine x 1      RTSS/AS01 x 1           RTSS/AS01 x 1 
                                   SMC x 4                 SMC placebo x 4          SMC x 4 
Year3: Aug-Nov  HepA vaccine  x 1      RTSS/AS01 x 1           RTSS/AS01 x 1 
                                 SMC x 4                   SMC placebo x 4          SMC x 4 
Seasonal vaccination with either RTS,S/AS01 or control vaccine was 
undertaken approximately one month before the start of the malaria 
transmission season and the first administration of SMC. 
Study site 
The trial is being conducted in Houndé district, Burkina Faso and in 
Bougouni district, Mali, sites of  a previous trial of the impact on mortality 
and hospital admissions of adding azithromycin to the SP + AQ used for 
SMC.   
 
Study population 
Children of either sex, 5-17 months of age on the scheduled date of 
administration of the first dose of RTS,S/AS01 vaccine (April 2017) who 
are living permanently in the study area were eligible for inclusion in the 
trial provided the consent of a parent or legally acceptable representative 

 
 
7 
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 received a malaria vaccine were excluded from the 
trial. Children known to have received SMC during the year prior to 
enrolment were not be excluded from the trial but  distributed equally 
between study groups at the time of randomisation. 
  
Group1 - SMC arm 
Children in the control group were given three doses of rabies vaccine 
(April-June) and four rounds of SMC (SP+AQ) (August to November) at 
monthly intervals in year 1. In years 2 and 3 they  received one dose of a 
control vaccine (Hepatitis A) in June and four rounds of SMC (August-
November) at monthly intervals.  
  
Group 2 – RTSS/ 
AS01 arm 
Children in this group were given three doses of RTSS/AS01 vaccine (April-
June) and four rounds of placebo-SMC (August to November) at monthly 
intervals in year 1. In years 2 and 3 they received one dose of a RTSS/AS)1 
vaccine in June and four rounds of placebo-SMC (August-November) at 
monthly intervals.  
  
Group 3 – SMC + 
RTS,S/AS01 arm  
Children in the intervention group two were given three doses of 
RTS,S/AS01 vaccine (April-June) and four rounds of SMC in year 1 (August 
to November) at monthly intervals. In years 2 and 3 they were given one 
dose of RTS/AS01 vaccine (June) and four rounds of SMC (August-
November) at monthly intervals. 
   
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 
per µl or more.   
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. This activity to cease for the final 3 months of the study, 
April – June 2020 due to the COVID-19 outbreak. 

 
 
8 
d. The prevalence of malaria parasitaemia, including gametocytaemia, 
moderate and severe anaemia and malnutrition at the end of the 
malaria transmission season.  
e. Serious adverse events (SAEs), including any deaths, occurring at any 
time during the study with special reference to any cases of meningitis 
and cerebral malaria (WHO case definition). 
f. Anti-CSP antibody concentrations obtained before and after priming 
and before and after each booster dose, determined in a sub-sample 
of children.   
g. The presence of molecular markers of resistance to SP and AQ in 
parasite positive samples collected at the final l cross-sectional survey  
h. The 28-day treatment outcome in children with asymptomatic malaria 
parasitaemia treated with SP+AQ.  
 
Secondary end-point (b) will be important for the economic evaluation of 
the interventions. Particular attention will be paid to the occurrence of 
any cases of meningitis and cerebral malaria [end-point (e)] as an increase 
in the incidence of these conditions was identified as a potential safety 
signal in the phase 3 RTS,S/AS01 trial. The trial will not be large enough to 
measure an impact on mortality but all deaths will be recorded and 
investigated. Evaluation of the clinical and immunological response to a 
fifth dose of RTS,S/AS01 is highlighted as an important research objective 
by the WHO.  
Sample size 
 Approximately 3000 children have been recruited in Burkina Faso and in 
Mali (total 6,000) and these children will be followed for three years.  
Study duration 
October 2016 – June 2020 
 
 
 

 
 
9 
TABLE OF CONTENTS 
1 
BACKGROUND AND RATIONALE ............................................................................................................10 
2 
STUDY OBJECTIVES ................................................................................................................................12 
3 
STUDY AREA ..........................................................................................................................................12 
4 
COMMUNITY SENSITISATION ................................................................................................................14 
5 
TRIAL POPULATION ...............................................................................................................................15 
6 
SAMPLE SIZE .........................................................................................................................................16 
7 
THE INTERVENTIONS .............................................................................................................................17 
8 
IMPLEMENTATION OF THE INTERVENTIONS ..........................................................................................20 
9 
CONTRAINDICATIONS TO SUBSEQUENT VACCINATION .........................................................................21 
10 FOLLOW-UP AND MEASUREMENT OF OUTCOMES ................................................................................21 
11 LABORATORY PROCEDURES ..................................................................................................................24 
12 SOCIO-ECONOMIC STUDIES ...................................................................................................................25 
13 DATA MANAGEMENT ............................................................................................................................26 
14 STUDY OUTCOMES ................................................................................................................................27 
15 ANALYSIS ..............................................................................................................................................28 
16 ETHICS ..................................................................................................................................................29 
17 TRIAL MANAGEMENT ............................................................................................................................29 
18 ROLES OF THE INVESTIGATORS AND COLLABORATORS ..........................................................................30 
19 DISSEMINATION PLANS .........................................................................................................................31 
20 REFERENCES ..........................................................................................................................................32 
21 ACTIVITIES SCHEDULE ...........................................................................................................................34 
22 APPENDICES ..........................................................................................................................................36 
 

 
 
10 
1 
BACKGROUND AND RATIONALE 
The RTS,S/AS01 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/AS01 induces a strong 
antibody response to the P. falciparum CSP and high titres of anti-CSP antibody are associated with 
protection [2]. Following a long process of development, a phase 3 study of RTS,S/AS01 conducted 
in 15,439 children in 7 countries in Africa showed that three doses of RTS,S/AS01 given with a one 
month interval between doses, followed by a fourth dose 18 months post 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 given to infants at the age of 6-
12 weeks [3]. RTS,S/AS01 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 [4].  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, 4]. In July 2015, the 
European Medicines Agency reviewed efficacy and safety data on RTS.S/AS01 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 [5]. These 
included the pilot implementation of RTS,S/AS01 in children aged 5-17 months in 3-5 settings with 
moderate-to-high malaria transmission intensity, with a preference for areas where SMC is not 
being delivered, and evaluation of alternative approaches to deployment of the vaccine.  Recent 
evidence [6] from challenge studies conducted in American adult volunteers suggests that a higher 
level of protection can be obtained when the third dose of the priming schedule is reduced to one 
fifth of the usual amount and delayed until approximately 6 months post dose 2, and when a 
reduced dose is used for boosting. In these studies, a vaccine efficacy of 86% was achieved three 
weeks following priming and 90% efficacy following boosting with a fractional dose. This 
encouraging result is now being followed in further studies. 
 

 
 
11 
SMC involves monthly administration of an antimalarial drug or drug combination in a full 
therapeutic course to children on three of four occasions during the period of highest risk of 
malaria infection. Studies undertaken in several countries in West Africa, including Burkina Faso and 
Mali, have shown 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% [7-9]. 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 [10]. Recent studies have defined the areas where SMC would be an appropriate 
intervention based on the seasonality and incidence of malaria [11]. These include most of the 
Sahel and sub-Sahel, population approximately 200 million, and possibly other areas in southern 
and eastern Africa. 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. 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 implementation of SMC at scale is in progress in many 
countries in this region through the UNITAID supported SMC ACCESS programme and the support 
of other major donor organisations. 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).  
 
SMC is effective but its delivery is demanding on the recipient and provider, requiring four contacts 
each malaria transmission season if anti-malarials are given to mothers to administer at home and 
12 contacts if directly observed treatment is employed. In addition, SMC is threatened by the 
emergence of resistance to SP and AQ and there are currently no other combinations of licensed 
antimalarials that could be used to replace them. It is likely to be 5-10 years before novel 
antimalarials under development could be deployed for SMC.  In contrast to SMC, seasonal 
vaccination with RTS,S/AS01  would require only one visit each transmission season after priming. 
RTS,S/AS01 may be a little less effective than  SMC during the malaria transmission season but this 
may be balanced by provision of protection during the dry season, when some malaria transmission 
still occurs and when SMC would provide no benefit. There is, therefore, a need for a comparative 
study of these two interventions. In some areas where SMC is currently being deployed, and other 

 
 
12 
malaria control interventions such as long-lasting insecticide treated nets are used widely, the 
incidence of malaria in young children remains high (0.4 episodes per year in children under the age 
of five years in SMC recipients in Burkina Faso).  Thus, determining whether RTS,S/AS01 would 
provide added, useful protection to SMC in such situations is also important. It might also be able to 
protect some children who, because of side effects, are unable or unwilling to take SMC.    
 
Although the EMA has given a positive opinion on RTS,S/AS01, it is not yet certain how this partially 
effective malaria vaccine can be used most effectively [12]. Three, large-scale pilot implementation 
studies are now underway in Ghana, Kenya and Malawi. The WHO recommendations on 
RTS,S/AS01 indicate the need for research on  alternative approaches to the delivery of this vaccine 
[13]. Exploration of the potential of the vaccine to prevent seasonal malaria, taking advantage of its 
high but rapidly waning efficacy, meets this recommendation and is, therefore, timely. 
2 
STUDY OBJECTIVES  
The trial seeks to determine whether –  
a. Seasonal vaccination following priming with the RTS,S/AS01 malaria vaccine would be non-
inferior to  SMC SP + AQ in preventing malaria in children in the areas of the Sahel and sub-
Sahel of Africa where malaria is still a major public health challenge and whether it would be 
easier to deliver.  
b. RTS,S/AS01 would provide additional, useful and cost effective protection against malaria if 
given together with SMC in areas with highly seasonal malaria transmission and reduce the 
risk of the emergence of resistance to the antimalarials used for SMC. 
3 
STUDY AREA 
The trial will be conducted in Houndé health district, Burkina Faso and in Bougouni Koulikoro 
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 IRSS.   The study site in Mali is the district of Bougouni in the 
region of Sikasso, Mali, 150 km south of Bamako where the MRTC is based.  
 
 
 

 
 
13 
Figure 1 : Map of Mali and Burkina Faso showing the two sites. 
 
 
 
 
 
 
 
 
 
 
 
 
 
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 Plasmodium 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 2015 was 53% in 
Bugouni and 62% in Houndé.  The main malaria vector in each study area is Anopheles gambiae ss. 
A high proportion of children sleep under an ITN in Bougouni (96%) but the percentage is less in 
Houndé. 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. During the  SMC + AZ trial the 
incidence of clinically suspected meningitis in children aged 3 - 59 months in Bougouni was 
approximately 0.11 per 1,000 per year and that of cerebral malaria 0.88 per 1,000 per year. 
 
During   2014-2017, Houndé and Bougouni districts were the sites of a trial, involving approximately 
20,000 children aged 3-59 months, which  evaluated the impact of adding azithromycin to the SP + 
Houndé 
Bougouni 

 
 
14 
AQ used for SMC in preventing overall mortality and hospital admissions with non-traumatic 
illnesses. The trial found that addition of azithromycin to the antimalarials used for SMC did not 
reduce the incidence of death or admission to hospital with a condition not due to trauma, its 
primary end-point, although it led to a modest reduction in the incidence of clinical attendances 
with respiratory, gastrointestinal or skin infections [14].  This trial finished at the end of 2016  
allowing a smooth transition to the new trial, incorporating many of the well trained staff who had 
conducted the previous study. Field laboratories, which are equipped to undertake parasitological 
and haematological investigations, have been established at each district hospital and efficient data 
management systems set up.   
 
In Mali, vaccines are stored at MRTC, Bamako, Mali. The MRTC has a long history (> 12 years) of 
testing vaccines including  collaboration with GSK, the WRAIR, the NIAID/NIH and Sanaria. 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.  In Burkina 
Faso, vaccines are stored at IRSS, in Bobo-Dioulasso where there is a dedicated storage room for 
drugs that 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 average with temperature and humidity controllers.  
4 
COMMUNITY SENSITISATION 
The objectives of the study and the way in which it would be conducted  were discussed with staff 
of the national malaria control progammes (NMCPs) and expanded infant immunisation 
programmes (EPIs) in both Burkina Faso and Mali and their support for the trial obtained in 
principle.  These discussions have been continued. Community approval was sought through 
meetings with leaders of the study communities and through open meetings held in the study 
communities. Community leaders were consulted prior to the start of the intervention on the best 
ways of achieving high compliance with vaccination, drug delivery and follow-up. 

 
 
15 
5 
TRIAL POPULATION 
After obtaining permission from the community leaders for the trial, a household census was 
conducted in February – March 2017 and all households within the study areas with children 5-17 
months of age on April 1st 2017 were enumerated. At the census, a preliminary screening of 
potentially eligible children was undertaken. Potentially eligible children and their caretakers were 
visited again and written informed consent was obtained from their caretakers for their inclusion in 
the trial before the administration of the first dose of study vaccines. Children entered into the trial 
were assigned a unique ID number and their demographic data (date of birth and/or age, and 
gender), use of insecticide treated nets (ITN) and history of receiving SMC during the last 
transmission season were collected. The census data was updated in April/May 2018 and 2019 prior 
to the administration of the booster doses of vaccine.  Eligible children were allocated randomly to 
one of the three study arms in permuted blocks of 12 using standard randomisation procedures 
(table). Children who had a history of receiving SMC in the previous years were distributed equally 
between the three study groups 
 
A child was eligible for inclusion in the trial if -   
a. 
The child was a permanent resident of the study area and likely to remain a resident for the 
duration of the trial. 
b. 
The child was 5 - 17 months of age at the time of first vaccination.   
c. 
A parent or legally recognised guardian provided informed consent for the child to join the 
trial.   
A child was ineligible for inclusion in the trial if -  
a. 
The child was a transient resident in the study area. 
b. 
The child was in care. 
c. 
The age of the child was outside the stipulated range.  
d. 
The child had a history of an adverse reaction to SP or AQ. 
e. 
The child had a serious underlying illness, including known HIV infection, unless this was well 
controlled by treatment, or severe malnutrition (weight for age or mid arm circumference Z 
scores < 3 SD). 
f. 
The child was known to have an immune deficiency disease or was receiving an 
immunosuppressive drug. 

 
 
16 
g. 
The child had previously received a malaria vaccine. 
h. 
The child was enrolled in another malaria intervention trial. 
i. 
The parents or guardians did not provide informed consent.  
 
Table. Study groups                                          
                                         Group1 (SMC alone)      Group2 (RTS,S alone)      Group3 (RTS,S+SMC) 
Year1: April-June                  Rabies vaccine x 3              RTSS/AS01 x 3                 RTSS/AS01 x 3 
            Aug-Nov                         SMC x 4                      SMC placebo x 4                    SMC x 4 
Year2: Aug-Nov                     HepA vaccine x 1            RTSS/AS01 x 1                    RTSS/AS01 x 1 
                                                      SMC x 4                   SMC placebo x 4                      SMC x 4 
Year3: Aug-Nov                   HepA vaccine  x 1            RTSS/AS01 x 1                      RTSS/AS01 x 1 
                                                    SMC x 4                      SMC placebo x 4                      SMC x 4 
SMC or placebo is given at monthly intervals on four occasions during the malaria transmission 
season. 
6 
SAMPLE SIZE 
Approximately 3,000 children have been recruited in Burkina Faso and a similar number in Mali 
(total 6,000) and these children will be followed for three years. A low dropout rate of around 5 % 
per year (15% overall) is anticipated based on findings from the previous SMC+AZ study, although it 
is possible that the dropout rate may be higher during a vaccine trial as a vaccine trial has not been 
conducted previously in the study districts.  Since the relative efficacy of SMC and RTS,S/AS01 is 
uncertain, the conservative approach of recruiting equal numbers of children into each arm of the 
study (2,000 per arm) has been followed. Results obtained over a period of three years of 
observation in the two study sites will be combined for the primary analysis of the incidence of 
clinical malaria. An analysis will also be undertaken of efficacy in each year of the trial to estimate  
the efficacy of administration of a booster dose.  It is not anticipated that a formal unblinded 
interim analysis will be undertaken although a preliminary analysis by an independent statistician 
will be undertaken early in 2020 to provide guidance as to whether the trial should be extended.  
 

 
 
17 
During the  SMC study in the  trial sites, the incidence of clinical episodes of malaria during the first 
year of the study (2014) was 442 and 382 per 1000 children in Mali and in Burkina Faso 
respectively. Complete morbidity data was not available for the second year of the study (2016). 
For the sample size calculations,  a conservative incidence rate of 300 cases per 1000 children over 
a calendar year was assumed. Using a two sided 95% confidence interval, a three-arm study which 
enrolled 2000 children in each arm (1000 per arm per centre) would have, for the non-inferiority 
comparisons, 90% power to exclude a relative difference in incidence between RTS,S/AS01 and SMC 
given alone of 16.1% over the three-year study period and of 28.6% or more for each individual 
year. The study would also have 80% power to exclude a relative difference in incidence of 13.9% 
over the whole study period and of 24.7% in each year. The differences that could be detected will 
be smaller if, as suspected, the incidence of clinical malaria is greater than 300 cases per 1,000 
children per year. For the superiority comparisons of the combined interventions with SMC alone, 
using a two-sided 95% confidence interval, the study would have 90% power to detect a difference 
greater than 11.1% over the three years of the study and of 19.2% in each year.  
 
For the analysis of the serological response to RTS,S/AS01, comparisons will be made between 
mean anti-CSP antibody titres pre and post the primary series of vaccination and before and after 
the two subsequent booster doses. Based on the standard deviations in antibody titres observed in 
children enrolled in the RTS,S/AS01 phase 2 and phase 3 trials, inclusion of around 160 individuals 
in each group (pre and post vaccination at each of the three time points) will give a study with 
approximately 80% power to detect a difference of 25% - 30% in mean titre between children who 
receive RTS,S/AS01 with or without co-administration of SMC.  
7 
THE INTERVENTIONS 
RTS,S/AS01 vaccine: Three doses of RTS,S/AS01 were given to children allocated to the RTSS or 
RTSS+SMC groups at  approximately monthly intervals (window 3-8 weeks) in April – June 2017 
before the SMC administration period followed by a fourth and fifth dose at the beginning of the 
2018 and 2019 malaria transmission seasons.  A decision was made by the trial’s steering 
committee not to use a fractional dose for the booster immunisation.   
 
The efficacy of RTS,S/AS01 against both severe and uncomplicated malaria in children vaccinated at 
the ages of 5 - 17 months and subsequently given a booster dose has been assessed by both the 

 
 
18 
EMA and WHO to outweigh the vaccines side effects. The vaccine causes 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 common following vaccination. Fever post vaccination 
occurs in about 10% of children and febrile convulsions occurred in about 1% of 5-17 month old 
recipients enrolled in the phase 3 trial [3] but no persistent neurological effects were recorded. The 
main safety issue related to administration of RTS,S/AS01 is the unexplained, statistically significant 
increase in meningitis observed in children given the vaccine at the age of 5-17 months during the 
phase 3 trial (about 4 per 1,000 during a four-year period of follow up);  this was not observed 
when the  vaccine was given at the age of 6-12 weeks.  
 
 Meningitis was caused by a variety of organisms and did not show any temporal relationship to 
vaccination and more than 40% of cases occurred at one centre in Malawi.   The EMA concluded 
that the increase in cases of meningitis was probably a chance finding and that the benefits of the 
vaccine exceeded any safety issues. However, the agency recommended further evaluation of the 
incidence of meningitis in vaccine recipients when RTS,S/AS01 was deployed and this will be done 
in this study. Any study children admitted to hospital with suspected meningitis are being 
investigated as fully as possible and CSF samples obtained for microbiological diagnosis by PCR.  In 
addition, there was a suggestion that the proportion of cases of severe malaria which were 
classified as cerebral malaria was increased in the RTS,S/AS01 recipients, although the incidence of 
severe malaria overall was reduced.  Therefore, the incidence of cases of severe malaria meeting 
the WHO definition is monitored carefully. 
 
RTS,S/AS01 malaria vaccine should not be administered to subjects with known hypersensitivity to 
any component of the vaccine, to a previous dose of RTS,S/AS01 E malaria vaccine or who is known 
to be hypersensitive to hepatitis B vaccine. 
 
Control vaccines: Rabies vaccine has been used as the control vaccine for the primary series of 
vaccinations during the dry season in year 1 (2017) for those allocated to the SMC group. The rabies 
vaccine used  was  a licensed, WHO approved vaccine RabipurR, previously produced by Novartis 
but now by GSK. This vaccine is produced in chick embryo cells and contains polygeline and residues 
of chicken proteins, and it may contain traces of neomycin, chlortetracycline and amphotericin and 
it is, therefore, contraindicated in subjects with a history of a severe hypersensitivity to any of the 

 
 
19 
ingredients in the vaccine. Minor local and systemic reactions are common (>1:100, <1.10)   after 
vaccination with rabies vaccine and neurological complications including Guillain Barré syndrome 
have been described but are very rare (<1:10.000). 
 
Hepatitis A vaccine (HAVRIXR), a licensed inactivated hepatitis A vaccine produced by GSK, is being 
used for the control booster dose in years 2 and 3. HAVRIXR is contraindicated in subjects who have 
had an allergic reaction to prior administration of the vaccine or who are sensitive to neomycin. 
This vaccine may also cause minor local and systemic reactions ((>1:100, <1.10). Severe reactions, 
including anaphylaxis have been described but are very rare (<1.10,000).   
 
Seasonal malaria chemoprevention: Four courses of SMC with SP+AQ will be given at monthly 
intervals during the malaria transmission season in line with WHO’s recommendation and national 
policy [8]. 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. Infants  receive half 
of these doses. SP and AQ and matching placebo have been obtained from Guilin Pharamceuticals, 
Shanghai, Co),  a GMP certified supplier. All treatments are given under observation.   
 
Approximately 5 million children have received SMC with SP + AQ during the 2015 rainy season 
across the Sahel and sub-Sahel (http://www.malariaconsortium.org/pages/access-smc.htm)  
 and this drug combination has been shown to be remarkably safe [10]. 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. The possibility that SMC with SP+AQ might induce resistance to these drugs in P. 
falciparum has been investigated in a number of trials of SMC. Selection of parasites carrying 
mutations which confer resistance to pyrimethamine or sulphadoxine has been demonstrated in 
some but not all studies [7]. However, because the prevalence of parasitaemia in children who 
received SMC was substantially less than in the control group, the total number of parasites 
carrying resistance markers was less in children who had received SMC than in control children. 
Extensive use of SP for intermittent preventive treatment in pregnant women has not accelerated 
resistance to SP in West Africa.   Preliminary analysis of samples obtained from children in an SMC 
trial in the study sites at the end of 2014 did not show levels of mutations in dhfr and dhps genes 

 
 
20 
likely to be associated with in vivo resistance to SP.   The potential risk of inducing resistance to SP 
or AQ by SMC was reviewed carefully by a WHO Technical Expert Group and WHO’s Malaria Policy 
Advisory Group and considered to be an acceptable risk in light of the major benefits conveyed by 
the intervention. Malaria parasites isolated at the end of the transmissions season will be tested for 
resistance markers to SP. 
8 
IMPLEMENTATION OF THE INTERVENTIONS 
Vaccination.  RTS,S/AS01 is provided in  a two-doses glass vial of lyophilized RTS,S antigen to be 
reconstituted with a two-dose glass vial of AS01 Adjuvant System. The final product for 
administration is prepared by reconstitution of the lyophilized antigen with the liquid adjuvant to 
deliver two doses (1.0 ml). A single dose consists of 0.5 ml of RTS,S/AS01 final preparation. All vials 
of vaccine provided in this study are intended for single use only. After reconstitution the vaccine 
was   administered by slow IM injection, using a fresh 25G needle with length of one inch (25 mm), 
in the left deltoid. Vaccine was injected within four hours of reconstitution (storage at +2°C to 
+8°C). Syringes containing RTS,S/AS01 or the control vaccine  were prepared by a pharmacist who 
took no other part in the trial. Loading of syringes with vaccines and masking with tape to blind the 
person administering the vaccine as to its nature was done by a person who took no further part in 
the trial.  Vaccines were administered by a nurse or other category of health worker trained to give 
vaccines. 
Rabies vaccine, 1.0 ml in volume was given by intramuscular injection. The paediatric dose of     
HAVRIX R is 0.5 ml and it was also administered by intramuscular injection. 
SMC. SMC drugs are 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 four cycles of 
treatment required for one full malaria transmission season appropriate for the child’s age. 
Treatment with each dose of SMC is given by trained, paid volunteers at a central point in each 
study community under observation. Study children have been given an 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.   

 
 
21 
                                                                                                                                                                                
All children were given an ITN at the commencement of the 2017 rainy season.  
9 
CONTRAINDICATIONS TO SUBSEQUENT VACCINATION 
Contraindications to administration of a further dose of vaccine in any child included –  
a. Anaphylaxis following administration of the first dose of the vaccine. 
b.  Any condition that in the judgment of the investigator would make intramuscular injection 
unsafe.  
c. The occurrence of a new adverse event (AE) or the exacerbation of an existing AE that, in 
the opinion of the investigator, exposed the subject to an unacceptable risk from 
subsequent vaccination.    
d. An acute disease and/or fever at the time of vaccination.  Fever is defined as temperature 
 37.5°C/99.5°F for oral, axillary or tympanic measurements, or  38.0°C/100.4°F for rectal 
temperature. Subjects whose fever resolved with treatment were vaccinated provided that 
revaccination fell within the stipulated time period. 
Subjects with a minor illness (such as mild diarrhea, mild upper respiratory infection) without fever 
were vaccinated. 
10 
FOLLOW-UP AND MEASUREMENT OF OUTCOMES 
The following follow-up procedures required to measure the study outcomes are being undertaken  
a. Passive surveillance for cases of 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 are responsible for 
identifying and documenting all cases of malaria who present to these health facilities. In 
Mali, cases of uncomplicated malaria may also be treated by community health workers 
who have been taught to diagnose malaria with a RDT and to treat RDT positive cases, and 
these cases are also being recorded if they meet the inclusion criteria. Cases of suspected 
malaria (fever, history of fever within 48 hours or any other symptom/sign suggestive of 
malaria) are tested with a RDT and managed on the basis of their RDT result and blood films 

 
 
22 
and filter paper strips  are obtained from all these cases for subsequent confirmation of the 
diagnosis.  
b. 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 has an RDT done and those who are positive are treated with a full 
course of an ACT. This activity to cease for the final 3 months of the study, April – June 2020 
due to the COVID-19 outbreak. 
c. Prevalence of malaria parasitaemia and anaemia. A survey of all study children is 
undertaken one month after the last round of SMC administration at the end each malaria 
transmission season. Temperature is measured and any child who is febrile or who has 
other features suggestive of a diagnosis of malaria has an RDT done; those who are positive 
are treated with a full course of an ACT. Finger prick blood samples are collected for 
preparing blood slides and blood spots on filter paper from all children. The prevalence of 
parasitaemia, including the presence of gametocytes, is detected by microscopy. 
d. Serious Adverse Events. Project staff based at the district hospitals are responsible for the 
identification of any child in the trial admitted to hospital and 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.  Definitions for meningitis and cerebral malaria, currently 
being developed by WHO for use in the pilot RTS,S/AS01 implementation trials are being 
used. 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 is done.  
Surveillance of all children will be maintained throughout the study period for any Serious 
Adverse Events (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 are thoroughly investigated 
and reported to GSK and to the DSMB within 72 hours of their detection. All SAEs, whether 

 
 
23 
considered related to the study interventions or not are tabulated in a blinded fashion and 
provided to the DSMB and to GSK at the times requested by the DSMB.   Details of 
definitions of adverse events and SAEs and of reporting procedures are described in 
appendix 1. 
e.    Immune response to the vaccine.  Blood samples (2ml)  have been collected from 
approximately 160 children in each of the groups (80 per country) who received 
RTS,S/AS01  prior to administration of  the first dose of vaccine and one month after third 
dose of the primary series of  vaccination, and then in year 2 and 3 before  and one month 
after administration of the fourth and fifth doses of vaccine for measurement of anti-CSP 
antibodies.  
f.    Drug resistance: Dried blood spots from children who have malaria parasitaemia detected 
by microscopy at each annual cross-sectional survey will be used for analysis of molecular 
markers of resistance to SP and AQ at MRTC, Bamako.  
g.    In vivo measurement of drug sensitivity to SP+AQ:  At the end of the 2019 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, an important factor in evaluation of the 
outcome of the trial.  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, from  children 
sequentially until the required number of RDT positive children to meet the sample size at 
each site has been recruited.   Blood smears will be examined the same day, or as soon as 
possible afterwards, for any child who has positive RDT test. Children who are parasitaemic 
but 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 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 (AL), the 
national first line treatment for uncomplicated malaria.  Infections after day 7 (late 
parasitological failures) will be investigated to determine if the recurrent infection was due 
to a reinfection or a recrudescence. DNA from dried blood spots will be used for molecular 
characterisation of the parasite using polymorphisms in the msp1, msp2 and CA1 
polymerase genes.    

 
 
24 
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 155.. We anticipate 
that about 7.5% of 3000 children will carry malaria parasite at end transmission season in 
2019, leading to 225 potentially eligible children per site.  
h.   Malaria endemicity in the study area and the trend in the distribution of molecular SP 
resistance markers: A survey of schoolchildren was conducted in Year 2 and  will be 
repeated in Year 3 of the trial to assess the malaria parasite prevalence at the end of the 
transmission and to monitor the trend in the distribution of molecular SP resistance 
markers. 200 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 parasitemia at the end of each malaria 
transmissions season and to determine the overall level of malaria transmission and (ii)  by  
PCR to assess the trend in the distribution of molecular markers of resistance to SP in the 
study area during the period of the trial. The sample size for this study is based on the 
following assumptions: (1) the prevalence of malaria parasite will be 50% and intra-cluster 
correlation coefficient (ICC) will be 0.1 (this is based on the observations from the surveys 
done in 2014, 2015 and 2016 as part of the recently completed seasonal malaria 
chemoprevention plus azithromycin trial); (2) the design effect will be 1.9 if the cluster size 
10 children; (3) refusal to take part in the survey will be <5%; (3) the acceptable 95% 
precision of the prevalence will be +/-10%.  
11 
LABORATORY PROCEDURES 
a. Detection of malaria. A histidine rich protein (HRP2) based Rapid Diagnostic Test (RDT) is be 
used for the initial diagnosis of malaria and to guide treatment. Blood films collected at the 
same time are read subsequently by two microscopists. All slides are read twice by two 
separate readers following the guidelines developed for the phase 3 RTS.S/AS01 trial [15].  
Slides which are judged to be discordant for either positivity or parasite density are read by 
a third reader.  For slides with high or medium density parasitaemia (> 400/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 

 
 
25 
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 applies. 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. 
b. Detection of markers of resistance to SP.  Parasite DNA is extracted from dried blood spots 
and nested PCR reactions are 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  [16-18]. 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.  
c. Measurement of haemoglobin concentration. Haemoglobin concentration is measured 
colorimetrically using a Hemocue colorimeter (Hemocue AB, Angelholm, Sweden). 
d. Measurement of anti-CSP concentration. Antibodies to CSP will be measured by a 
standardised ELISA at the University of Ghent in the laboratory of Professor Leroux-Roels as 
used in many previous trials of RTS,S/AS01. 
12 
SOCIO-ECONOMIC STUDIES 
Objectives. The objectives of the socio-economic component of the trial are to determine: 
a. The cost-effectiveness of a) RTS, S vaccine versus SMC and b) the combination of both 
versus SMC alone. 
b. The acceptability of the two interventions (separately and combined) to the health care 
deliverers and to the study communities. 
c. The feasibility of introducing two malaria control strategies simultaneously from the health 
system perspective. 
Economic Evaluation. Data on the costs of a clinical case of malaria and of a hospital admission with 
malaria to both the health care deliverers and recipients have already been collected in the two 
study areas during the course of the SMC+AZ trial and this information will be updated. The costs of 
adverse events, in particular of meningitis, will be estimated using published literature.  Information 
on the costs of delivery of SMC outside an intervention study has also been gathered by the SMC 

 
 
26 
ACCESS team and this will facilitate determination of the costs of this intervention.  The costs of 
adding RTS,S/AS01 to the routine vaccination programme will require specific study through 
observations, key stakeholder interviews and review of relevant documents  and files. The delivery 
costs will be estimated through a combination of a step-down and ingredients-approach costing 
methodology. A cost effectiveness analysis of the two interventions and their use in combination 
will incorporate the results from the trial, the costs collected during this study as well as during 
previous studies and then model the cost effectiveness of the interventions from a societal 
perspective in a decision tree model. The final outcomes used in the model will be costs/DALYs 
averted. The costs of scaling up the optimum intervention at regional/ national level will be 
investigated. 
Acceptability. The acceptability of the two interventions to the health care deliverers and the 
preference of study communities for each of the interventions will be investigated through focus 
group discussions involving both the families of trial participants and those involved in 
administration of the trial interventions.  
Feasibility. Investigating the feasibility of delivery of RTS,S/AS01 outside the routine EPI programme 
will be a major objective of the large pilot studies being planned by WHO. Experience gained on the 
logistic challenges posed to the national immunisation programme of delivering RTS,S/AS01 as a 
seasonal vaccine will be shared with both the national immunisation programmes in Burkina Faso 
and Mali and also with WHO.   
13 
DATA MANAGEMENT  
To ensure data is fit for purpose, questionnaires were tested prior to use and staff  received training 
prior to and at regular intervals during data collection. Data are managed using the DataFax system 
which was set up for the SMC + AZ trial and which is working well. This system is based on 
electronic transfer of the CRFs from the research sites where the data are automatically captured 
and validated. The MRTC data management team are responsible for the training and support of 
the IRSS data management team, with overall support from the NIAID/NIH central data 
management team.  Data is uploaded to the DataFax system at the earliest opportunity to enable 
queries to be validated and issues resolved as soon as possible after data collection. Automatic 
checks are performed on clinical and laboratory forms to ensure they are complete and contain 
valid responses prior to uploading by the local data managers at both sites. 
 

 
 
27 
An experienced independent GCP monitor (Raouf Osseni) monitors the trial to ensure the quality of 
the data collected and that GCP standards are met. The monitor  conducted a trial initiation visit,  and 
at least one additional visit each year. He will also conduct a 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 
STUDY OUTCOMES 
Primary outcome: The primary outcome measure of the trial is the incidence of clinical 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 outcomes: Secondary outcomes include: - 
a. Blood slide or rapid diagnostic test (RDT) positive malaria defined as a clinical episode 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 parasitemia) or a positive RDT. 
b. Hospital admissions with malaria, including cases of severe malaria which meet WHO 
criteria for a diagnosis of severe malaria.  
c. The prevalence of malaria infection (symptomatic or asymptomatic parasitaemia) 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 the malaria transmission season.  
e. Serious adverse events (SAEs), including any deaths, occurring at any time during the study 
with special reference to any cases of meningitis, cerebral malaria or immune deficiency 
illness. 
f. Anti-CSP concentrations obtained after priming and after each booster dose, determined in 
a sub-sample of children. 
g. Comparison of the anti-CSP concentrations among children who received co-administration 
of SMC and RTS,S/AS01 versus those who received RTS,S/AS01 alone.   

 
 
28 
h. The presence of molecular markers of resistance to SP and AQ in parasite positive samples 
collected at  the final cross-sectional survey. 
i. The 28-day treatment response of subjects with asymptomatic P. falciparum malaria to 
treatment with SP+AQ. 
Particular attention will be paid to the occurrence of any cases of meningitis as this was identified 
as a potential safety signal in the phase 3 RTS,S/AS01 trial. The trial will not be large enough to 
measure an impact on mortality but all deaths will be recorded and investigated, including by 
verbal autopsy if these occur at home. Safety data are provided to GSK in the format requested by 
the company. Evaluation of the clinical and immunological response to a fifth dose of RTS,S/AS01 is 
highlighted as an important research objective by the WHO.  
15 
ANALYSIS  
The primary endpoint of incidence of all episodes of clinical malaria over the study period will be 
analysed using Cox regression models with a robust standard error to account for clustering of 
episodes within individuals (i.e. the Andersen-Gill extension of the Cox model). This will estimate 
the total effect of the interventions. The proportion of children in each group who experience one 
episode of malaria will be compared as a secondary outcome using Kaplan-Meier estimates: 
evidence for provision of complete protection through the combination of SMC and vaccination 
each year will be explored using recently published methods [19, 20]. For the comparison of 
RTS,S/AS01 plus SMC to the other interventions, a standard superiority comparison will be 
performed, calculating two-sided 95% confidence intervals for the hazard ratio. For the non-
inferiority comparison of RTS,S/AS01 to SMC, the two-sided 95% confidence interval for the hazard 
ratio will be compared to a pre-specified non-inferiority margin. The trial is powered to have more 
than 80% power to exclude a difference of 15% in the incidence of clinical malaria over the study 
period, a difference considered to be clinically important. Demonstration of a smaller difference 
would require a substantially larger trial and is considered to be not clinically relevant.  If the lower 
limit of the 95% does not overlap zero (i.e. it is clear that RTS,S is superior to SMC) then a 
superiority comparison will be performed as for the combined intervention. 
 
A formal analysis plan will be prepared and approved by the Data Safety Monitoring Board (DSMB) 
appointed for the trial before the study code is broken. Both intention to treat and per protocol 
analyses will be undertaken. Children who received any dose of SMC or vaccine will be included in 

 
 
29 
the intention to treat analysis. Children who received all scheduled doses of SMC, or 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 but the study is only powered to meet its primary 
and major secondary end-points if results from both countries are combined. Additional sub-
analyses will include analysis by age, gender, bed net use during the transmission season, as 
determined by the history obtained at the cross-sectional surveys, and socio-economic status as 
determined by the educational level and occupation of the child’s family. 
 
A formal interim analysis will not be undertaken.  However, a preliminary analysis of the key trial 
clinical trial end-points will be undertaken by an independent statistician early in 2020 in order to 
decide whether the trial should be continued until children reach the age of five years, the age at 
which SMC is no longer given.   
16 
ETHICS    
Inclusion in the trial of an RTS,S/AS01 alone group even though SMC is recommended policy is 
justified on the grounds that RTS,S/AS01 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 has been obtained from the family or legally recognised 
guardian of each child entered into the trial.  Ethical approval has been 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.   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 the district hospitals in the study areas. 
17 
TRIAL MANAGEMENT 
The London School of Hygiene & Tropical Medicine  acts as the main sponsor for the trial. Delegated 
responsibilities may be assigned locally.  The London School of Hygiene & Tropical Medicine holds 
Public Liability (‘’negligent harm’’) and Clinical Trial (non-negligent harm’’) insurance policies which 

 
 
30 
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. 
 
An independent trial steering committee provides scientific oversight and holds regular meetings 
either through  teleconferencing or face-face meetings .  
 
A DSMB  oversees the safety of subjects in the trial and a clinical trial monitor  ensures that the trial 
is conducted to GCP standards.  In order to ensure that children in the RTS,S/AS01 group are not 
put at any increased risk compared to children who receive SMC, the DSMB will monitor regularly 
the incidence of  clinical cases of malaria in the three study groups. The  DSMB has met on several 
occasions during the trial and at its meeting held in February2019 members of the board reviewed 
unblinded data on the occurrence of deaths, deaths due to malaria, hospital admissions overall and 
hospital admissions due to malaria in the three study groups and gave their permission for the trial 
to continue in 2019 retaining all three study groups.  Membership of the trial steering committee 
and the DSMB is shown in appendix 2. 
The trial management committee  includes the LSHTM PIs, trial site PIs and the trial administrator.  
The trial management committee is responsible for overseeing the trial and its members 
communicate regularly by teleconferences.  
 
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 trial has 
been registered on clinicaltrials.gov (NCT03143218).  
18 
ROLES OF THE INVESTIGATORS AND COLLABORATORS 
The team from the IRSS, Burkina Faso which includes Jean Bosco Ouédraogo, Halidou Tinto and 
Issaka Zongo are 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 will be 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, Irene Kuepfer, Matthew Cairns, Paul Milligan, Karen Slater) provides 
epidemiological, statistical, administrative and financial management support. Silke Fernandes and 

 
 
31 
Kara Hanson from the LSHTM are responsible for the economic aspects of the study. A consultant 
may be recruited to assist the design of the acceptability and feasibility studies. 
19 
DISSEMINATION PLANS 
Results from the trial will be presented at national and international conferences and in peer 
reviewed journals 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 in connection with the implementation of SMC and these 
links will facilitate the incorporation of RTS,S/AS01 vaccine into SMC/EPI programme  if this is found 
to be a useful intervention. The study team has established good links with many other 
organisations involved in the delivery of SMC trials, including the SMC ACCESS programme 
coordinated by the Malaria Consortium and with the WHO staff responsible for conducting the 
RTS,S/AS01 implementation studies Thus, if it is found that RTS,S/AS01 vaccine is a useful 
replacement or addition to SMC regimens, routes have already been established through which this 
knowledge could be disseminated rapidly.  
 
 
 

 
 
32 
20 
REFERENCES 
[1]  Cohen J, Nussenzweig V, Nussenzweig R et al.  From the circumsporozoite protein to the 
RTS,S/AS candidate vaccine. Human Vaccin 2010; 6:90-6.  
[2]  White MT, Verity R, Griffin JT  et al. Immunogenicity of the RTS,S/AS01 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/AS01 malaria vaccine with or 
without a booster dose in infants and children in Africa: final results of a phase 3, individually 
randomised, controlled trial. Lancet 2015; 386: 31-45.  
 [4]  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;358:1927-34.  
[5]  http:www.who. int/mediacentre/news/release/2015/sage/en  
[6]  Regules JA, Cicatelli SB, Bennett JW et al. Fractional third and fourth dose of RTS,S/AS01 malaria 
candidate vaccine: a phase 2a controlled human malaria parasite infection and immunogenicity 
study.  J Infect Dis 2016; 214:762-771. 
[7]  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.  
[8]  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.  
[9]  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. 
[10] 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, in press.   
[11] 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.  
[12] Penny MA, Verity R, Bever CA et al. The public health impact and   cost-effectiveness of malaria 
vaccine candidate RTS,S/AS01: a systematic comparison of predictions from four mathematical 
models. Lancet 2016; 387: 367-375. 
[13] World Health Organization. Weekly Epidemiol Rec 2015; 90:681-700.  
[14} Chandramohan, D  Dicko A, Zongo I, et al. Effect of adding azithromycin to seasonal malaria 
chemoprevention. N Engl J Med 2019; 380:2197-2206 

 
 
33 
[15] 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/AS01 candidate malaria vaccine. Mal J 2011; 
10: e223.    
                                                                                           
[16] Plowe CV, Djimde A, Bouare M et al. Pyrimethamine and proguanil reistance-conferring 
mutations in plasmodium falciparum dihydrofolate reductase: polymerase chain reaction 
methods for surveillance in Africa. Am J Trop Med Hyg 1995; 52:565-568.   
[17] Djimde A, Doumbo OK, Cortese JF et al. A molecular marker for chloroquine-resistant 
falciparum malaria   N Engl J Med 2001; 344: 257-63. 
[18] 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.      
[19] Cairns M, Cheung YB, Xu Y et al.  Analysis of preventive interventions for malaria: exploring 
partial and complete protection, and total and direct intervention effects.  AmerJ Epidemiol 
2015; 181:1008-17. 
[20] Xu Y, Cheung YB, Lam KF et al.  Estimation of summary protective efficacy using a frailty 
mixture model for recurrent event time data. Stat Med 2012; 31: 4023-39. 
 
 

 
 
34 
21 
ACTIVITIES SCHEDULE 
 
Activities 
2016 
2017 
2018 
2019 
2020 
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 J 
F 
M A M J 
J 
A S 
O N D 
J-
M 
A-
J 
Protocol submission for 
ethics approval 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Ethics and regulatory 
approval 
 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Staff recruitment  
 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Trial registration 
 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Order study drugs 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Vaccine shipment 
 
 
 
 
 
 
x 
x 
x 
 
 
 
 
 
 
 
 
 
 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
x 
 
 
 
 
 
 
 
 
Steering committee meeting 
x 
 
 
 
 
x 
 
 
 
 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
x 
 
 
 
x 
 
DSMB meeting 
 
 
 
 
 
x 
 
 
 
 
x 
 
x 
 
 
 
 
 
x 
 
 
 
 
 
 
 
 
x 
 
 
 
 
 
 
x 
 
 
 
 
x 
x 
 
Census and consenting of 
children 
 
 
 
 
 
 
x 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Randomisation  
 
 
 
 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Printing IDcards, labels 
(vaccine & drugs) 
 
 
 
 
 
 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
x 
 
 
 
 
 
 
 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
Vaccination 
 
 
 
 
 
 
 
x 
x 
x 
 
 
 
 
 
 
 
 
 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
x 
 
 
 
 
 
 
 
 
Administration of SMC 
 
 
 
 
 
 
 
 
 
 
 
x 
x 
x 
x 
 
 
 
 
 
 
 
 
x 
x 
x 
x 
 
 
 
 
 
 
 
 
x 
x 
x 
x 
 
 
 
Health facility based 
surveillance of  malaria 
morbidity and mortality 
 
 
 
 
 
 
 
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 
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 
 
 
End of transmission surveys 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
x 
 
 
Repeat household census 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
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 
x 
x 
x 
 
PCR assays 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
x 
x 
 
Serology 
 
 
 
 
 
 
 
x 
 
 
x 
 
 
 
 
 
 
 
 
 
 
x 
x 
 
 
 
 
 
 
 
 
 
 
x 
x 
 
 
 
 
 
 
 
GCP monitoring 
 
 
 
 
x 
 
 
 
 
 
 
x 
 
 
 
 
 
 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
x 
 
 

 
 
35 
 
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 
x 
x 
x 
x 
 
Blinded analysis & report for 
committees 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
Locking of data 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
x 
 
Data analysis 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
xx 
xx 
Dissemination  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  x 

 
 
36 
22 
APPENDICES 
 
Appendix 1: Definitions of adverse and serious adverse events and reporting schedule 
a. Definition of an adverse event and serious adverse event 
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 serious adverse event (SAE) is any clinical condition that fulfils at least one of the following 
criteria: 
a. 
results in death, 
b. 
results in admission to hospital, 
c. 
is life-threatening (the child was at risk of death at the time of the adverse event), 
d. 
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: Patients 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.   

 
 
37 
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: 
 
Completely recovered : 
The child has fully recovered with no observable 
residual effects 
 
Not yet completely 
recovered : 
The child’s condition has improved, but still has 
some residual effects 
 
Deterioration : 
The child’s overall condition has worsened 
 
Permanent damage : 
The AE has resulted in a permanent impairment 
 
Death : 
The child died due to the AE 
 
Ongoing : 
The AE remains the same as at onset 
 
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 and provided to the DSMB and GSK as 
requested. Any SAE potentially related to the vaccine or drug administration will be reported to the 
DSMB and institutional Ethics Committees within 72 hours and GSK within 96 hours.  

 
 
38 
Appendix 2.  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) 
Morven Roberts  MRC Head Office, London, (donor representative) 
Feiko ter Kuile, LSTM, Liverpool, UK (independent member) 
Mahamdou Thera (MRTC, Bamako, Mali (independent member) 
 
Data, Safety and Monitoring Board 
Members of the Data Safety and Monitoring Board are -        
Sheick Oumar Coulibaly, University de Ouagadougou, Burkina Faso and WHO, Brazzaville. 
Umberto D’Alessandro, MRC Unit, The Gambia. 
Blaise Genton, Swiss Tropical and Public Health Institute, Basle, Switzerland (chair). 
Francesca Little, University of Capetown, Capetown, South Africa. 
Malcolm Molyneux, MLW Research Programme, College of Medicine, Blantyre, Malawi. 

RTS,S + SMC – Protocol amendments 
 
Original Protocol dated December 2016 was approved by the LSHTM Ethics Committee on 5 January 
2017. 
 
Protocol Amendment 1 – May 2017 
The following amendments to the protocol were approved by the LSHTM Ethics committee on 21 
July 2017: 
1. to include an interim, blinded analysis of the incidence of malaria in the three trial groups to be 
undertaken at the middle of the 2017 malaria transmission season as well as at the end to be 
reviewed by the DSMB; 
2. to change the starting date for primary vaccination from February to April 
3. to allow a window of 3-8 weeks between doses as opposed to the previous ‘approximately one 
month’ between doses  
4. clarification that episodes of malaria that occur at least two weeks after the third dose of vaccine 
will contribute to the trial end-point. 
 
Protocol Amendment 2 - December 2017 
The following amendment to the protocol was approved by the LSTHM Ethics committee on 1 
February 2018: 
To continue the active surveillance during the dry season throughout the study period. 
 
Protocol Amendment 3 – September 2018 
The following amendment to the protocol was approved by the LSTHM Ethics committee on 26 
October 2018: 
To include a survey of schoolchildren aged 6-12 years to assess the malaria parasite prevalence at 
the end of the transmission season in Year 2 and Year 3 of the trial. 
 
Protocol Amendment 4 – August 2019 
The following amendments to the protocol was approved by the LSTHM Ethics committee on 18 
October 2019 
1. to include of a sub-study to measure the in vivo efficacy of the SMC drugs used for SMC 
2. to include a preliminary analysis in Jan Feb 2020 at the request of the donors. 
3. the decision to consider a fractionated dose was removed from the protocol 
4. sub study to detect vaccine escape mutants dropped due to lack of funding. 

Protocol amendment 5 - April 2020 (final protocol) 
The following amendment to the protocol approved by the LSHTM Ethics Committee 7 April 2020 
a) cessation of the home visits required for active detection of malaria infection during the 
remaining three months of the first phase of the study (April – June 2020) due to the COVID-19 
outbreak. 
 

Statistical Analysis Plan 
 
RTS,S-SMC Study 
2 
 
 
 
 
RTS,S-SMC:  
  
A comparative trial of  
seasonal vaccination with the RTS,S/AS01 malaria vaccine,  
seasonal malaria chemoprevention,  
and of the two interventions combined 
 
 
Revisions to the Statistical Analysis Plan: 
  
Revision 
Number 
Date of 
Revision 
Page 
Number(s) 
Details 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Statistical Analysis Plan 
 
RTS,S-SMC Study 
2 
 
 
Study title 
A comparative trial of seasonal vaccination with the malaria vaccine RTS,S/AS01, seasonal 
malaria chemoprevention, and of the two interventions combined. 
 
Clinical trial registration 
Clinicaltrials.gov Identifier: NCT03143218 
 
Study duration 
 
April 2017 – March 2020 
 
Study site 
The trial is being conducted in Houndé district, Burkina Faso and in Bougouni district, Mali, sites 
of a previous trial (NCT02211729), which evaluated the effectiveness of adding azithromycin to 
the drug combination used for seasonal malaria chemoprevention.   
 
Study objectives 
This trial seeks to determine  
 
1. Whether seasonal vaccination with RTS,S/AS01 is 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 and SMC with SP+AQ) is superior to RTS,S/AS01 alone or SMC alone in 
preventing clinical malaria and other adverse outcomes. 
 
These aspects are of equal priority. 
 
 
Study groups 
This is a double-blind, individually-randomised trial with three study arms.   
 
The study groups, and abbreviated names used for ease of reference, are shown in the table 
below.  
  
 
Group 
 
 
1) SMC  
‘SMC alone’ 
 
2) RTS,S/AS01  
‘RTS,S alone’ 
 
3) RTS,S/AS01+SMC 
‘Combined group’ 
April/May – June/July 2017 
Rabies vaccine x 3 
RTS,S/AS01 x 3 
RTS,S/AS01 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/AS01 x 1 
RTS,S/AS01 x 1 
July-Oct 2018 
SMC x 4 
SMC placebo x 4 
SMC x 4 
 
 
 
 
June 2019 
Hep A vaccine x 1    RTS,S/AS01 x 1 
RTS,S/AS01 x 1 
July-Oct 2019 
SMC x 4 
SMC placebo x 4 
SMC x 4 
 
 
 
 

Statistical Analysis Plan 
 
RTS,S-SMC Study 
2 
 
Group 1 – SMC arm - ‘SMC alone’   
In 2017, children in the SMC group were given three doses of rabies vaccine 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.  
  
Group 2 – RTSS/ AS01 arm – ‘RTS,S alone’ 
In 2017, children in the RTS,S/AS01 group were given three doses of RTSS/AS01 vaccine and 
four rounds of placebo-SMC at monthly intervals.   In 2018 and 2019, children received one 
dose of RTS,S/AS01 vaccine and four rounds of placebo-SMC at monthly intervals.  
  
Group 3 – RTS,S/AS01+SMC arm – ‘Combined arm’  
 
In 2017, children in the combined group were given three doses of RTS,S/AS01 vaccine and four 
rounds of SMC with SP+AQ at monthly intervals.   In 2018 and 2019, children received one dose 
of RTS,S/AS01 vaccine and four rounds of SMC with SP+AQ at monthly intervals. 
 
 
Schematic of Key Study Activities  
 
 
 
 
 
Precise timing of interventions 
Administration of RTS,S/AS01 or control vaccine:  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/AS01 or control vaccine were given in the first two weeks of June.   
 
Placebo
Placebo 
Placebo
SMC
SMC
SMC
SMC
SMC
SMC
2017
2018
2019
Transmission season
Control vaccinations
RTS,S Vaccine
Booster 1
Booster 2
RTS,S Vaccine
Census
~6000 children
Booster 1
Booster 2
Randomisation
2000 per group
End of study
Dry season 2020

Statistical Analysis Plan 
 
RTS,S-SMC Study 
2 
 
Administration of SMC or placebo SMC:  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. 
 
 
Study population 
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. 
 
 
Sample size and Study Design 
Based on the calculations described below, the trial aimed to recruit approximately 3,000 
children in Burkina Faso and 3,000 in Mali (total 6,000) who would be followed for three years. A 
low dropout rate of around 5% per year (15% overall) was anticipated based on findings from 
the previous trial conducted in the same study areas.   
 
In Burkina Faso and Mali, 2777 and 3143 children, respectively, received the first dose of study 
vaccine.  
 
Superiority comparison: The trial was designed to compare the two interventions combined 
with either used alone.  The study is powered to i) assess the statistical evidence against the null 
hypothesis of no difference between the combined group and either the SMC alone or RTS,S 
alone group,  and ii) estimate the efficacy of the combined group relative to the single 
intervention groups with a relatively high degree of precision. This latter aspect is important 
because if the combined intervention is to be used in practice, it is necessary to show that 
adding RTS,S/AS01 to SMC has a clinically significant benefit. 
 
After two years of follow-up, incidence of clinical malaria, as defined below, was approximately 
200 cases per 1000 child-years at risk. With approximately 2000 individuals in each arm, if the 

Statistical Analysis Plan 
 
RTS,S-SMC Study 
2 
 
incidence is at least 30% lower in the combined group, there is very high power over the three 
years of the study (close to 100%) to reject the null hypothesis of no difference between the 
combined group and the single treatment groups.  If efficacy of the combined group relative to 
either of the other groups is at least 30%, there will be 90% power for the lower limit of the 95% 
confidence interval to exclude 15%, i.e. to address point ii) above, establishing that the 
protection from the combined group is at least 15% better than SMC or vaccination alone.  
 
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 
potential advantages of RTS,S over SMC in terms of ease of delivery and the potential to sustain 
high levels of coverage. A reduction in annual efficacy from 50% to 40% was considered the 
largest difference that would be likely to be acceptable. This translates to a 20% greater 
incidence in the RTS,S/AS01-alone arm compared to the SMC-alone arm. With the anticipated 
incidence rates, there is adequate power to reject a smaller margin, as detailed in the protocol. 
However, 20% was considered the largest difference that would be considered unimportant.   
 
The study has 80% power to exclude, at the 2.5% significance level, a relative difference in the 
incidence of clinical malaria of 20% over the three year study period between the RTS,S/AS01 
and SMC alone groups, if these two interventions were equally effective.  Evidence of the 
effectiveness of the reference group – SMC alone - in these areas come from clinical trials in 
Mali and Burkina Faso in 2009 (1, 2), and more recently in case-control studies conducted in 
both countries in 2016.  In the previous AZ-SMC trial in these two districts (3), the prevalence of 
molecular markers of resistance to SP and AQ were very low, and the curative efficacy of SP+AQ 
over 28 days was above 95% in both areas (Cairns et al., submitted).   
 
Hypothesis testing 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.  
 
For the non-inferiority comparison 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. 
 
 
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 weekly and 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 

Statistical Analysis Plan 
 
RTS,S-SMC Study 
2 
 
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 burned to CD and a copy sent to the chair of the DSMB for 
archiving. 
 
The randomisation codes will only be released 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. 
 
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 temperature ≥ 
37.5°C, or a history of fever within the past 48 hours), and a positive blood film, with a P. 
falciparum parasite density of 5,000 per µl or more.  This cut-off has been used in previous 
studies of SMC in Burkina Faso and Mali, as well as 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.  However, 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 the 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). 
 
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 

Statistical Analysis Plan 
 
RTS,S-SMC Study 
2 
 
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 surgery 
 
2.2.3  Hospital admissions due to malaria.  Defined as hospital admissions where malaria 
was the primary diagnosis, supported by a positive blood smear, or positive RDT if no blood 
smear result was 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 
 
2.2.5 
Deaths due to any cause  
 
2.2.6 
Deaths due to any cause excluding external causes and surgery 
 
2.2.7 
Deaths due to malaria.  Defined as hospital admissions resulting in death, where 
malaria was recorded as the primary cause of death, and where parasitology results did not 
exclude malaria (i.e. a positive blood smear, if a slide result was available, or a positive RDT if no 
slide was done).  Deaths in the community will also be included when malaria is assigned as the 
primary cause of death by verbal autopsy. 
 
2.3 
Outcomes measured at weekly surveys 
A subset of children were selected to be sampled for parasitaemia during weekly surveys. 
Systematic random sampling, from lists sorted on age, was used to allocate children to be 
sampled in not more than one week per year, such that the sample of 24 children in each week, 

Statistical Analysis Plan 
 
RTS,S-SMC Study 
2 
 
in each country was balanced with respect to age and treatment group.  Results from the 
weekly surveys will be analysed and presented separately for each malaria transmission season 
(2017, 2018 and 2019). 
 
2.3.1 
The prevalence of asexual stage P. falciparum infection of any density detected during 
the weekly home visits.  
2.3.2 
The prevalence of asexual stage P. falciparum infection with a density ≥ 5000 per ul 
detected during the weekly home visits. 
2.3.3 
The arithmetic mean P. falciparum parasite density, including samples which are parasite 
negatives as having a density of zero. 
 
2.4 
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 each 
malaria transmission season (2017, 2018 and 2019). 
  
2.4.1 
The prevalence of asexual stage P. falciparum infection of any density 
2.4.2 
The prevalence of asexual stage P. falciparum infection with density ≥ 5000 per ul  
2.4.3  The prevalence of sexual stage P. falciparum infection (i.e. gametocytes) 
2.4.4 
The arithmetic mean P. falciparum parasite density, including samples which are parasite 
negatives as having a density of zero.  
2.4.5 
The prevalence of asexual stage infection of non-falciparum Plasmodium species. 
2.4.6 
The prevalence of sexual stage infection (i.e. gametocytes) of non-falciparum 
Plasmodium species. 
2.4.7 
The mean haemoglobin concentration in g/dL. 
2.4.8  The prevalence of anaemia, defined as measured Hb < 10 g/dL.   
2.4.9 
The prevalence of moderate anaemia, defined as measured Hb < 7 g/dL.   
2.4.10 The prevalence of severe anaemia, defined as measured Hb < 5 g/dL.  
 
2.5 
Other secondary outcomes 
2.5.1 
The prevalence of molecular markers of resistance to SP and AQ in children with P. 
falciparum  infection, among samples collected at the final cross-sectional survey in December 
2019.  These 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.     
2.5.2 
The percentage of children with asymptomatic P. falciparum parasitaemia at the cross-
sectional survey in December 2019, who, when treated with SP+AQ, have an adequate clinical 
and parasitological response (ACPR) after 28 days.  
 
 
 

Statistical Analysis Plan 
 
RTS,S-SMC Study 
2 
 
 
Serious Adverse Events 
In addition to the comparison of incidence rates described above (section 2.2), 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.   
 
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 up to the 30th 
November 2019.  A 95% confidence interval for the incidence rate of meningitis among children 
vaccinated with RTS,S/AS01 will be calculated. 
The incidence of cerebral malaria among children vaccinated with RTS,S/AS01 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. 
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. The subset of febrile convulsions that occurred within 7 days of vaccination 
will also be analysed.  
An exploratory analysis will investigate if there is any evidence that RTS,S/AS01 increases 
mortality in girls.  This will compare the incidence of deaths using Cox regression, with an 
interaction between a dummy variable indicating receipt of RTS,S/AS01 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 female: male mortality ratio and its 95% confidence interval to be calculated separately for 
RTS,S/AS01 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.  
 
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.  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 
3.3 
The arithmetic mean P. falciparum parasite density, including samples which are parasite 
negatives as having a density of zero. 
 
Analysis populations and person-years at risk 
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/AS01 or control 

Statistical Analysis Plan 
 
RTS,S-SMC Study 
2 
 
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 
1) the date observation formally ended (31st March, 2020), 2) the date last seen if lost-to-follow-
up (LTFU), 3) the date of permanent exit from the study area, or 4) 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).   
 
As a secondary analysis, the primary outcome will also be analysed per protocol (PP).  The PP 
population will be defined separately for each year of the study.  Children who were seen at all 
scheduled vaccination contacts in a particular year (3 in 2017, 1 in 2018, 1 in 2019) 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 study drug 
administration but who did not receive SMC because they had malaria and were referred for 
treatment will be included in the per protocol analysis.   
 
All secondary outcomes will be analysed by modified intention to treat as described above.   
 
Trial profile 
The trial profile will show 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.  
Reasons that children seen at the census were not eligible to join the study will be tabulated.    
 
The profile will also show, 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.  Reasons that children did not attend the first study contact for 
vaccination will be tabulated.  
 
The number of children who exited the study population by the end of the first, second and 
third year of the study will be 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 will be produced for each of the two trial centres. 
 

Statistical Analysis Plan 
 
RTS,S-SMC Study 
2 
 
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/AS01 alone, and the combined group.  
Comparisons will also be made between RTS,S/AS01 alone and the combined group. 
 
Primary endpoint  
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, starting 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. 
 
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. 
 
As recommended in the updated CONSORT guidelines (8), the incidence rate differences (IRD) 
will also be calculated, as this gives an indication of the reduction in incidence attributable to 
the interventions, i.e. the absolute public health impact in similar contexts.  The IRD will be 
calculated using ordinary least squares regression of transformed variables, as described by Xu 
et al. (9). This method uses a robust standard error and controls for unequal follow-up time, as 
well as 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.   
 
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.   
 

Statistical Analysis Plan 
 
RTS,S-SMC Study 
2 
 
Covariates 
All analyses (primary outcomes and secondary outcomes) will adjust for 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 
 
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.   
 
Investigation of differences between study groups in successive years of the study is also pre-
specified, because it is possible that the efficacy of RTS,S/AS01 booster doses is different to the 
primary series.  This will be assessed by exploring evidence for an interaction between 
intervention group and study year (2017-18, 2018-19, 2019-20). 
  
Finally, 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.  Of 
particular interest are participants who were young infants at the time of the first vaccination in 
2017, as they may have had no exposure to malaria prior to enrolment in the trial. 
 
 
Pre-specified Secondary analyses  
As this is the first trial of seasonal vaccination, a number of secondary analyses are planned to 
investigate the effect of malaria event history, completeness of protection and protection over 
time. Lexis expansion will be used to stratify person-time since vaccination. This will enable 
regression splines to be fitted to obtain smooth estimates of protection over time from 
RTS,S/AS01.  
 
Further analyses will explore the changing relative benefits of SMC and RTS,S/AS01 with age 
and transmission intensity (by comparing efficacy profiles with age between Burkina Faso, which 
has higher incidence rates, with Mali).   
 
 
 
 

Statistical Analysis Plan 
 
RTS,S-SMC Study 
2 
 
 
Ancillary studies to be reported separately 
1.  Evaluation of anti-CSP antibody concentrations obtained before and after priming and after 
each booster dose, determined in a sub-sample of children, and the relation of antibody 
concentration following vaccination to the subsequent risk of malaria.    
2.  The effect of the intervention on nutritional status at the end of season cross-sectional 
surveys. 
 
Analysis of the preference of participants for an injection of vaccine or for multiple rounds of 
SMC was scheduled to take place during the last few months of this phase of the study. 
However, this has had to be postponed because of travel restrictions imposed by the COVID-19 
crisis.  
 
On the advice of the steering committee and one of the trial funders (PATH) the economic 
analysis of the two approaches to malaria control will be undertaken in the second year of an 
extension study. The extension study will observe children up to the age of five years when they 
will no longer be eligible to receive SMC, rather than stopping follow-up after three years, as 
had been proposed initially.     
 
 
 
 

Statistical Analysis Plan 
 
RTS,S-SMC Study 
2 
 
Planned main tables for published report  
 
Table 1: Incidence of the primary outcome: 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 
 
RTS,S-SMC Study 
2 
 
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 1 
 
 
 
 
 
 
 
 
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) 
- 
- 
- 
 
 
 
 
 
 
 
 
 
 
Study Year 2 
 
 
 
 
 
 
 
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) 
- 
- 
- 
 
 
 
 
 
 
 
 
 
 
Study Year 3 
 
 
 
 
 
 
 
 
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.   
Similar tables will be used to report the incidence of passively detected secondary outcomes. 
 

Statistical Analysis Plan 
 
RTS,S-SMC Study 
2 
 
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 - 2017 
 
 
 
 
 
 
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 - 2018 
 
 
 
 
 
 
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 - 2019 
 
 
 
 
 
 
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 Study 
2 
 
Figure 1.  This will show i) cumulative hazards of malaria, by treatment group and ii) the risk of malaria, by treatment group.

Statistical Analysis Plan 
 
RTS,S-SMC Study 
2 
 
 
References 
 
1. 
Konate AT, Yaro JB, Ouedraogo AZ, Diarra A, Gansane A, Soulama I, et al. Intermittent reventive 
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(2):e1000408. 
2. 
Dicko A, Diallo AI, Tembine I, Dicko Y, Dara N, Sidibe Y, 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(2):e1000407. 
3. 
Chandramohan D, Dicko A, Zongo I, Sagara I, Cairns M, Kuepfer I, et al. Effect of Adding 
Azithromycin to Seasonal Malaria Chemoprevention. N Engl J Med. 2019. 
4. 
Piaggio G, Elbourne DR, Altman DG, Pocock SJ, Evans SJ. Reporting of noninferiority and 
equivalence randomized trials: an extension of the CONSORT statement. Jama. 2006;295(10):1152-
60. 
5. 
RTS S Clinical Trials Partnership. First results of phase 3 trial of RTS,S/AS01 malaria vaccine in 
African children. N Engl J Med. 2011;365(20):1863-75. 
6. 
RTS S Clinical Trials Partnership. A phase 3 trial of RTS,S/AS01 malaria vaccine in African infants. N 
Engl J Med. 2012;367(24):2284-95. 
7. 
RTS S Clinical Trials Partnership. Efficacy and safety of RTS,S/AS01 malaria vaccine with or without 
a booster dose in infants and children in Africa: final results of a phase 3, individually randomised, 
controlled trial. Lancet. 2015;386(9988):31-45. 
8. 
Schulz KF, Altman DG, Moher D. CONSORT 2010 Statement: updated guidelines for reporting 
parallel group randomised trials. Trials. 2010;11:32. 
9. 
Xu Y, Cheung YB, Lam KF, Tan SH, Milligan P. A simple approach to the estimation of incidence rate 
difference. Am J Epidemiol. 2010;172(3):334-43. 
10. Zou G. A modified poisson regression approach to prospective studies with binary data. Am J 
Epidemiol. 2004;159(7):702-6. 
11.  Cheung YB, Xu Y, Cairns M, Milligan P. Evaluation of the impact of disease prevention measures: a 
methodological note on defining incidence rates. BMC medical research methodology. 
2017;17(1):72. 
 
 
 

Statistical Analysis Plan 
 
RTS,S-SMC Study 
1 
 
 
 
 
RTS,S-SMC:  
  
A comparative trial of  
seasonal vaccination with the RTS,S/AS01 malaria vaccine,  
seasonal malaria chemoprevention,  
and of the two interventions combined 
 
 
Revisions to the Statistical Analysis Plan: 
  
Revision 
Number 
Date of 
Revision 
Page 
Number(s) 
Details 
1 
02-Jun-
2020 
6 
 
 
10 
Clarification of passive case detection of the primary 
outcome. 
 
Clarification of the rationale for the definition of the 
‘per protocol’ population 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Statistical Analysis Plan 
 
RTS,S-SMC Study 
2 
 
 
Study title 
A comparative trial of seasonal vaccination with the malaria vaccine RTS,S/AS01, seasonal 
malaria chemoprevention, and of the two interventions combined. 
 
Clinical trial registration 
Clinicaltrials.gov Identifier: NCT03143218 
 
Study duration 
 
April 2017 – March 2020 
 
Study site 
The trial is being conducted in Houndé district, Burkina Faso and in Bougouni district, Mali, sites 
of a previous trial (NCT02211729), which evaluated the effectiveness of adding azithromycin to 
the drug combination used for seasonal malaria chemoprevention.   
 
Study objectives 
This trial seeks to determine  
 
1. Whether seasonal vaccination with RTS,S/AS01 is 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 and SMC with SP+AQ) is superior to RTS,S/AS01 alone or SMC alone in 
preventing clinical malaria and other adverse outcomes. 
 
These aspects are of equal priority. 
 
 
Study groups 
This is a double-blind, individually-randomised trial with three study arms.   
 
The study groups, and abbreviated names used for ease of reference, are shown in the table 
below.  
  
 
Group 
 
 
1) SMC  
‘SMC alone’ 
 
2) RTS,S/AS01  
‘RTS,S alone’ 
 
3) RTS,S/AS01+SMC 
‘Combined group’ 
April/May – June/July 2017 
Rabies vaccine x 3 
RTS,S/AS01 x 3 
RTS,S/AS01 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/AS01 x 1 
RTS,S/AS01 x 1 
July-Oct 2018 
SMC x 4 
SMC placebo x 4 
SMC x 4 
 
 
 
 
June 2019 
Hep A vaccine x 1    RTS,S/AS01 x 1 
RTS,S/AS01 x 1 
July-Oct 2019 
SMC x 4 
SMC placebo x 4 
SMC x 4 
 
 
 
 

Statistical Analysis Plan 
 
RTS,S-SMC Study 
3 
 
Group 1 – SMC arm - ‘SMC alone’   
In 2017, children in the SMC group were given three doses of rabies vaccine 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.  
  
Group 2 – RTSS/ AS01 arm – ‘RTS,S alone’ 
In 2017, children in the RTS,S/AS01 group were given three doses of RTSS/AS01 vaccine and 
four rounds of placebo-SMC at monthly intervals.   In 2018 and 2019, children received one 
dose of RTS,S/AS01 vaccine and four rounds of placebo-SMC at monthly intervals.  
  
Group 3 – RTS,S/AS01+SMC arm – ‘Combined arm’  
 
In 2017, children in the combined group were given three doses of RTS,S/AS01 vaccine and four 
rounds of SMC with SP+AQ at monthly intervals.   In 2018 and 2019, children received one dose 
of RTS,S/AS01 vaccine and four rounds of SMC with SP+AQ at monthly intervals. 
 
 
Schematic of Key Study Activities  
 
 
 
 
 
Precise timing of interventions 
Administration of RTS,S/AS01 or control vaccine:  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/AS01 or control vaccine were given in the first two weeks of June.   
 
Placebo
Placebo 
Placebo
SMC
SMC
SMC
SMC
SMC
SMC
2017
2018
2019
Transmission season
Control vaccinations
RTS,S Vaccine
Booster 1
Booster 2
RTS,S Vaccine
Census
~6000 children
Booster 1
Booster 2
Randomisation
2000 per group
End of study
Dry season 2020

Statistical Analysis Plan 
 
RTS,S-SMC Study 
4 
 
Administration of SMC or placebo SMC:  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. 
 
 
Study population 
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. 
 
 
Sample size and Study Design 
Based on the calculations described below, the trial aimed to recruit approximately 3,000 
children in Burkina Faso and 3,000 in Mali (total 6,000) who would be followed for three years. A 
low dropout rate of around 5% per year (15% overall) was anticipated based on findings from 
the previous trial conducted in the same study areas.   
 
In Burkina Faso and Mali, 2777 and 3143 children, respectively, received the first dose of study 
vaccine.  
 
Superiority comparison: The trial was designed to compare the two interventions combined 
with either used alone.  The study is powered to i) assess the statistical evidence against the null 
hypothesis of no difference between the combined group and either the SMC alone or RTS,S 
alone group,  and ii) estimate the efficacy of the combined group relative to the single 
intervention groups with a relatively high degree of precision. This latter aspect is important 
because if the combined intervention is to be used in practice, it is necessary to show that 
adding RTS,S/AS01 to SMC has a clinically significant benefit. 
 
After two years of follow-up, incidence of clinical malaria, as defined below, was approximately 
200 cases per 1000 child-years at risk. With approximately 2000 individuals in each arm, if the 

Statistical Analysis Plan 
 
RTS,S-SMC Study 
5 
 
incidence is at least 30% lower in the combined group, there is very high power over the three 
years of the study (close to 100%) to reject the null hypothesis of no difference between the 
combined group and the single treatment groups.  If efficacy of the combined group relative to 
either of the other groups is at least 30%, there will be 90% power for the lower limit of the 95% 
confidence interval to exclude 15%, i.e. to address point ii) above, establishing that the 
protection from the combined group is at least 15% better than SMC or vaccination alone.  
 
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 
potential advantages of RTS,S over SMC in terms of ease of delivery and the potential to sustain 
high levels of coverage. A reduction in annual efficacy from 50% to 40% was considered the 
largest difference that would be likely to be acceptable. This translates to a 20% greater 
incidence in the RTS,S/AS01-alone arm compared to the SMC-alone arm. With the anticipated 
incidence rates, there is adequate power to reject a smaller margin, as detailed in the protocol. 
However, 20% was considered the largest difference that would be considered unimportant.   
 
The study has 80% power to exclude, at the 2.5% significance level, a relative difference in the 
incidence of clinical malaria of 20% over the three year study period between the RTS,S/AS01 
and SMC alone groups, if these two interventions were equally effective.  Evidence of the 
effectiveness of the reference group – SMC alone - in these areas comes from clinical trials in 
Mali and Burkina Faso in 2009 (1, 2), and more recently in case-control studies conducted in 
both countries in 2016.  In the previous AZ-SMC trial in these two districts (3), the prevalence of 
molecular markers of resistance to SP and AQ were very low, and the curative efficacy of SP+AQ 
over 28 days was above 95% in both areas (Cairns et al., submitted).   
 
Hypothesis testing 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.  
 
For the non-inferiority comparison 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. 
 
 
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 weekly and 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 

Statistical Analysis Plan 
 
RTS,S-SMC Study 
6 
 
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 burned to CD and a copy sent to the chair of the DSMB for 
archiving. 
 
The randomisation codes will only be released 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. 
 
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 temperature ≥ 
37.5°C, or a history of fever within the past 48 hours), and a positive blood film, with a P. 
falciparum parasite density of 5,000 per µl or more.  This cut-off has been used in previous 
studies of SMC in Burkina Faso and Mali, as well as 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). Morbidity detected at contacts for which study 
children were visited at home (for serological sampling, and for the weekly parasitaemia survey) 
are excluded, as is vaccination, because some children were visited at home and brought to the 
clinic to be vaccinated.  
 
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 the order of priority - include the following: 
 

Statistical Analysis Plan 
 
RTS,S-SMC Study 
7 
 
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). 
 
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 surgery 
 
2.2.3  Hospital admissions due to malaria.  Defined as hospital admissions where malaria 
was the primary diagnosis, supported by a positive blood smear, or positive RDT if no blood 
smear result was 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 
 
2.2.5 
Deaths due to any cause  
 
2.2.6 
Deaths due to any cause excluding external causes and surgery 
 
2.2.7 
Deaths due to malaria.  Defined as hospital admissions resulting in death, where 
malaria was recorded as the primary cause of death, and where parasitology results did not 

Statistical Analysis Plan 
 
RTS,S-SMC Study 
8 
 
exclude malaria (i.e. a positive blood smear, if a slide result was available, or a positive RDT if no 
slide was done).  Deaths in the community will also be included when malaria is assigned as the 
primary cause of death by verbal autopsy. 
 
2.3 
Outcomes measured at weekly surveys 
A subset of children were selected to be sampled for parasitaemia during weekly surveys. 
Systematic random sampling, from lists sorted on age, was used to allocate children to be 
sampled in not more than one week per year, such that the sample of 24 children in each week, 
in each country was balanced with respect to age and treatment group.  Results from the 
weekly surveys will be analysed and presented separately for each malaria transmission season 
(2017, 2018 and 2019). 
 
2.3.1 
The prevalence of asexual stage P. falciparum infection of any density detected during 
the weekly home visits.  
2.3.2 
The prevalence of asexual stage P. falciparum infection with a density ≥ 5000 per ul 
detected during the weekly home visits. 
2.3.3 
The arithmetic mean P. falciparum parasite density, including samples which are parasite 
negatives as having a density of zero. 
 
2.4 
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 each 
malaria transmission season (2017, 2018 and 2019). 
  
2.4.1 
The prevalence of asexual stage P. falciparum infection of any density 
2.4.2 
The prevalence of asexual stage P. falciparum infection with density ≥ 5000 per ul  
2.4.3  The prevalence of sexual stage P. falciparum infection (i.e. gametocytes) 
2.4.4 
The arithmetic mean P. falciparum parasite density, including samples which are parasite 
negatives as having a density of zero.  
2.4.5 
The prevalence of asexual stage infection of non-falciparum Plasmodium species. 
2.4.6 
The prevalence of sexual stage infection (i.e. gametocytes) of non-falciparum 
Plasmodium species. 
2.4.7 
The mean haemoglobin concentration in g/dL. 
2.4.8  The prevalence of anaemia, defined as measured Hb < 10 g/dL.   
2.4.9 
The prevalence of moderate anaemia, defined as measured Hb < 7 g/dL.   
2.4.10 The prevalence of severe anaemia, defined as measured Hb < 5 g/dL.  
 
2.5 
Other secondary outcomes 
2.5.1 
The prevalence of molecular markers of resistance to SP and AQ in children with P. 
falciparum  infection, among samples collected at the final cross-sectional survey in December 
2019.  These 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.     
2.5.2 
The percentage of children with asymptomatic P. falciparum parasitaemia at the cross-
sectional survey in December 2019, who, when treated with SP+AQ, have an adequate clinical 
and parasitological response (ACPR) after 28 days.  
 
 

Statistical Analysis Plan 
 
RTS,S-SMC Study 
9 
 
 
Serious Adverse Events 
In addition to the comparison of incidence rates described above (section 2.2), 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.   
 
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 up to the 30th 
November 2019.  A 95% confidence interval for the incidence rate of meningitis among children 
vaccinated with RTS,S/AS01 will be calculated. 
The incidence of cerebral malaria among children vaccinated with RTS,S/AS01 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. 
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. The subset of febrile convulsions that occurred within 7 days of vaccination 
will also be analysed.  
An exploratory analysis will investigate if there is any evidence that RTS,S/AS01 increases 
mortality in girls.  This will compare the incidence of deaths using Cox regression, with an 
interaction between a dummy variable indicating receipt of RTS,S/AS01 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 female: male mortality ratio and its 95% confidence interval to be calculated separately for 
RTS,S/AS01 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.  
 
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.  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 
3.3 
The arithmetic mean P. falciparum parasite density, including samples which are parasite 
negatives as having a density of zero. 
 
Analysis populations and person-years at risk 
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/AS01 or control 

Statistical Analysis Plan 
 
RTS,S-SMC Study 
10 
 
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 
1) the date observation formally ended (31st March, 2020), 2) the date last seen if lost-to-follow-
up (LTFU), 3) the date of permanent exit from the study area, or 4) 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).   
 
As a secondary analysis, the primary outcome 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 2018, 1 in 2019) 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.   
 
Trial profile 
The trial profile will show 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.  
Reasons that children seen at the census were not eligible to join the study will be tabulated.    
 
The profile will also show, 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.  Reasons that children did not attend the first study contact for 
vaccination will be tabulated.  
 
The number of children who exited the study population by the end of the first, second and 
third year of the study will be 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;  

Statistical Analysis Plan 
 
RTS,S-SMC Study 
11 
 
- 
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 will be produced for each of the two trial centres. 
 
 
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/AS01 alone, and the combined group.  
Comparisons will also be made between RTS,S/AS01 alone and the combined group. 
 
Primary endpoint  
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, starting 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. 
 
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. 
 
As recommended in the updated CONSORT guidelines (8), the incidence rate differences (IRD) 
will also be calculated, as this gives an indication of the reduction in incidence attributable to 
the interventions, i.e. the absolute public health impact in similar contexts.  The IRD will be 
calculated using ordinary least squares regression of transformed variables, as described by Xu 
et al. (9). This method uses a robust standard error and controls for unequal follow-up time, as 
well as 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.   
 
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).  
 

Statistical Analysis Plan 
 
RTS,S-SMC Study 
12 
 
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 adjust for 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 
 
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.   
 
Investigation of differences between study groups in successive years of the study is also pre-
specified, because it is possible that the efficacy of RTS,S/AS01 booster doses is different to the 
primary series.  This will be assessed by exploring evidence for an interaction between 
intervention group and study year (2017-18, 2018-19, 2019-20). 
  
Finally, 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.  Of 
particular interest are participants who were young infants at the time of the first vaccination in 
2017, as they may have had no exposure to malaria prior to enrolment in the trial. 
 
 
Pre-specified Secondary analyses  
As this is the first trial of seasonal vaccination, a number of secondary analyses are planned to 
investigate the effect of malaria event history, completeness of protection and protection over 
time. Lexis expansion will be used to stratify person-time since vaccination. This will enable 
regression splines to be fitted to obtain smooth estimates of protection over time from 
RTS,S/AS01.  
 
Further analyses will explore the changing relative benefits of SMC and RTS,S/AS01 with age 
and transmission intensity (by comparing efficacy profiles with age between Burkina Faso, which 
has higher incidence rates, with Mali).   
 

Statistical Analysis Plan 
 
RTS,S-SMC Study 
13 
 
 
 
Ancillary studies to be reported separately 
1.  Evaluation of anti-CSP antibody concentrations obtained before and after priming and after 
each booster dose, determined in a sub-sample of children, and the relation of antibody 
concentration following vaccination to the subsequent risk of malaria.    
2.  The effect of the intervention on nutritional status at the end of season cross-sectional 
surveys. 
 
Analysis of the preference of participants for an injection of vaccine or for multiple rounds of 
SMC was scheduled to take place during the last few months of this phase of the study. 
However, this has had to be postponed because of travel restrictions imposed by the COVID-19 
crisis.  
 
On the advice of the steering committee and one of the trial funders (PATH) the economic 
analysis of the two approaches to malaria control will be undertaken in the second year of an 
extension study. The extension study will observe children up to the age of five years when they 
will no longer be eligible to receive SMC, rather than stopping follow-up after three years, as 
had been proposed initially.     
 
 
 
 

Statistical Analysis Plan 
 
RTS,S-SMC Study 
14 
 
Planned main tables for published report  
 
Table 1: Incidence of the primary outcome: 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 
 
RTS,S-SMC Study 
15 
 
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 1 
 
 
 
 
 
 
 
 
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) 
- 
- 
- 
 
 
 
 
 
 
 
 
 
 
Study Year 2 
 
 
 
 
 
 
 
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) 
- 
- 
- 
 
 
 
 
 
 
 
 
 
 
Study Year 3 
 
 
 
 
 
 
 
 
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.   
Similar tables will be used to report the incidence of passively detected secondary outcomes. 
 

Statistical Analysis Plan 
 
RTS,S-SMC Study 
16 
 
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 - 2017 
 
 
 
 
 
 
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 - 2018 
 
 
 
 
 
 
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 - 2019 
 
 
 
 
 
 
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 Study 
17 
 
Figure 1.  This will show i) cumulative hazards of malaria, by treatment group and ii) the risk of malaria, by treatment group.

Statistical Analysis Plan 
 
RTS,S-SMC Study 
18 
 
 
References 
 
1. 
Konate AT, Yaro JB, Ouedraogo AZ, Diarra A, Gansane A, Soulama I, et al. Intermittent reventive 
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(2):e1000408. 
2. 
Dicko A, Diallo AI, Tembine I, Dicko Y, Dara N, Sidibe Y, 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(2):e1000407. 
3. 
Chandramohan D, Dicko A, Zongo I, Sagara I, Cairns M, Kuepfer I, et al. Effect of Adding 
Azithromycin to Seasonal Malaria Chemoprevention. N Engl J Med. 2019. 
4. 
Piaggio G, Elbourne DR, Altman DG, Pocock SJ, Evans SJ. Reporting of noninferiority and 
equivalence randomized trials: an extension of the CONSORT statement. Jama. 2006;295(10):1152-
60. 
5. 
RTS S Clinical Trials Partnership. First results of phase 3 trial of RTS,S/AS01 malaria vaccine in 
African children. N Engl J Med. 2011;365(20):1863-75. 
6. 
RTS S Clinical Trials Partnership. A phase 3 trial of RTS,S/AS01 malaria vaccine in African infants. N 
Engl J Med. 2012;367(24):2284-95. 
7. 
RTS S Clinical Trials Partnership. Efficacy and safety of RTS,S/AS01 malaria vaccine with or without 
a booster dose in infants and children in Africa: final results of a phase 3, individually randomised, 
controlled trial. Lancet. 2015;386(9988):31-45. 
8. 
Schulz KF, Altman DG, Moher D. CONSORT 2010 Statement: updated guidelines for reporting 
parallel group randomised trials. Trials. 2010;11:32. 
9. 
Xu Y, Cheung YB, Lam KF, Tan SH, Milligan P. A simple approach to the estimation of incidence rate 
difference. Am J Epidemiol. 2010;172(3):334-43. 
10. Zou G. A modified poisson regression approach to prospective studies with binary data. Am J 
Epidemiol. 2004;159(7):702-6. 
11.  Cheung YB, Xu Y, Cairns M, Milligan P. Evaluation of the impact of disease prevention measures: a 
methodological note on defining incidence rates. BMC medical research methodology. 
2017;17(1):72. 
 
 
 

Details of Amendments to the Statistical Analysis Plan 
 
The Original SAP (dated 09-Apr-2020) was amended on one occasion (02-Jun-2020) and approved by 
the DSMB prior to locking and archiving of the trial database.  In the amendment, two edits for 
clarification were made as detailed below. 
 
Edit 1 - Clarification of passive case detection of the primary outcome (Page 6): 
The text in bold italics was inserted as below. 
 
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 temperature ≥ 37.5°C, or 
a history of fever within the past 48 hours), and a positive blood film, with a P. falciparum parasite 
density of 5,000 per µl or more.  This cut-off has been used in previous studies of SMC in Burkina 
Faso and Mali, as well as 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). Morbidity detected at contacts for which study children were 
visited at home (for serological sampling, and for the weekly parasitaemia survey) are excluded, as 
is vaccination, because some children were visited at home and brought to the clinic to be 
vaccinated.  
 
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). 
 
 
 
 
 

Edit 2 – Clarification of the rationale for the definition of the ‘per protocol’ population 
The text in italics was inserted as below: 
 
Page 10:  
As a secondary analysis, the primary outcome 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 2018, 1 in 2019) 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.