Molecular Detection and Genotyping of Plasmodium Falciparum Parasites in Young African Children After Immunization With RTS,S/AS01E Malaria Vaccine. An Ancillary Study Protocol to GlaxoSmithKline Phase IIb RTS,S/AS01E Malaria Vaccine Trial (Study Number 204889) Entitled, "Efficacy, Safety and Immunogenicity Study of GSK Biologicals' Candidate Malaria Vaccine (SB257049) Evaluating Schedules With or Without Fractional Doses, Early Dose 4 and Yearly Doses, in Children 5-17 Months of Age.
Conditions: Malaria
Sponsor: GlaxoSmithKline

Protocol
Study ID: 206213
Official Title of Study: An Ancillary Study Protocol to GlaxoSmithKline Phase IIb 
RTS,S/AS01E Malaria Vaccine Trial (Study Number 204889 [MALARIA-094]) entitled, 
“Efficacy, safety and immunogenicity study of GSK Biologicals’ candidate malaria 
vaccine (SB257049) evaluating schedules with or without fractional doses, early Dose 4 
and yearly doses, in children 5-17 months of age
NCT ID: NCT03281291
Date of Document: 8 May 2017

C O N FI D E N TI A L
0 8- M a y -2 0 1 7
- 1 -
M olec ul ar Detecti o n a n d Ge n ot y pi n g of Pl as m o di u m  f alci p ar u m
P ar asites i n Y o u n g Afric a n C hil dre n after I m m u niz ati o n wit h 
R T S, S/ A S 0 1 E M al ari a V acci ne 
Pr ot oc ol N u m ber: 2 0 6 2 1 3 ( M A L A RI A - 0 9 5)
“ A n A ncillar y St u d y Pr ot oc ol t o Gla x o S mit h Kli ne P hase II b R T S, S/ A S 01 E Malaria 
Vacci ne Trial ( St u d y N u m ber 2 0 4 8 8 9 [ M A LA RI A - 0 9 4]) e ntitle d, “ Efficac y, safety a n d 
i m m u n o ge nicity st u d y of G S K Bi ol o gicals’ ca n di date malaria vacci ne ( S B 2 5 7 0 4 9) 
e val uati n g sc he d ules wit h or wit h o ut fracti o nal d oses, earl y D ose 4 a n d ye arl y d oses, i n 
c hil dre n 5 -1 7 m o nt hs of a ge.”
Pri nci p al I n vesti g at or:
D y a n n Wirt h 1, 2
S u b -I n vesti g at ors: 
C o ntri b uti n g A ut h ors:
Affili ati o ns:
1 De part me nt of I m m u n ol o g y  a n d I nfecti o us Diseases, Har var d T. H. C ha n Sc h o ol of 
P u blic Healt h , U S A
2 T he Br oa d I nstit ute, U S A
3 Vacci ne a n d I nfecti o us Disease Di visi o n, Fre d H utc hi ns o n Ca ncer Researc h Ce nte r, 
U S A
4 De part me nt of Bi ostatistics, U ni versit y  of Was hi n gt o n, U S A
5 U ni versit y  of Calif or nia, Ber kele y, U S A 
6 P A T H M alaria Vacci ne Initiat i ve ( M VI), U S A
7 Gla x o S mit h Kli ne Bi ol o gicals, Bel gi u m
P P D
P P D

Ancillary Genotyping Protocol in support of 204889 (MALARIA-094) Protocol
Harvard T. H. Chan School of Public Health
Protocol Number: 206213 (MALARIA-095)
Final
CONFIDENTIAL
08-May-2017
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SYNOPSIS
The goal of the proposed amplicon sequencing and genotyping study (hereafter referred 
to as ‘genotyping study’) is to assess vaccine efficacy against clinical and asymptomatic 
malaria infection using ultra-sensitive molecular amplification and sequencing 
methodology to detect Plasmodium falciparum (P. falciparum) parasites from serial 
blood samples to be collected from children immunized with the primary and yearly 
booster immunizations of the RTS,S/AS01E vaccine as part of their participation in 
Protocol MALARIA-094. Genomic analysis will be performed on parasites from blood 
spot samples collected from children aged 5-17 months immunized with RTS,S/AS01E
on different dosage and schedule regimens under the parent clinical study protocol.
Specifically, the aims of this protocol are:
Primary Objective: To estimate and compare vaccine efficacy (VE) against new malaria 
infection(s) across all genotypes among the vaccine arms of the trial, using amplicon 
sequencing and genotyping to detect new infections. Asymptomatic P falciparum 
infections will be detected in monthly cross-sectional samples and clinical infection will 
be detected by passive case detection.
Two independent measures of VE will be determined:
a.
Vaccine efficacy against first new infection
VE against first new P. falciparum infection (i.e., time-to-infection; clinical case or 
cross-sectional) in all vaccine groups for the first 12 months following the third 
immunization in each vaccine group. 
b.
Vaccine efficacy against all new infections
VE against all new P. falciparum infections (clinical case or cross-sectional) in all 
vaccine groups for the first 12 months following the third immunization. The 
comparator group (rabies) window of follow-up will differ depending on the vaccine 
group to which it is compared.
Exploratory Objectives: The following exploratory objectives will be addressed using 
data generated for the primary objective:
a.
Exploratory Objective 1: To assess whether VE against new malaria infection 
based on blood slides (passive case detection) and molecular detection (active case 
detection) depends on genotypic characteristics of the malaria parasites, based on 
cross-sectional and clinical case samples.
b.
Exploratory Objective 2: To study whether prior infection or vaccination has a 
relationship to subsequent infection by measuring the molecular Force of Infection 
(mol[FOI]) determining the relationship between (mol)FOI and subsequent malaria 
infection risk.
c.
Exploratory Objective 3: To determine whether prior infection by a particular 
parasite genotype, not necessarily the vaccine strain, reduces the likelihood of re-
infection by a parasite with the same genotype.

Ancillary Genotyping Protocol in support of 204889 (MALARIA-094) Protocol
Harvard T. H. Chan School of Public Health
Protocol Number: 206213 (MALARIA-095)
Final
CONFIDENTIAL
08-May-2017
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d.
Exploratory Objective 4: To understand how gaps in time as measured by 
consecutive monthly dried blood spots impact the evaluation of VE against new 
infections.
Study outcomes to be evaluated:
Time to first new malaria infection - Infection is defined as sequence positive status. 
As there is no guarantee of complete clearance of asymptomatic infections by drug 
treatment, a new infection may originate from a sample that goes from negative to 
positive, or from a positive genotype X to a positive genotype Y.
Sequence-based parasite positive/negative status - Positive status defined by the 
minimal threshold number of sequencing reads (defined in the Statistical and Analysis 
Plan) for at least one amplicon following filtration of polymerase chain reaction (PCR) 
and sequencing errors. 
Genotype data for the P. falciparum circumsporozoite (CS) C-terminus and SERA2 
loci - Defined as the haplotype(s) observed at each amplicon following filtration of PCR
and sequencing errors.
New infections - New infection(s) for the primary objective is defined as a haplotypic 
genotype not previously detected in any sample that is different from baseline sample 
collected prior to immunization # 1.
This study is proposed to be an ancillary study of the GlaxoSmithKline (GSK)
RTS,S/AS01E phase IIb randomized trial (Study Number 204889) and is to be performed 
across all study sites. The original randomized trial (MALARIA-094) will be vaccinating 
approximately 1,500 subjects 5-17 months of age in multi-centers according to the 
following groups: 
i.
Control group, i.e., rabies vaccine at Month 0, Month 1, Month 2; or 
ii.
Group R012-20 - a course of RTS,S/AS01E full dose at Month 0, Month 1, 
Month 2 + a full dose at Month 20; or 
iii.
Group R012-14-mD - a course of RTS,S/AS01E full dose at Month 0, Month 1, 
Month 2 + yearly full doses at Month 14, Month 26, Month 38; or 
iv.
fractionated (Fx) Group Fx012-14-mFxD - a course of RTS,S/AS01E full dose at 
Month 0, Month 1 + RTS,S/AS01E 1/5th dose at Month 2, + RTS,S/AS01E 1/5th
dose at Month 14, Month 26, and Month 38; 
v.
Group Fx017-mFxD - a course of RTS,S/AS01E full dose at Month 0, Month 1 
+ RTS,S/AS01E 1/5th dose at Month 7, + RTS,S/AS01E 1/5th dose at Month 20,
and Month 32. 
A total of 300 subjects will be allocated to each group. 
This genotyping study of malaria parasites collected from serial blood samples following
RTS,S/AS01E immunization will assess VE using molecular genetic data to identify first 
infections following vaccination, and to distinguish new from existing infections, using 

Ancillary Genotyping Protocol in support of 204889 (MALARIA-094) Protocol
Harvard T. H. Chan School of Public Health
Protocol Number: 206213 (MALARIA-095)
Final
CONFIDENTIAL
08-May-2017
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an amplicon sequencing-based strategy: deep sequencing of small, highly variable 
regions of the parasite genome to allow for both:
i.
highly sensitive detection of parasitemia (analogous to conventional PCR-based 
detection), and 
ii.
identification of genetically distinct parasite populations within or between 
affected individuals. 
Given the similarities between this project and the ancillary genotyping study performed 
for the phase 3 MALARIA-055 study (MALARIA-066), there is confidence to perform 
the necessary data generation and analyses to accomplish the listed Specific Aims. The 
following sequential approach is proposed for sample evaluation to investigate the 
described Specific Aims, with Tier 1 sampling essential to addressing the Primary 
Objective, and Tier 2 sampling to be performed after primary immunization series and 
booster immunization series. Two analyses will be performed:
Tier 1:
All cross-sectional dried blood spot samples collected monthly from all subjects 
during the first 20 months (approximately 31,500 samples) and clinical malaria 
cases (approximately 14,400) of the primary series from all arms of the study.
Tier 2:
All cross-sectional dried blood spot samples collected for three consecutive 
months and then quarterly thereafter for one year following the first booster 
RTS,S dose (approximately 9,000 samples) and clinical malaria cases in all 
arms of the study up through Month 32 (approximately 8,640 samples). 

Ancillary Genotyping Protocol in support of 204889 (MALARIA-094) Protocol
Harvard T. H. Chan School of Public Health
Protocol Number: 206213 (MALARIA-095)
Final
CONFIDENTIAL
08-May-2017
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TABLE OF CONTENTS
PAGE
LIST OF ABBREVIATIONS.............................................................................................7
1.
OBJECTIVES...........................................................................................................9
1.1.
Rationale for the MALARIA-094 Study Design..............................................9
1.2.
MALARIA-095: Genetic Analysis of MALARIA-094 Infections.....................10
1.3.
Statement of Specific Objectives ................................................................11
1.3.1.
Primary Objective ........................................................................11
1.3.2.
Exploratory Objectives.................................................................11
2.
BACKGROUND AND SIGNIFICANCE...................................................................12
2.1.
RTS,S Vaccine-Induced Immune Responses .............................................12
2.2.
Immunity and Genetic Diversity ..................................................................13
2.3.
Genotyping Technologies ...........................................................................14
2.4.
Study Objectives.........................................................................................15
2.4.1.
Primary objective .........................................................................15
2.4.2.
Exploratory objective 1.................................................................16
2.4.3.
Exploratory objective 2.................................................................16
2.4.4.
Exploratory objective 3.................................................................18
2.4.5.
Exploratory objective 4.................................................................18
3.
RESEARCH DESIGN AND METHODS..................................................................18
3.1.
Study Design Overview...............................................................................18
3.2.
Outcomes, Predictors, and Other Covariates of Interest .............................19
3.2.1.
Outcomes ....................................................................................19
3.2.2.
Main exposures ...........................................................................19
3.2.3.
Candidate confounders................................................................19
3.3.
Study Cohort...............................................................................................20
3.3.1.
Overview......................................................................................20
3.3.2.
Endpoints.....................................................................................20
3.3.3.
Total vaccinated cohort (TVC) and according-to-protocol 
(ATP) analyses ............................................................................20
3.4.
Parasite Genotyping Methodology ..............................................................20
3.5.
Conduct of the Study ..................................................................................21
3.5.1.
Follow-up and identification of cases ...........................................21
3.5.2.
Laboratory assays .......................................................................22
3.5.3.
Additional study procedures.........................................................22
4.
INVESTIGATIONAL PRODUCTS AND ADVERSE EVENTS.................................22
5.
STUDY TIMELINE..................................................................................................22
6.
STATISTICAL CONSIDERATIONS........................................................................22
6.1.
Analytical Plan ............................................................................................22
6.2.
Sample Size and Power..............................................................................23
7.
DATA MANAGEMENT METHODS.........................................................................24
8.
PROTECTION OF HUMAN RESEARCH SUBJECTS............................................24

Ancillary Genotyping Protocol in support of 204889 (MALARIA-094) Protocol
Harvard T. H. Chan School of Public Health
Protocol Number: 206213 (MALARIA-095)
Final
CONFIDENTIAL
08-May-2017
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9.
STUDY ORGANIZATION.......................................................................................24
10. REFERENCES.......................................................................................................25

Ancillary Genotyping Protocol in support of 204889 (MALARIA-094) Protocol
Harvard T. H. Chan School of Public Health
Protocol Number: 206213 (MALARIA-095)
Final
CONFIDENTIAL
08-May-2017
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LIST OF ABBREVIATIONS
AS01E
GlaxoSmithKline’s proprietary Adjuvant System containing MPL, 
QS-21 and liposome (25 µg MPL and 25 µg QS-21)
ATP
According to protocol
BAM
Binary alignment/map
CDC
Center for Disease Control
CHMI
Controlled human malaria infection
CI
Confidence interval
COI
Complexity of infection
CRF
Case report form
CRO
Clinical research organization
CS
Circumsporozoite
CSP
Circumsporozoite protein of Plasmodium falciparum
csp
DNA seq for circumsporozoite gene
C-terminus
C-terminal domain from CSP protein
DNA
Deoxyribonucleic acid
EMA
European Medicines Agency
FOI
Force of infection
Fx
Fractionated / fractional
GP
Genomics platform
GSK
GlaxoSmithKline
HLA
Human leukocyte antigen
LIMS
Laboratory information management system
LL
Lower limit
mol(FOI)
Molecular force of infection

Ancillary Genotyping Protocol in support of 204889 (MALARIA-094) Protocol
Harvard T. H. Chan School of Public Health
Protocol Number: 206213 (MALARIA-095)
Final
CONFIDENTIAL
08-May-2017
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MVI
Malaria Vaccine Initiative
NANP
Amino acid repeat region of P. falciparum circumsporozoite 
protein
NCBI
National Center for Biotechnology Information
NVDP
Non-repetitive island
PCR
Polymerase chain reaction
P. falciparum
Plasmodium falciparum
PI
Principal investigator
POC
Proof of concept
pyr
Per year
rcdfs
Reverse cumulative distribution functions
RTS,S
Particulate antigen, containing both RTS and S (hepatitis B surface 
antigen) proteins
SAP
Statistical Analysis Plan
SERA2
Serine repeat antigen 2 from P. falciparum
SNP
Single nucleotide polymorphism
SQL
Structured query language
TVC
Total vaccinated cohort
VE
Vaccine efficacy

Ancillary Genotyping Protocol in support of 204889 (MALARIA-094) Protocol
Harvard T. H. Chan School of Public Health
Protocol Number: 206213 (MALARIA-095)
Final
CONFIDENTIAL
08-May-2017
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1.
OBJECTIVES
GlaxoSmithKline (GSK) Biologicals and the PATH-Malaria Vaccine Initiative (MVI) are 
committed to develop a malaria vaccine for reduction of malaria disease burden in 
children and contribution to the malaria elimination goal. Characterization of an optimal 
dosing regimen and boosting schedules will be critical.
The results of the phase 3 RTS,S/AS01E malaria vaccine trial (MALARIA-055) 
previously reported, including the long term follow-up data and efficacy of the fourth 
dose, and the preliminary results of the controlled human malaria challenge study, 
MALARIA-071, were reviewed by the European Medicines Agency (EMA) during the 
assessment of the initial application of the RTS,S/AS01E candidate vaccine. The EMA 
asked GSK to address the following points in ongoing or future studies:  1) the timing of 
the fourth dose and evaluation of the safety and efficacy of an early fourth dose; 2) the 
efficacy and safety of multiple yearly doses and whether the vaccine predisposes to some 
degree of hyporesponsiveness to sequential doses; and 3) the plans to further investigate 
the potential utility of a delayed and fractionated third dose schedule in the target age 
range.
The primary protocol MALARIA-094 is designed to address these points in the pediatric 
population. It aims to:

Establish proof of concept (POC) for a fractional (Fx) dose schedule under 
conditions of natural exposure,

Establish the role of third dose spacing in a Fx dose schedule,

Describe the effect of an earlier full fourth dose at Month 14, and 

Describe the effect of multiple Fx or full yearly doses.
1.1.
Rationale for the MALARIA-094 Study Design
The study intends to establish POC for a Fx dose schedule under conditions of natural 
exposure. The study will be conducted in children 5-17 months of age at first vaccination 
living in areas of mid to high malaria transmission. In addition to assessing vaccine 
efficacy (VE) against clinical malaria disease, this study will for the first time determine 
VE against asymptomatic and sub-microscopic parasitemia in dried blood spots collected 
in monthly cross-sectional surveys from every child enrolled in this study using ultra-
sensitive molecular genotyping methods. This ancillary protocol, addressing RTS,S VE
against infection as defined by genetic methods, is in support of the MALARIA-094 
clinical protocol entitled, “Efficacy, safety and immunogenicity study of GSK 
Biologicals’ candidate malaria vaccine (SB257049) evaluating schedules with or without 
fractional doses, early Dose 4 and yearly doses, in children 5-17 months of age”.

Ancillary Genotyping Protocol in support of 204889 (MALARIA-094) Protocol
Harvard T. H. Chan School of Public Health
Protocol Number: 206213 (MALARIA-095)
Final
CONFIDENTIAL
08-May-2017
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The study proposed herein will include the five study groups according to the 
MALARIA-094 study:
a.
Control group will receive three doses of rabies vaccine at Month 0, Month 1 and 
Month 2.
b.
Group R012-20 will receive the standard schedule consisting of RTS,S/AS01E full 
dose given at Month 0, Month 1, Month 2 and a fourth dose at Month 20. This 
corresponds to the schedule that received a positive scientific opinion from EMA. 
The four other groups will receive alternative schedules as follows:
c.
Group R012-14-mD will receive RTS,S/AS01E full dose at Month 0, Month 1, 
Month 2, an early fourth dose at Month 14 and yearly full doses at Month 26 and 
Month 38.
d.
Group Fx012-14-mFxD will receive RTS,S/AS01E full dose at Month 0 and Month 
1, RTS,S/AS01E Fx dose (1/5th dose) at Month 2 and yearly Fx doses at Month 14, 
Month 26, Month 38.
e.
Group Fx017-mFxD will receive RTS,S/AS01E full dose at Month 0 and Month 1, 
RTS,S/AS01E delayed Fx third dose (1/5th dose) at Month 7 and yearly Fx doses at 
Month 20 and Month 32.
Results from this study will be critical in forming future possibilities for the development 
of vaccine-based strategies which, in combination with other interventions, may 
contribute to the malaria elimination agenda.
1.2.
MALARIA-095: Genetic Analysis of MALARIA-094 Infections
Genomic and statistical analysis approaches can be applied to the MALARIA-094 trial to 
help understand and improve the efficacy of RTS,S, forming the basis of the ancillary 
protocol MALARIA-095 described here. Whereas traditional polymerase chain reaction
(PCR)-based infection detection methods yield a simple binary outcome of positive or 
negative infection status, amplicon sequencing data will provide information of the 
complexity of infection (COI), a measure of the number of genetically distinct parasite 
lineages in each participant sample in the trial, and indicate the rate with which new 
infections occur and parasite lineages turn over. The resulting data will allow assessment 
of VE of each vaccine study arm to prevent new genetically distinct malaria infection by 
yielding not only the infection status of each sample, but also information on the 
incidence rates of new and distinct malaria infection.
In addition to supporting the primary objective of evaluating differences in overall VE 
against new malaria infection among the arms of the MALARIA-094 trial, amplicon 
sequencing data from MALARIA-095 will enable investigations into the nature of 
immunity generated by vaccination and natural disease exposure. The dense cross-
sectional sampling design will allow for exploration of the impact of prior infection 
history on susceptibility to new infections from the perspective of parasite genotype. 
There can also be evaluation of the degree to which allele-specific protection contributes 
to overall VE, and how VE depends on genetic distance of malaria infections from the 
vaccine strain, as were performed in the MALARIA-066 ancillary genotyping study. The

Ancillary Genotyping Protocol in support of 204889 (MALARIA-094) Protocol
Harvard T. H. Chan School of Public Health
Protocol Number: 206213 (MALARIA-095)
Final
CONFIDENTIAL
08-May-2017
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sampling schedule of this trial, in combination with the unique data generation capacity 
developed at the Broad Institute for previous RTS,S work, creates an opportunity to 
significantly increase the understanding of RTS,S efficacy, and to investigate the 
dynamics of naturally acquired immunity to malaria infection.
In summary, this proposed ancillary genotyping study is an evaluation of the 
RTS,S/AS01E VE when administered as different immunization schedules for the
prevention of P. falciparum malaria infection upon molecular detection by amplicon 
sequencing and parasite genotyping. Sample collection methodology is described in the 
MALARIA-094 trial (Study Number 204889). 
1.3.
Statement of Specific Objectives
1.3.1.
Primary Objective
Primary Objective:  To estimate and compare vaccine efficacy (VE) against new 
malaria infection(s) across all genotypes among the vaccine arms of the trial, using 
amplicon sequencing and genotyping to detect new infections. Asymptomatic P 
falciparum infections will be detected in monthly cross-sectional samples and clinical 
infection detected by passive case detection.
Two independent measures of VE will be determined:
a.
Vaccine efficacy against first new infection
VE against first new P. falciparum infection (i.e., time-to-infection; clinical case or 
cross-sectional) in all vaccine groups. The comparator group (rabies) window of 
follow-up will differ depending on the vaccine group to which it is compared. 
b.
Vaccine efficacy against all new infections
VE against all new P. falciparum infections (clinical case or cross-sectional) in all 
vaccine groups. The comparator group (rabies) window of follow-up will differ 
depending on the vaccine group to which it is compared.
1.3.2.
Exploratory Objectives
The following exploratory objectives will be addressed using data generated for the 
primary aim:
a.
Exploratory Objective 1: To assess whether VE against new malaria infection 
based on blood slides (passive case detection) and molecular detection (active case 
detection) depends on genotypic characteristics of the identified malaria parasites, 
based on cross-sectional and clinical case samples.
b.
Exploratory Objective 2: To study whether prior infection or vaccination has a 
relationship to subsequent infection by measuring the molecular Force of Infection 
(mol[FOI]) determining the relationship between (mol)FOI and subsequent malaria 
infection risk.

Ancillary Genotyping Protocol in support of 204889 (MALARIA-094) Protocol
Harvard T. H. Chan School of Public Health
Protocol Number: 206213 (MALARIA-095)
Final
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08-May-2017
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c.
Exploratory Objective 3: To determine whether prior infection by a particular 
parasite genotype, not necessarily the vaccine strain, reduces the likelihood of re-
infection by a parasite with the same genotype.
d.
Exploratory Objective 4: To understand how gaps in time as measured by 
consecutive monthly dried blood spots impact the evaluation of VE against new 
infections. 
2.
BACKGROUND AND SIGNIFICANCE
2.1.
RTS,S Vaccine-Induced Immune Responses
Previous investigations have identified candidate mechanisms of both natural and 
vaccine-induced immune response to the CS protein (CSP) [Nardin and Nussenzweig, 
1993]. Natural malaria infection induces a non-sterilizing protective immune response
against blood stage merozoites (that do not express csp) targeted towards epitopes that 
bind antibodies as well as human leukocyte antigen (HLA) I and HLA II molecules. The 
mechanism of RTS,S/AS01E vaccine-induced protection remains unresolved as there is 
not a clear immunological surrogate of protection which is predictive of outcome. The 
efficacy of the vaccine has been most closely associated with anti-CSP antibody 
responses, more specifically, antibody response to the NANP amino acid repeat region. 
The NANP repeat region is interrupted by a non-repetitive island (NVDP), and variably 
exhibits 10-17 NANP repeats to the left of this island and 17-23 repeats to the right of 
this island. Early studies found that antibodies bind to CSP at two identical B cell 
epitopes [Zavala et al., 1983], suggesting that each NANP repeat region may 
independently bind antibodies. Antibodies elicited against epitopes within the amino acid 
NANP repeat region of the CS antigen correlate with protection (while not predictive) as
demonstrated in the controlled human malaria infection (CHMI) challenge model [Stoute, 
et al., 1998; Kester, et al., 2001; Kester, et al., 2009, Ockenhouse, et al., 2015] and in 
field studies [Olotu, et al., 2011; White, et al., 2013]. Recently, Ockenhouse, et al. have 
identified antibodies in human immune sera detected after immunization with 
RTS,S/AS01 that react with conformational strain specific epitopes present in the C-
terminal domain of the CSP (unpublished).
Studies in the Gambia [Bojang et al., 2001; Alloueche et al., 2003], Mozambique [Enosse 
et al., 2006] and Kenya [Waitumbi et al., 2009] comparing RTS,S and comparator 
vaccinated subjects could not identify any particular polymorphism or variants targeted 
by the vaccine in the csp gene and association with protection.
The RTS,S/AS02 vaccine is capable of eliciting CD4+ T-cell production, and IFN-γ 
secretory CD4+ T-cell responses, particularly of the Th1 type [Lalvani et al., 1999]. It has 
been hypothesized that polymorphism observed in the Th2R and Th3R CD4 epitopes 
[Weedall et al., 2007] may indicate that T-cell response to these regions of CSP may 
provide some degree of protective immunity against infection by providing T-cell help.
The vaccine is also known to induce a strong CD4+ T-cell response to the non-variable 
CST3 epitope [Bojang et al., 2001].

Ancillary Genotyping Protocol in support of 204889 (MALARIA-094) Protocol
Harvard T. H. Chan School of Public Health
Protocol Number: 206213 (MALARIA-095)
Final
CONFIDENTIAL
08-May-2017
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2.2.
Immunity and Genetic Diversity
The malaria parasite is one of the most widespread and deadly human pathogens, causing 
an estimated 214 million cases per year with 88% of cases occurring in sub-Saharan 
Africa where approximately 300,000 malaria deaths occur in children annually [World 
Malaria Report 2015]. The parasite has demonstrated great evolutionary plasticity, and 
has developed mechanisms to evade the host immune response and intervention strategies 
such as chemotherapeutics. Sequence analysis of csp demonstrates significant genetic 
diversity in the extant parasite populations consistent with immune-mediated selection at 
this locus. It is therefore anticipated that vaccination with a monovalent vaccine such as 
RTS,S/AS01E may provide differential protection across haplotypic variants for csp and 
possibly select those variants that can evade clearance by the vaccinated host.
Investigations of the immune responses to RTS,S in malaria-naïve subjects suggested that 
the protection conferred by RTS,S/AS02 could be allele-specific. For instance, Lalvani et 
al. [1999] have found that the CD4+ T-cell response to the Th2R and Th3R epitopes did 
not provide cross-reactive immunity to non-vaccine epitope haplotypes in malaria-naïve 
subjects. However, studies conducted with cells primed through natural infection or with 
subjects previously exposed to malaria could detect responses against non-vaccine 
parasite variants [Pinder et al., 2004].
Results of three studies comparing RTS,S and comparator vaccinated subjects in endemic 
areas also suggested that protection mediated by this vaccine is not allele specific. 
Investigators in the Gambia [Bojang et al., 2001; Alloueche et al., 2003], Mozambique 
[Alonso et al., 2004; Enosse et al., 2006] and Kenya [Waitumbi et al., 2009] could not 
find differences in parasite diversity in the Th2R and Th3R regions between isolates from 
RTS,S recipients and isolates from subjects receiving comparator vaccines, either in the 
occurrence of Th2R and Th3R vaccine haplotypes or in the occurrence of subject point 
mutations in those regions.
Next generation (Illumina MiSeq, PacBio) sequencing of PCR amplicons from infected 
participant samples from the phase 3 RTS,S clinical study (MALARIA-055) was used to 
conduct more sensitive genetic investigations of allele-specific protection, and to perform 
analyses at a potentially more immunologically relevant level: epitope haplotypes, rather 
than individual polymorphic residues. A sieve analysis methodology was used to analyze 
the CS data for differential VE at three levels: 1) the entire C-terminal domain from CSP 
protein (C-terminus) amplicon haplotype (95 residues), 2) defined haplotypic regions of 
the C-terminus (10-17 residues), and 3) individual polymorphic residues, for two defined 
trial endpoints in the two age cohorts [Neafsey et al., 2015]. PCR amplicon directed next-
generation sequencing of deoxyribonucleic acid (DNA) extracted from 4,985 participant 
samples was used to survey polymorphisms in CS and evaluate the impact of 
polymorphic CS residues and several haplotypic regions on VE to prevent first or only 
episodes of clinical malaria within a year of vaccination. VE was significantly greater 
against clinical malaria with infections matching the vaccine strain in several haplotype 
regions and individual residues of the CS C-terminus in the 5-17 month old per-protocol 
cohort of 4,557 vaccinated and 2,328 control vaccinated participants. For matched versus 
mismatched malaria based on the entire CS C-terminus, VE cumulatively through 12 

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months was 50.3% (95% confidence interval [CI], 34.6 to 62.3) versus 33.4% (95% CI, 
29.3 to 37.2), P = 0.04 for differential VE; and VE based on a hazard ratio was 62.7% 
(95% CI, 51.6 to 71.3) versus 54.2% (95% CI, 49.9 to 58.1), P = 0.06. VE did not vary 
with CS C-terminus haplotypes for the 6-12 week old per-protocol cohort. Given the low 
frequency of parasites matching the vaccine strain at many of the phase 3 trial study sites, 
these results partially explain the incomplete protective efficacy of RTS,S/AS01 
vaccination.
2.3.
Genotyping Technologies
A high-throughput pipeline was developed at the Broad Institute to generate PCR 
amplicon-based next generation sequencing data for the MALARIA-066 ancillary 
genotyping study from over 5,000 dried blood spot samples (see Appendix from Neafsey 
et al., 2015 for details on sequencing methodology). This capacity yielded highly useful 
information about parasite samples that could not be obtained via any other practical 
means. By generating deep sequencing coverage of hundreds to thousands of sequencing 
reads from a collection of short (~300 nucleotide) PCR amplicons spanning highly 
variable antigenic regions from the P. falciparum genome, complete phasing information 
(i.e., ordering of the variable positions within each amplicon for each patient sample), 
was recovered. From this information, the haplotypes of common to extremely rare (~1% 
abundance) parasite strains within complex infections were reconstructed. Because the 
sequenced regions exhibit a high diversity of haplotypes at the parasite population level, 
it could be expected that genetically distinct parasites would only very rarely exhibit the 
same haplotype at more than one amplicon locus by chance, so the data therefore allowed 
detection of all infections within patient samples with high sensitivity.
In the current study it is proposed to replace conventional 18S PCR-based detection of 
parasitemia with the amplicon sequencing approach. Parasite positivity will be defined as 
≥200 sequence reads aligning to at least one amplicon. This is based on findings of the 
MALARIA-066 study, in which 200 reads was found to be the threshold below which 
true parasite detection could not be resolved from background signal, and at which 
parasitemia was detected in 90% of samples. In MALARIA-066, where samples did not 
yield a positive result there was no correlation with parasitemia; the lack of sequence 
reads was likely due to poor sample quality or other technical issues. For the filtration of 
PCR and sequencing errors within samples meeting the standard of parasite positivity, we 
will require that filtered haplotypes be supported by a minimum of 1% and with at least 5 
reads associated with a given sample-amplicon. There will be a continuance to optimize 
this methodology and further benchmark it against standard PCR-based parasite 
detection.
There has been a reduction in the cost of the DNA extraction and amplicon sequencing, 
and tailoring it to new study designs and sequencing platforms. Simulations conducted 
based on data collected in the MALARIA-066 ancillary protocol indicate that with two 
sequenced amplicons per sample (specifically, the CS C-terminus and serine repeat 
antigen 2 from Pf [SERA2] amplicons), there will be greater than 99% power to detect 
new superinfections in samples containing a single initial strain, and approximately 95% 

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power to detect new superinfections in samples already containing five parasite strains
(see Figure 1).
2.4.
Study Objectives
2.4.1.
Primary objective

To estimate and compare vaccine efficacy (VE) against new malaria infection(s) 
across all genotypes among the vaccine arms of the trial, using amplicon sequencing 
and genotyping to detect new infections. Asymptomatic P falciparum infections will 
be detected in monthly cross-sectional samples and clinical infection detected by 
passive case detection.
To achieve this aim it is proposed to use data generation methodology very similar to 
what was employed in the MALARIA-066 ancillary genotyping study (see 
Supplementary appendix, Neafsey et al., 2015 for details). We will be using two of the 
same PCR amplicons analyzed for MALARIA-055, one spanning 288 base pairs in the 
C-terminus of the CS locus (coding sequence nucleotide coordinates 878–1165) and a 
second amplicon spanning 286 base pairs in the N-terminus of the SERA2 locus (coding 
sequencing nucleotide coordinates 72–357).
In the MALARIA-066 trial sequence data were successfully generated from 
approximately 90% of the samples received. No association between sequence read 
coverage and parasite density (Pearson correlation = 0.15) was observed, suggesting that 
PCR performs robustly on samples with a parasite density of at least 200/ul. The 
sensitivity of the CS and SERA2 amplicons against traditional PCR assays for 
Plasmodium detection, such as 18S amplicons, will be benchmarked.
Execution of the primary objective will require the capacity to distinguish new infections 
from existing infections in cross-sectional samples. Analysis of data gathered from the 
MALARIA-066 trial to estimate the power to detect new infections as a function of the 
initial COI and the number of PCR amplicon loci sequenced per sample (one or two) 
have been performed. Because power for this application is contingent on the local 
haplotypic diversity of the amplicon regions in the parasite population, simulation 
analyses have been conducted using MALARIA-066 data in both a West African site 
(Agogo) and an East African site (Siaya), given the differences in population haplotypic 
composition in those two regions. Infections were simulated of varying degrees of initial 
COI by randomly sampling haplotypes from the MALARIA-066 dataset according to 
their frequency in the parasite populations in Agogo and Siaya, and evaluated how often 
the addition of one more haplotype (also sampled according to population frequency) 
resulted in a distinct genotypic composition. One thousand simulations for each 
combination of parameters were performed.
As indicated by Figure 1, sequence data from two amplicons (CS and SERA2) yield 
much greater power to detect new infections than data from a single amplicon (CS), in 
both Agogo and Siaya. For infections that start with a COI of 5, data from two amplicons 
is sufficient to detect the addition of a sixth super-infecting strain at a frequency over 
95%. There is confidence that, using data from two amplicons, new infections will be 

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resolved from existing infections in cross-sectional samples to address the key questions 
of the Primary and Exploratory Aims.
The Statistical Analysis Plan (SAP) (see Section 6.1) for the Primary Objective will 
assess VE against each of the two primary endpoints listed in Section 3.3.2 and for each 
of the ATP and TVC cohorts (see Section 3.3.3).   
2.4.2.
Exploratory objective 1 

To assess whether VE against new malaria infection based on blood slides (passive 
case detection) and molecular detection (active case detection) depends on genotypic 
characteristics of the identified malaria parasites, based on cross-sectional and 
clinical case samples.
2.4.3.
Exploratory objective 2

To study whether prior infection or vaccination has a relationship to subsequent 
infection by measuring the molecular Force of Infection (mol[FOI]) determining the 
relationship between (mol)FOI and subsequent malaria infection risk.

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Molecular FOI, the number of parasite infections accrued over a given time period 
detected using molecular tools, has been demonstrated to be an excellent measure of 
malaria epidemiology and an informative parameter to measure in intervention studies 
[Mueller et al., 2012]. Molecular FOI can be difficult to measure outside the context of a 
longitudinal trial, but the structure of the MALARIA-094 trial offers the perfect 
opportunity to study mol(FOI). In this study mol(FOI) will be calculated for the 
individual participants of the trial using amplicon sequence data generated for the 
Primary Objective from cross-sectional and clinical malaria samples, and used to explore 
the relationship between that parameter and subsequent infection risk across the arms of 
the trial. By capturing variation among individuals in mol(FOI), understanding the impact 
of various booster dosages and scheduling regimens on VE will be refined and improved.
In contrast to molecular studies utilizing 18S PCR or other non-antigenic markers for 
infection detection, amplicon sequencing data will be taken a step further to evaluate 
whether prior infection by a given haplotype reduces the likelihood of subsequent 
infection by the same haplotype. Though only two antigens out of hundreds in the P. 
falciparum proteome with amplicon sequencing data will be surveyed, the CS and 
SERA2 are assumed to be highly polymorphic due to selection to evade an acquired 
immune response. Each locus exhibits high haplotype diversity, but harbors at least four 
common haplotypes with a population frequency of at least 10% at most of the 
MALARIA-055 study sites. Given the density of cross-sectional sampling in the 
MALARIA-094 trial design and the expectation that 10 or more infections will be 
detected from many participants, reasonable power to detect decreased susceptibility to 
infection by parasite strains exhibiting common amplicon haplotypes given prior 
infection by the same haplotype is anticipated.
One of the major unknowns in the development of strategies to interrupt malaria 
transmission using vaccine approaches that target incident infections is how high VE
needs to be in order to drive R0 to < 1. This study will capture both the incidence and 
prevalence of malaria infection using two accepted methodologies that differ in the 
sensitivity of parasite detection by several log fold, namely positive blood smear (asexual 
stage and gametocyte parasites) determined by expert microscopy and molecular genetic 
sequence-based technology. This study will provide the first ever comparison of malaria 
incidence based both on smear-positive (asymptomatic and clinical) infection in non-
immune young children and also VE based on sub-microscopic parasite prevalence in 
asymptomatic children. Whether one believes that all parasites in any single individual 
need to be eliminated to achieve malaria elimination for a population or community is a 
matter of debate. Malaria has been eliminated from dozens of countries based solely on 
detection of new smear-positive clinical infections when the FOI was exceedingly low. 
The data collected here in conjunction with mathematical modeling (Gates Foundation 
Malaria Modeling Consortium) will provide for the first time an indication of the 
incidence of asymptomatic malaria infection after RTS,S immunization, the prolongation 
of VE with annual booster immunizations, the relative difference in the sensitivity of 
detection of parasites in the population, and VE as a measure of escape mutations in 
circulating strains that differ from vaccine strain.

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2.4.4.
Exploratory objective 3

To determine whether prior infection by a particular parasite genotype, not 
necessarily the vaccine strain, reduces the likelihood of re-infection by a parasite 
with the same genotype.
Since the first new infection in young children may be a clinical malaria episode 
(uncomplicated or severe malaria), it is presently not possible to predict how antimalarial 
therapy impacts the clearance of blood stage parasitemia in children. We assume that 
asexual parasites are cleared from the blood as typically defined by the absence of 
parasites on subsequent thick blood smears. However, we have no knowledge of whether 
parasites at levels only detectable by ultrasensitive molecular methodologies are 
persistently present following treatment. Furthermore, we know that stage 5 gametocytes 
are not affected by antimalarial treatments that are aimed at clearing parasites in clinical 
malaria, and therefore we cannot exclude the possibility that such low levels of 
circulating gametocytes may be detected by the DNA amplification methodology used in 
this study. Hence we aim to understand how antimalarial treatment impacts the detection 
and definition of a new genotype in a subject previously identified as having an identical 
haplotype detected at a previous time point prior to antimalarial treatment. In geographic 
areas that have low parasite transmission, we predict that the complexity of infection will 
be reduced to less than 2 genotypes and therefore any recurrent haplotype may indeed be 
the basis for a new infection from a new mosquito bite (we do not assume that this is 
operative in Siaya, Kenya or Agogo, Ghana).
2.4.5.
Exploratory objective 4

To understand how gaps in time as measured by consecutive monthly dried blood 
spots impact VE against new infections. 
Since parasites are not necessarily present in the peripheral circulation continuously over 
time because of parasite sequestration in the deep vascular beds, the absence of the same 
genotype(s) detected on successive monthly blood sampling does not exclude the 
possibility that the same parasite genotype may be still present in the subject, although 
undetected. For example, genotype A is present at Month 1, absent at Month 2, but 
genotype A is detected again at the Month 3 time point and may represent either 
persistent parasites or a new infection from a mosquito bite occurring in the interval 
between Month 2 and Month 3. We will explore whether such genotypes represent a new 
infection or represent persistent but undetected parasites from an antecedent infection.
3.
RESEARCH DESIGN AND METHODS
3.1.
Study Design Overview
This study is proposed to be an ancillary of the GSK RTS,S/AS01E phase IIb randomized 
trial MALARIA-094 (Study Number 204889) to be performed across all study sites. The 
incidence of P. falciparum infection defined by positive amplicon sequencing detection, 
and subsequent parasite genotyping, will be evaluated using samples collected as 
described in the MALARIA-094 trial.

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The following hierarchical approach is proposed for sample evaluation to investigate the 
study objectives, with Tier 1 sampling essential to addressing the Primary Objective, and 
Tier 2 sampling to be performed after the primary immunization series and booster 
immunization series. Two analyses will be performed:
Tier 1:
All cross-sectional dried blood spot samples collected monthly from all subjects 
during the first 20 months (approximately 31,500 samples) and clinical malaria 
cases (approximately 14,400) of the primary series from all arms of the study.
Tier 2:
All cross-sectional dried blood spot samples collected for three consecutive 
months and then quarterly thereafter for one year following the first booster 
RTS,S dose (approximately 9,000 samples) and clinical malaria cases in all 
arms of the study up through Month 32 (approximately 8,640 samples). 
In this genomic study interests include: i) assessing VE using molecular genetic data to 
identify first infections following vaccination, and to distinguish new from existing 
infections, ii) determining parasite positivity in cross-sectional samples from all children 
and distinguish new versus existing infections using an amplicon sequencing-based 
strategy: deep sequencing of small, highly variable regions of the parasite genome to 
allow for highly sensitive detection of parasitemia (analogous to conventional PCR-based 
detection), and iii) identification of genetically distinct parasite populations within or 
between affected individuals.
3.2.
Outcomes, Predictors, and Other Covariates of Interest
3.2.1.
Outcomes
The outcomes of interest will be: a) amplicon sequence-based parasite positive/negative 
status - positive status will be defined as ≥200 sequence reads aligning to at least one 
amplicon; and b) genotype data for the CS C-terminus and SERA2 loci – haplotypes 
observed at each amplicon following filtration of PCR and sequencing errors must be 
supported by 1% and at least 5 sequence reads aligned. 
3.2.2.
Main exposures
The main predictor of interest will be vaccination group as defined in MALARIA 094. 
3.2.3.
Candidate confounders
When studying all aims, additional predictors may be considered such as study site, age, 
date of infection or disease, parasitemia, time of detection from baseline, bednet use,
number of antimalarial treatments, and relevant clinical characteristics, as available. 

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3.3.
Study Cohort
3.3.1.
Overview
Information about the study cohort(s) can be found in the main trial protocol MALARIA-
094 (Study Number 204889). 
3.3.2.
Endpoints
Cohorts of analyses include According-To-Protocol (ATP) and Total Vaccinated Cohorts 
(TVC) which are described in the MALARIA-094 protocol. Analyses as per this study 
will be conducted on the same cohort of study participants with the following Primary 
Endpoints:
1. Time to the first new malaria infection
2. Number of new malaria infections during a specified follow-up period
A new malaria infection endpoint is defined by a detected new infection from genomic 
analysis of blood spots detected from active screening of infection or passive detection of 
clinical malaria (secondary case definition [no parasitemia threshold] as defined in the 
MALARIA-094 protocol); thus it measures either an asymptomatic or clinical malaria 
infection of a new genotype (primary objective) not previously detected either at baseline 
or in a prior sampling timepoint. Exploratory endpoints may define a “new” genotype 
less conservatively than the definition above. 
Any participant may be excluded from the ATP analysis according to the criteria outlined 
in the main trial protocol (204889; MALARIA-094). Furthermore, exclusion of samples 
from the final analysis may occur if the sample: is not collected; is not collected with full 
or clear identification; does not yield any parasite genetic material upon process; or does 
not yield a clear and unambiguous genotyping result for any reason.
3.3.3.
Total vaccinated cohort (TVC) and according-to-protocol (ATP) 
analyses
VE against each primary endpoint will be assessed using both a TVC and ATP analysis. 
For the primary endpoints these analyses will be based on data collected up to Month 20.  
For primary endpoint 1, the time to event is measured from the date of first vaccination in 
the TVC analysis and from the date of Dose 3 plus 14 days in the ATP analysis. For 
primary endpoint 2, the TVC analysis estimates the number of new malaria infections 
occurring from the date of first vaccination until Month 20, and the ATP analysis 
estimates the number of new malaria infections occurring from the date of Dose 3 plus 14 
days until Month 20.  
3.4.
Parasite Genotyping Methodology
The Broad Institute Genomics Platform implements strict quality control guidelines to 
reduce the likelihood of sample contamination. Appropriate and rigorous data cleaning 
for all single nucleotide polymorphisms (SNPs) called will be performed. Unreliable 

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sequencing or genotyping data will be excluded from the analysis. All unused or 
discarded samples will be noted and reported as described in the main trial protocol 
MALARIA-094.
Current technology exists to accomplish the specific aims of this proposal and has been 
optimized and validated on samples from the MALARIA-055 phase 3 study (see 
Supplementary Appendix, Neafsey et al., 2015 for details). Briefly, custom workflows 
developed at the Broad Institute will be used to extract DNA from filter paper samples, 
PCR amplify the DNA, and sequence it via the Illumina MiSeq platform. Whatman FTA 
blood spot cards will be batched and shipped according to an agreed schedule from the 
GSK-designated clinical research organization (CRO) in Africa to the Broad Institute 
Genomics Platform (GP) under a Center for Disease Control (CDC) import permit. GSK 
barcodes will be registered in the GP laboratory information management system 
(LIMS), and the cards will be stored securely in desiccator cabinets at room temperature 
until further processing.
Genomic DNA extraction will be performed in batches of 96 FTA cards, including one 
blank FTA card as a control. For each FTA card, disks will be punched out of the blood 
spot, using an automated laser guided hole puncher, into a distinct well of 96 well plates. 
Genomic DNA will be extracted from the punches using the automated Chemagen 
Chemagic bead-based DNA extraction platform using standard protocols. DNA samples 
will be registered in LIMS and stored in barcoded tubes. The DNA concentration of each 
sample will be quantified using standard automated PicoGreen quantification. 
Plasmodium PCR amplicons; “CS C-terminus” and “SERA2”, will be amplified as 
previously described [Neafsey et al., 2015]. Samples will be batched into groups, 
typically 384 (4 sets of 96), with a negative control sample (originating from a blank 
blood spot card) included within each set of 96. For each batch, CS C-terminus and 
SERA2 PCR2 product pools will be normalized and combined into a single pool for 
MiSeq sequencing. Sequencing data will be processed through the Broad Picard 
sequencing analysis pipeline generating standard sequencing metrics (e.g. read counts) 
and demultiplexed sample specific sequencing read Binary Alignment/Map (BAM) files. 
BAM files will be screened and filtered for human contaminating sequences, and 
packaged for submission to the National Center for Biotechnology Information (NCBI)
short read archive database. All steps will be tracked in the LIMS. 
3.5.
Conduct of the Study
3.5.1.
Follow-up and identification of cases
Study visits, cross-sectional surveys and surveillance for clinical and severe malaria cases 
are detailed in the main trial protocol (204889; MALARIA-094). Blood smears and filter 
paper materials (Whatman FTA Cards) will be obtained within the current protocol as 
detailed in the main trial protocol (204889; MALARIA-094). Additionally, blood smears 
and filter paper materials will be collected from any participant diagnosed with clinical 
malaria disease or severe malaria disease throughout the trial period, according to the 
case definitions defined in the main trial protocol (204889; MALARIA-094).   

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3.5.2.
Laboratory assays
All parasitological and clinical patient assessments will be performed as part of the 
vaccine trial procedures in compliance with the main trial protocol (204889; MALARIA-
094). 
3.5.3.
Additional study procedures
Subject identification, sample handling, and safety monitoring will be performed in 
compliance with the main trial protocol (204889; MALARIA-094). All processes used to 
transfer information to analysis sites will be defined in collaboration with GSK.  
4.
INVESTIGATIONAL PRODUCTS AND ADVERSE 
EVENTS
This study will be using the same investigational products as the main trial protocol 
(204889; MALARIA-094) and adverse events will be collected under the MALARIA-
094 protocol.
5.
STUDY TIMELINE 
The study timeline will follow the main trial protocol (204889; MALARIA-094) for 
collection times; parasite genotyping and sequence analysis will occur during the trial 
period. Results of genotyping will be kept in a database in Boston. Only the Principal 
Investigators (PIs) listed in this protocol, sub-Investigators and other staff members of the 
Broad Institute and Harvard T. H. Chan School of Public Health, designated by the PIs,
will have access to the genotyping database before the end of the study. 
6.
STATISTICAL CONSIDERATIONS
Primary endpoints, TVC and ATP analyses are presented in Section 3.3.2 and Section 
3.3.3.
6.1.
Analytical Plan
A formal Statistical Analysis Plan (SAP) will be finalized and approved by all parties 
prior to data un-blinding. All genetic material and sequence analysis will be performed 
through the study period in a schedule agreed upon by GSK.
Briefly, an alpha level of 0.05 will be used to test statistical significance. All statistical 
tests will be two-sided. Baseline characteristics of interest will be summarized by the 
appropriate comparison group. The number of filter paper samples lost or not collected 
will also be tabulated by comparison groups, as well as PCR negative results or any other 
reason for excluding a sample from the analysis. In mixed infections, it is expected that 
sequencing will yield the genotype of the more prevalent strain(s) or a heterozygous call. 
All analyses across study sites will be adjusted for study site. Analysis stratified by study 
site will also be presented.

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Primary Endpoint 1. Instantaneous VE against the first new malaria infection endpoint 
observed during 12 months after the third immunization in each vaccine arm vs. the 
common control vaccine arm will be assessed using a proportional hazards model with 
vaccine group indicator as the lone covariate, with the model stratified by study site (i.e., 
allowing a separate unknown baseline hazard of the first new malaria infection at each 
study site). The same proportional hazards modeling approach will be used for comparing 
the hazard of the first new malaria infection between pairs of vaccine arms, except hazard 
ratios will be assessed instead of VE parameters. A secondary analysis of the first new 
malaria infection endpoint will estimate instantaneous VE over time for each vaccine arm 
versus the control vaccine arm using nonparametric kernel smoothing with pointwise and 
simultaneous confidence bands [Gilbert et al., 2002] and using flexible partial-likelihood 
based proportional hazards modeling, for example using time-varying coefficients in the 
Cox model modeled with smoothing splines or using smoothed Schoenfeld residuals 
[Durham et al., 1998]. 
Overall VE against the first new malaria infection (Primary Endpoint 1) and VE against 
genotype-specific new malaria infection (with genotype defined by full vaccine match, a 
mismatch, or a genetic distance) (Exploratory Endpoint 1) will be estimated using both 
cumulative incidence and hazard ratio methodologies, with multiple outputation 
[Follmann et al., 2003] to analyze data from complex infections, as in MALARIA-066. 
Primary Endpoint 2. Plots of estimates of reverse cumulative distribution functions 
(rcdfs) of the number of new malaria infections by study arm will be used to summarize 
the distributions of the number of new infections. VE against all new infections for a 
given vaccine arm vs. the control arm will be assessed in two ways. First, VE will be
analyzed by negative binomial regression allowing for interdependence between episodes 
within the same participant (non-linear mixed model with overdispersion parameter 
estimated from the random effect and follow-up time entered into the model as an offset) 
and adjusting for study site. Second, VE against all new infections will be assessed by the 
additive difference in the mean number of all new malaria infections in each vaccine arm 
vs. the mean number of all new malaria infections in the control vaccine arm.  
6.2.
Sample Size and Power
For the MALARIA-094 study’s primary endpoint, demonstrating incremental VE against 
clinical malaria of a Fx dose schedule over the standard schedule, the study has at least 
90% power to show a significant incremental VE (lower limit [LL] of 95% CI above 0), 
assuming 250 evaluable subjects per group and an incidence of at least 0.5 episodes/per 
year (pyr) in the Control group, 0.29 episodes/pyr in the standard schedule groups and 
0.16 episodes/pyr in the Fx dose schedule. This corresponds to an incremental efficacy 
(1-Hazard Rate) of 50% of the Fx dose schedule over the standard schedule. The 
MALARIA-094 study’s primary endpoint compares the R012-20+R012-14-mD groups 
versus the Fx012-14-mFxD group (2:1). For the MALARIA-094 study’s secondary 
endpoints evaluating efficacy against P. falciparum infections, it is anticipated that 
incidence of infection in the study will be higher than incidence of clinical malaria 
resulting in adequate power.

A n cill ar y G e n ot y pi n g Pr ot o c ol i n s u p p ort of 2 0 4 8 8 9 ( M A L A RI A -0 9 4 ) Pr ot o c ol
H ar v ar d T. H. C h a n S c h o ol of P u bli c H e alt h
Pr ot o c ol N u m b er: 2 0 6 2 1 3 ( M A L A RI A -0 9 5 )
Fi n al
C O N FI D E N TI A L
0 8- M a y -2 0 1 7
- 2 4 -
7.
D A T A  MA N A G E M E N T M E T H O D S
All data c ollecte d f or t his st u d y  will be part of t he case re p ort f or ms ( C R Fs) ori gi nally 
desi g ne d f or t he mai n trial pr ot oc ol M A L A RI A -0 9 4. Sa m ples will be trac ke d usi n g t he 
LI M S s ys te m i n t he Br oa d Ge n o mics Platf or m; ra w a n d filtere d data will be st ore d i n a 
Str uct ure d Q u er y La n g ua ge (S Q L ) data base o n a sec ure Br oa d ser ver wit h user 
per missi o ns restricte d t o desi g nate d st u d y  pers o n nel. T he c li nical data ma na ge me nt 
s yste m will be o versee n b y G S K. E xc ha n ges of cli nical a n d ge netic data will be 
perf or me d acc or di n g t o a m ut ual l y a gree d sc he d ule a n d ter ms. 
8.
P R O T E C TI O N O F H U M A N  R E S E A R C H S U B J E C T S
N o a nal ys is of h u ma n D N A will be perf or me d i n t his st u d y . Sa m ples will be c ollecte d 
e xcl usi vel y f or parasite ge n ot ypi n g. T heref ore, t his st u d y  will n ot res ult i n a n y ris ks, le gal 
or et hic al c o ncer ns f or s u bjects ot her t ha n t he o nes disc usse d i n t he o ri gi nal st u d y 
pr ot oc ol ( M A L A RI A -0 9 4). S u bjects’ disc o nti n uati o n of t he st u d y will als o f oll o w 
pr oce d ures s pecifie d i n t he mai n pr ot oc ol. I nf or me d c o nse nt f or t he ge n ot y pi n g st u d y will 
be i ncl u d e d i n t he i nf or me d c o nse nt of t he mai n pr ot oc ol. 
9.
S T U D Y O R G A NI Z A TI O N

Dr.
, t he lea der of t he Har var d/Br oa d Malaria Ge n o me pr oject will 
o versee t he w h ole pr oject, pla y  a s u per vis or y r ole a n d pr o vi de scie ntific g ui da nce. 

Dr. 
 will ha ve a ke y r ole i n t he de vel o p me nt a n d c h oice of 
a p pr o priate tec h n ol o g y f or e val uati n g parasite ge netics a n d i n b ot h parasite 
ge n ot y pi n g a n d statistical a nal ys is.

Dr. 
 will o versee all parasite ge n ot ypi n g a n d will be res p o nsi ble 
f or c o m m u nicati o ns wit h ot her i n vesti gat ors.

Dr. 
will pr o vi de c o ns ultati o n o n la b orat or y  pr ocesses a n d scie ntific 
g ui da nce.

Dr. 
, Dr. 
a n d Dr.
will desi g n a n d 
o versee t he statistical a nal y sis of t he ge n oty pi n g da ta.

Mr. 
 will c o ntri b ute t o scie ntific disc ussi o ns, as nee de d. 

Dr. 
 will facilitate c o m m u nicati o ns, a n d e xc ha n ge of material 
a n d data a m o n g site i n vesti gat ors, G S K me m bers, t he Har var d T. H. C ha n Sc h o ol of 
P u blic Healt h a n d Br oa d I nstit ute , a n d t he Fre d H utc hi ns o n Ca ncer Researc h Ce nter.

Mr. 
 will ha ve a n a d vis or y r ole i n t he Ge n oty pi n g st u d y t hat i n v ol ves 
statistical desi g n a n d a nal ysis of t he ge n oty pi n g i nfecti o n e n d p oi nts.    
P P D
P P D
P P D
P P D
P P D
P P D
P P D
P P D
P P D
P P D

Ancillary Genotyping Protocol in support of 204889 (MALARIA-094) Protocol
Harvard T. H. Chan School of Public Health
Protocol Number: 206213 (MALARIA-095)
Final
CONFIDENTIAL
08-May-2017
- 25 -
10.
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Harvard T. H. Chan School of Public Health
Protocol Number: 206213 (MALARIA-095)
Final
CONFIDENTIAL
08-May-2017
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Ancillary Genotyping Protocol in support of 204889 (MALARIA-094) Protocol
Harvard T. H. Chan School of Public Health
Protocol Number: 206213 (MALARIA-095)
Final
CONFIDENTIAL
08-May-2017
- 27 -
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