Evaluation of RTS,S/AS02A and RTS,S/AS01B in Adults in a High Malaria Transmission Area

Background This study advances the clinical development of the RTS,S/AS01B candidate malaria vaccine to malaria endemic populations. As a primary objective it compares the safety and reactogenicity of RTS,S/AS01B to the more extensively evaluated RTS,S/AS02A vaccine. Methodology A Phase IIb, single centre, double-blind, controlled trial of 6 months duration with a subsequent 6 month single-blind follow-up conducted in Kisumu West District, Kenya between August 2005 and August 2006. 255 healthy adults aged 18 to 35 years were randomized (1∶1∶1) to receive 3 doses of RTS,S/AS02A, RTS,S/AS01B or rabies vaccine (Rabipur®; Chiron Behring GmbH) at months 0, 1, 2. The primary objective was the occurrence of severe (grade 3) solicited or unsolicited general (i.e. systemic) adverse events (AEs) during 7 days follow up after each vaccination. Principal Findings Both candidate vaccines had a good safety profile and were well tolerated. One grade 3 systemic AE occurred within 7 days of vaccination (RTS,S/AS01B group). No unsolicited AEs or SAEs were related to vaccine. A marked increase in anti-CS antibody GMTs was observed post Dose 2 of both RTS,S/AS01B (31.6 EU/mL [95% CI: 23.9 to 41.6]) and RTS,S/AS02A (16.7 EU/mL [95% CI: 12.9 to 21.7]). A further increase was observed post Dose 3 in both the RTS,S/AS01B (41.4 EU/mL [95% CI: 31.7 to 54.2]) and RTS,S/AS02A (21.4 EU/mL [95% CI: 16.0 to 28.7]) groups. Anti-CS antibody GMTs were significantly greater with RTS,S/AS01B compared to RTS,S/AS02A at all time points post Dose 2 and Dose 3. Both candidate vaccines produced strong anti-HBs responses. Vaccine efficacy in the RTS,S/AS01B group was 29.5% (95% CI: −15.4 to 56.9, p = 0.164) and in the RTS,S/AS02A group 31.7% (95% CI: −11.6 to 58.2, p = 0.128). Conclusions Both candidate malaria vaccines were well tolerated over a 12 month surveillance period. A more favorable immunogenicity profile was observed with RTS,S/AS01B than with RTS,S/AS02A. Trial Registration Clinicaltrials.gov NCT00197054


Final, 13 June 2006
Title A Phase IIb randomized, double-blind, controlled study of the safety, immunogenicity and proof-of-concept of RTS,S/AS02A, and RTS,S/AS01B, two candidate malaria vaccines in malaria-experienced adults living in Western Kenya.

BB-IND 11220
Title A Phase IIb randomized, double-blind, controlled study of the safety, immunogenicity and proof-ofconcept of RTS,S/AS02A, and RTS,S/AS01B, two candidate malaria vaccines in malaria-experienced adults living in Western Kenya. I agree: • To assume responsibility for the proper conduct of the study at this site.
• To conduct the study in compliance with this protocol, any mutually agreed future protocol amendments, and with any other study conduct procedures provided by GlaxoSmithKline Biologicals (GSK Biologicals). • Not to implement any changes to the protocol without agreement GSK Biologicals and prior review and written approval from the Institutional Review Board (IRB) or Independent Ethics Committee (IEC), except where necessary to eliminate an immediate hazard to the subjects, or for administrative aspects of the study (where permitted by all applicable regulatory requirements). • That I am thoroughly familiar with the appropriate use of the vaccines, as described in this protocol, and any other information provided by the sponsor (USAMMDA) and GSK Biologicals, including, but not limited to, the following: the current Investigator's Brochure (IB) or equivalent document, IB supplement (if applicable), prescribing information (in the case of a marketed vaccine) and/or Master Data Sheet (if the Master Data Sheet exists and serves as reference document for the vaccine in the case of a marketed vaccine). • That I am aware of, and will comply with, "Good Clinical Practices" (GCP) and all applicable regulatory requirements. • That I have been informed that certain regulatory authorities require the sponsor (USAMMDA) to obtain and supply, as necessary, details about the investigator's ownership interest in the sponsor or the investigational product, and more generally about his/her financial ties with the sponsor. GSK Biologicals will use and disclose the information solely for the purpose of complying with regulatory requirements.
Hence I: • Agree to supply GSK Biologicals with any necessary information regarding ownership interest and financial ties (including those of my spouse and dependent children). • Agree to promptly update this information if any relevant changes occur during the course of the study and for 1 year following completion of the study.
• Agree that GSK Biologicals may disclose any information it has about such ownership interests and financial ties to regulatory authorities. • Agree to provide GSK Biologicals with an updated Curriculum Vitae and other FDA required documents.
A Local Safety Monitor and Safety Monitoring Group will provide safety oversight and guidance for this trial.
This product development plan is conducted under a CRADA between WRAIR and GSK Biologicals.

Objectives Primary: Safety & Reactogenicity
• To compare the safety and reactogenicity of RTS,S/AS01B vs RTS,S/AS02A, when administered as 3 doses intramuscularly on a 0, 1 and 2 months schedule to adult volunteers living in a malaria endemic region

Secondary: Safety
• To describe the safety and reactogenicity of RTS,S/AS02A and RTS,S/AS01B, when administered as 3 doses intramuscularly on a 0, 1 and 2 months schedule to adult volunteers living in a malaria endemic region.

Secondary: Immunogenicity
• To describe antibody responses to the circumsporozoite (anti-CS) antigen and hepatitis B surface antigen (anti-HBs) of RTS,S/AS02A and RTS,S/AS01B when administered as 3 doses intramuscularly on a 0, 1 and 2 months schedule to adult volunteers living in a malaria endemic region.

Secondary: Proof-of-concept
• To assess the efficacy of RTS,S/AS02A and RTS,S/AS01B against infection with P. falciparum malaria (defined as P. falciparum asexual parasitemia > 0) over a period of 14 weeks post Dose 3, in adults under conditions of natural transmission.

Amended 13 June 2006
Study design • Phase IIb, single-center, double blind (observer blind, participant blind), randomized controlled trial with three groups in one study site (randomization 1:1:1, RTS,S/AS01B: RTS,S/AS02A:rabies vaccine). • 255 adults will be enrolled. It is assumed that 90% will complete study procedures and be evaluable. • Prior to study start, a community information program will inform the local population of the study. Throughout the period of enrollment, study information will be presented at community centers. • Healthy male and female adults aged 18 to 35 years will be screened. Those determined to be eligible, based on the inclusion and exclusion criteria, will be enrolled in the study. • Route of administration: all vaccines will be administered by the intramuscular route to the deltoid of the non-dominant arm • Each subject will be observed for at least 30 minutes after vaccination to evaluate and treat any acute adverse events • After vaccination, the subjects will visit field workers at field stations for the detection of adverse events (AEs) occurring after vaccination for one week (days 1, 2, 3, 4, 5 and day 6 after each vaccination). Diary cards and thermometers will be provided for the field workers to record oral temperature and any local (at the injection site) or general adverse events. • One week prior to Dose 3, subjects will be treated with Malarone ® for presumptive clearance of malaria parasitemia (in the event that subjects are unable to tolerate Malarone ® , Coartem ® (artemether/lumefantrine, Novartis AG, Zurich, Switzerland) will be used as the alternative clearance treatment. • Surveillance for Active Detection of Infection (ADI) will begin 2 weeks after Dose 3. Subjects will visit field workers at field-stations near their homes weekly during the ADI period. The total period of surveillance for ADI is 14 weeks. Two Clinic Visits and 14 field-worker visits will contribute to the ADI. • All subjects will have a blood slide prepared and read to check for asexual P. falciparum parasitemia one week post Dose 3. Any subject who tests positive will be treated with Coartem ® • At each contact for ADI, history of fever will be recorded and oral temperature taken; a smear will be taken for detection of malaria parasites. • Infection with malaria parasites is defined as P. falciparum asexual parasitemia > 0 • Subjects who are symptomatic at the time of ADI contact (i.e. history of fever within previous 24 hours or oral temperature >37.5°C) will have a blood slide read and treated within the same day if the blood slide is positive. • Subjects will be followed passively for the occurrence of clinical malaria from Day 0 until 16 weeks post Dose 3. Cases will be detected at Kombewa Clinic. If a subject feels ill they can present to a field station near their home and be transported to Kombewa Clinic for evaluation. • DNA will be extracted from EDTA blood of all subjects with patent asexual parasitemia. Amended 13 June 2006 • There will be a 30 day follow-up period after each dose of vaccine for reporting unsolicited symptoms • Recording of serious adverse events will be throughout the study period. They will be captured through the morbidity surveillance system at Kombewa Clinic. • Anti-CS antibody titers will be determined at baseline, one month post Dose 2, 1 month post Dose 2, 1 month post Dose 3, 4 months post Dose 3 and 10 months post Dose 3. • Anti-HBs antibody titers will be determined at baseline, one month post Dose 3 and 10 months post Dose 3. • CS-specific CMI will be assessed at three four time points during the study; prevaccination, 1 month post Dose 3, and 4 months post Dose 3 and 9 months post Dose 3. Amended 13 June 2006 • Data collection will be by conventional CRF.
• Duration of the study will be approximately 12 months per subject.
• There will be a planned interim analysis on all safety humoral and immunogenicity data collected up to 30 days after Dose 3. • The final analysis of the study for all primary and secondary endpoints will take place on all data collected up to the end of the surveillance for ADI (16 weeks post Dose 3). • All subjects will be followed for safety for a period that extends to 10 months post Dose 3. Any data collected after the final analysis will be reported in an annex report.

Sample size
• Primary Objective: The primary objective will be assessed by comparing the proportion of subjects experiencing a Grade 3 solicited or unsolicited general reaction after each vaccination during a seven day follow-up period (day of vaccination and six subsequent days). In a previous study with RTS,S/AS02A in Africa (Malaria-008) the proportion affected was 10%. The proportion affected was higher in a recent study conducted in non-immune adults at WRAIR (Malaria-027); 39%. Therefore the table below has been generated for a wide range of hypothetical proportions of Grade 3 general symptoms. The table shows the potential of the study to detect a difference in the proportion of subjects affected by Grade 3 general symptoms assuming 90% power and a sample size of 255 at a significance level of p < 0.05. • Secondary Objective; Safety: The data set will be examined, comparing the rates of SAEs at the Medical Dictionary for Regulatory Activities (MedDRA) preferred term level. SAEs are expected to occur infrequently and the study has therefore the power to detect only large differences in the frequencies of SAEs compared to control. See • Occurrence of Grade 3 solicited or unsolicited general reactions after each vaccination during a seven day follow-up period (day of vaccination and 6 subsequent days)

Secondary endpoints: Safety
• Occurrence of SAEs until 4 months post Dose 3.
• Occurrence of unsolicited AEs after Dose 1, 2 and 3 of vaccine over a 30 day follow-up period (day of vaccination and 29 subsequent days) • Occurrence of solicited general and local reactions over a 7 day follow-up period (day of vaccination and 6 subsequent days) after Dose 1, 2 and 3 of vaccine. • Occurrence of parameters of hematological monitoring below normal range. • Occurrence of parameters of biochemical monitoring above normal range • The time to first malaria infection (first recording of infection of asexual stage falciparum parasites detected by the active case detection or passive case detection) over a period starting 14 days after Dose 3 and extending for 14 weeks. • For each subject, the frequency of CS-specific CD4+ and CD8+ Tcells, as measured by intracellular cytokine staining.

Glossary of Terms
Adverse event: Any untoward medical occurrence in a patient or clinical investigation subject, temporally associated with the use of a medicinal product, whether or not considered related to the medicinal product.
An AE can therefore be any unfavorable and unintended sign (including an abnormal laboratory finding), symptom, or disease (new or exacerbated) temporally associated with the use of a medicinal product. For marketed medicinal products, this also includes failure to produce expected benefits (i.e. lack of efficacy), abuse or misuse.
Active detection of Infection (ADI): The detection of malaria parasitemia on a blood sample taken at a scheduled or unscheduled visit during a defined ADI phase of the study, i.e. regardless of whether a subject has signs or symptoms of infection.

Blinding:
A procedure in which one or more parties to the trial are kept unaware of the treatment assignment in order to reduce the risk of biased study outcomes. In a single-blind trial, the investigator and/or his staff are aware of the treatment assignment but the subject is not. In an observer-blind study, the subject and the study personnel involved in the clinical evaluation of the subjects are blinded while other study personnel may be aware of the treatment allocation. When the investigator and sponsor staff who are involved in the treatment or clinical evaluation of the subjects and review/analysis of data are also unaware of the treatment assignments, the study is double blind. Partially blind is to be used for study designs with different blinding levels between different groups, e.g. double blinded consistency lots which are open with respect to the control group. The level of blinding is maintained throughout the conduct of the trial, and only when the data are cleaned to an acceptable level of quality will appropriate personnel be unblinded or when required in case of a serious adverse event.

Central Study
Coordinator: An individual assigned by and centrally located at GSK Biologicals at Rixensart who is responsible for assuring proper conduct of a clinical study. Evaluable: Meeting all eligibility criteria, complying with the procedures defined in the protocol, and, therefore, included in the accordingto-protocol (ATP) analysis (see Sections 4.3, 4.4 and 10.6 for details on criteria for evaluability).
Investigational product: A pharmaceutical form of an active ingredient or placebo being tested or used as a reference in a clinical trial, including a product with a marketing authorization when used in a way different from the approved form, or when used for an unapproved indication, or when used to gain further information about an approved use.
Local Safety Monitor (LSM): The overall role of the Local Safety Monitor, an experienced physician based in-country, will be to support the study investigators and to act as a link between the investigators and the Safety Monitoring Group (SMG) (see Section 5.1.3.2 for further details).

Medical
Monitor: An individual medically qualified to assume the responsibilities of the GSK Biologicals especially in regards to the ethics, clinical safety of a study and the assessment of adverse events.
Protocol amendment: ICH defines a protocol amendment as: "A written description of a change(s) to or formal clarification of a protocol". GSK Biologicals further details this to include a change to an approved protocol that affects the safety of subjects, scope of the investigation, study design, or scientific integrity of the study.
Protocol administrative change: A protocol administrative change addresses changes to only logistical or administrative aspects of the study.
N.B. Any change that falls under the definition of a protocol amendment (e.g. a change that affects the safety of subjects, scope of the investigation, study design, or scientific integrity of the study) MUST be prepared as an amendment to the protocol.

Randomization:
Process of random attribution of treatment to subjects in order to reduce bias of selection SMG: The SMG is a group of medical experts representing the institutions participating in the study, appointed to oversee safety aspects of the conduct of the study. See Section 5.1.3.2 for a full overview of the role and structure of the SMG.
Solicited adverse event: Adverse events (AEs) to be recorded as endpoints in the clinical study. The presence/occurrence/intensity of these events is actively solicited from the subject or an observer during a specified postvaccination follow-up period. Study Monitor: An individual assigned by the sponsor, USAMMDA or GSK Biologicals who is responsible for assuring proper conduct of a clinical study.
Subject: Term used throughout the protocol to denote an individual that has been contacted in order to participate in the clinical study, either as a recipient of the investigational product(s) or as a control.
Treatment: Term used throughout the clinical study to denote a set of investigational product(s) or marketed product(s) or placebo intended to be administered to a subject, identified by a unique number, according to the study randomization or treatment allocation.

Treatment number:
A unique number identifying a treatment to a subject, according to the study randomization or treatment allocation.
Unsolicited adverse event: Any adverse event (AE) reported in addition to those solicited during the clinical study. Also any "solicited" symptom with onset outside the specified period of follow-up for solicited symptoms will be reported as an unsolicited adverse event. INTRODUCTION GSK Biologicals and the Walter Reed Army Institute of Research (WRAIR) are developing a candidate antigen against malaria caused by Plasmodium falciparum, RTS,S. The antigen consists of sequences of the circumsporozoite (CS) protein and hepatitis B surface antigen (HBsAg).
When adjuvanted with AS02, a proprietary adjuvant made up of oil-in-water emulsion, MPL ® and QS21, RTS,S has been found to be highly immunogenic. In human challenge studies conducted at WRAIR, the RTS,S/AS02 candidate vaccine prevented or delayed infection.
Vaccines containing the RTS,S antigen are being developed by GSK Biologicals primarily for the prevention of disease in children in malaria-endemic countries, and therefore most of the data referring to the safety, immunogenicity and reactogenicity has, to date, been collected in the pediatric population in Africa. In a recent study in which 3 doses of RTS,S/AS02A (0.25 mL dose) were administered to approximately 1000 1 to 4 year old children in Mozambique, 30% Vaccine Efficacy (VE) was demonstrated against clinical malaria disease.
As a potential improvement to RTS,S/AS02A, another candidate vaccine, RTS,S/AS01B, is being developed in parallel. The AS01B adjuvant contains the same quantities of MPL ® and QS21 as in AS02A, but is formulated with liposomes rather than oil-in-water emulsion. Recent preliminary results of a human challenge study conducted at WRAIR (Malaria-027) have been encouraging, indicating that RTS,S/AS01B may be more efficacious than RTS,S/AS02A. As a next step, this study proposes to evaluate RTS,S/AS01B and RTS,S/AS02A in adults in a malaria-endemic region under conditions of natural challenge.

Malaria
Four species of the Plasmodium protozoan parasite are the etiologic agents of malaria in humans (P. falciparum, P. vivax, P. ovale and P. malariae). Of these four parasites, P. falciparum is the major cause of severe morbidity and mortality.
There can be no doubt of the importance of P. falciparum malaria as a major cause of human suffering and economic drain across sub-Saharan Africa [Breman 2001a, Gallup 2001. In this region, it causes the deaths of between 0.5 and 2.0 million children every year and is the most common reason for admission to hospital, leading each year to about 300 million clinical episodes in children under five years [Breman 2001a].
The incidence of malaria in much of Africa is increasing for a variety of reasons: changes in agricultural practices, armed conflicts, migration of refugees, increasing drug resistance to conventional anti-malarial drugs, and insecticide resistance of the anopheline mosquito vectors. It is estimated that the number of cases of clinical malaria will more than double over the next 20 years without effective control. The burden of malaria at the country level correlates closely with the rate of economic development

Rationale for vaccine development
The RTS,S malaria vaccines are hypothesized to reduce frequency and severity of clinical disease episodes through their impact on the initial sporozoite and liver stage parasite burden, leading to a significant reduction of the infectious inoculum of liver stage merozoites released into the blood stream. This hypothesis is fully consistent with the data observed in clinical trials of RTS,S/AS02A to date. In the homologous sporozoite challenge model the vaccine prevents approximately 40% of individuals from becoming infected and a delay in the prepatent period of non-protected individuals is observed. [Kester, 2001]. In the field efficacy study in children (Malaria-026; refer to Section 1.3.3.2.2) the point estimate of VE against clinical disease was 30% (95% CI 11% to 45%), and higher against severe malaria; 58% (95% CI 16% to 81%). A vaccine that induces partial immunity against pre-erythrocytic stages of disease may provide protection to vulnerable young children from the severe forms of the disease, whilst continuing exposure allows them to build up natural bloodstage immunity. Acquisition of natural immunity may be important to prevent a shift of severe disease burden from younger to older age groups upon waning of vaccine-induced immunity.
In parallel to the continued development of RTS,S/AS02A and RTS,S/AS02D in children in endemic countries, GSK Biologicals and WRAIR have continued to pursue alternate strategies to improve the VE and duration of efficacy. One such strategy is the combination of the RTS,S antigen with the AS01B adjuvant. Both adjuvant formulations have a number of similar key components (refer to Section 1.3.1, Section 1.3.2 and Table  1). Also refer to Table 1

AS01B Adjuvant
The pre-clinical evaluation of new adjuvant formulations has led to the selection of AS01B as a potential candidate possibly capable of prolonging persistence of the immune response. AS01B is a liposome-based adjuvant containing MPL ® and QS21 (see Table  1).
Preclinical studies in mice and rhesus monkeys show that the AS01B adjuvant induces significantly superior antigen-specific cytotoxic T lymphocyte (CTL) and interferon gamma (IFN-gamma) responses than does AS02A, while humoral responses generated by both formulations remain equivalent. In rhesus monkey studies, AS01B also induces a cell-mediated response characterized by a more Th1-biased profile: a higher proportion of IFN-gamma-secreting lymphocytes than observed with AS02A. In toxicology studies in rabbits and primates the vaccine has been shown to be safe.
Most of the previous clinical experience with AS01B comes from a study of AS01Badjuvanted hepatitis B vaccine. In this double blind Phase I/II immunogenicity and safety study in adults, AS01B was formulated with hepatitis B surface antigen (HBsAg). The HBsAg used in this study is encoded by the same gene as that used to express HBsAg in the RTS,S antigen, and the commercially available hepatitis B vaccine (Engerix-B). A total of 146 doses of HBsAg/AS01B have been administered to date to 49 healthy adults according to a 0, 1, 10 month vaccination schedule. No SAEs deemed causally related by the investigator to vaccination have been reported. No clinically significant abnormal hematological or biochemical laboratory test results have been reported. Most solicited symptoms reported after vaccination with HBsAg/AS01B were not severe in intensity and resolved in the course of the 7-day follow-up period after vaccination. All unsolicited symptoms that were causally related to HBsAg/AS01B vaccination and of intensity Grade 3 (symptoms that prevented normal everyday activity) were local (injection site) adverse events and resolved within 2 days. In conclusion, the AS01B adjuvant formulated with HBsAg is safe and induces an acceptable amount of reactogenicity.
In the evaluation of immunogenicity, HBsAg/AS01B induced a high and persistent humoral immune response as shown by the presence of seroprotective levels of HBs antibodies 40 weeks after the second dose of vaccine and dramatic boosting of geometric mean titers of anti-HBs antibodies to levels above 900 000 mIU/mL after Dose 3. The cell-mediated immune (CMI) responses to vaccination were characterized by a HBsspecific cytotoxic response, marked lymphoproliferation and IFN-gamma secretion but low interleukin-5 secretion. These CMI results are indicative of a Th1-like immune response and confirm the immunogenicity profile established for the AS01B adjuvant in preclinical studies with RTS,S/AS01B. The RTS,S/AS02A vaccine has progressed through sequential stages of evaluation in trials in adults. Evaluation commenced with a series of trials to assess safety and proofof-concept using the malaria sporozoite challenge model in malaria-naïve adults at the laboratories of WRAIR. Assessments of safety and immunogenicity have also been carried out on malaria-naïve adults in Belgium. Most relevant to this trial, the vaccine was subsequently assessed in malaria-experienced adults in malaria-endemic countries in Africa. An overview of the study design of each of the trials of RTS,S/AS02A in adults can be found in Table 4.
To date 14 trials in adults have been completed. During the course of these trials 607 volunteers have received 1608 doses of RTS,S/AS02A.

Safety experience of RTS,S/AS02A in malaria-experienced adults in Africa
Three studies of RTS,S/AS02A have been carried out in adults in a malaria-endemic region of The Gambia (Malaria-003, Malaria-004 and Malaria-005) and another in adults in a malaria-endemic region of Kenya (Malaria-008). The largest trial in malariaexperienced adults to date has been Malaria-005. In this double-blind, randomized, controlled Phase IIb study in a malaria-endemic region of The Gambia, 250 men received 3 doses of the vaccine on a 0, 1 and 5 month schedule and were followed for up to 15 weeks. Subsequently, 158 of the men received a fourth dose during the peak malaria season of the following year and were followed for up to 9 weeks. The Malaria-008 study in Kenya was an open Phase I trial to investigate safety and immunogenicity of RTS,S/AS02A in 20 adults on a 0, 1 and 6 month schedule.
In both of these studies, the vaccine was safe and well tolerated (refer to Table 2 and  Table 3). Other trials of RTS,S/AS02A in malaria-experienced adults yielded comparable results.  Pain at the injection site (almost all mild to moderate in intensity) has been the most frequently reported local solicited symptom in adults in all trials. In malaria-naïve populations, the most frequently reported general solicited symptoms causally related to vaccination have been myalgia and fatigue (a small number of transient severe cases have been observed). In studies in Africa, headache and malaise were the most frequently reported.
Only one serious adverse event has been suspected of being causally related to vaccination with RTS,S/AS02A in adults to date. A chronic carrier of HBsAg had a raised ALT during the vaccination course of Malaria-005. ALT levels returned to normal within 2 weeks. In the comparator group of the same study, four recipients of rabies vaccine had similarly deranged ALT levels. ALT levels for these subjects returned to normal ranges within 4 weeks.

Efficacy of RTS,S/AS02A in adults
Evidence of efficacy comes from both the malaria sporozoite challenge model in malarianaïve adults in the laboratory and from field studies in malaria-experienced adults. The RTS,S/AS02A vaccine has been shown to protect between 42% and 86% of healthy

Safety and immunogenicity of alternative vaccination schedules in adults
Various vaccination schedules have been assessed in adults. These include 0, 1, 5 and 19 months (Malaria-005); 0, 1, 6 and13 months (Malaria-002) and 0, 1, and 9 months (WRMAL-004). All these schedules showed the vaccine to be immunogenic with acceptable reactogenicity in adults. There has been no significant increase in reactogenicity with a fourth vaccine dose. Data from a study conducted in the USA to evaluate a 0, 1 and 3-month and a 0, 7 and 28-day vaccination schedule in malaria-naïve adults (Malaria-012) indicates that the immune response and efficacy following sporozoite challenge are comparable to that after a 0, 1 and 6-month vaccination schedule . No changes in the safety or reactogenicity profile of the vaccine were identified with either schedule.

Studies of RTS,S/AS02A in adults; conclusions
Evaluation of RTS,S/AS02A has gradually progressed from laboratory evaluation of safety and proof-of-concept in malaria-naïve adults to evaluation in the field in Africa in malaria-experienced adults. The vaccine has been shown to be safe, with acceptable reactogenicity. Efficacy against malaria in both the laboratory and the field have been demonstrated.   These studies were to aid in the selection of the dose level to be used in the future pediatric development of the RTS,S/AS02 candidate vaccine.

Malaria
The solicited symptoms are displayed in Table 6 and Table 7 (corresponding to Malaria-015 and Malaria-020 respectively). Pain at the injection site was a frequent symptom across all study groups. The incidence of swelling increased with dose level. The majority of general solicited symptoms were mild to moderate in intensity and short lasting. In children aged 6 to 11 years headache was the most frequently occurring general solicited symptom in subjects receiving RTS,S/AS02A (any dose level). In the younger children (aged 1 to 5 years), the most frequently occurring general symptom in subjects receiving 0.1 mL dose RTS,S/AS02A was fever, reported after 11% of doses compared to 26% of doses in the rabies control group. In subjects receiving 0.25 mL dose RTS,S/AS02A, loss of appetite was the most frequent symptom, being reported after 12% of doses, which compared to 20% of doses in the control group. In subjects receiving 0.5 mL dose RTS,S/AS02A, irritability/fussiness was most frequently reported, occurring after 27% of doses compared to 12% of doses in the control group. Grade 3 general solicited symptoms were infrequent in both studies and resolved or decreased in intensity within 24 hours.
Unsolicited symptoms were recorded with similar (6 to 11 year-olds) or lower frequency (1 to 5 year-olds) in the study vaccine groups compared to the control vaccine groups. The majority of unsolicited symptoms were mild to moderate in intensity and unrelated to vaccination.
Hematocrit values were generally low but comparable between study groups. Six children in the RTS,S/AS02A groups (one in 0. to 30 days post Dose 3. There were no cases of anemia in the 0.5 mL or control groups. All these children had documented malaria episodes before or at the time that anemia was recorded.
All children were monitored for liver function. Among the 6 to 11 year-olds, two children in the RTS,S/AS02A groups experienced a transient rise in ALT levels judged not to be related to vaccination and not clinically relevant. In the younger children, increases in ALT levels from pre-vaccination were observed in 2 subjects in RTS,S/AS02A groups and 2 subjects in the rabies groups. Of these, only one case in the rabies control group was judged to be clinically relevant and vaccination was discontinued after Dose 1. No other clinically relevant abnormalities of hematological or biochemical laboratory parameters were observed.
In the interval to 30 days post Dose 3, 6 serious adverse events (SAEs) in total occurred. One SAE occurred in the 6 to 11 year old study; a case of bronchopneumonia was reported in a subject in the rabies-control group. The event was not considered causally related to vaccination and the subject made a full recovery. Five SAEs were reported in the 1 to 5 year old study. Among the RTS,S/AS02A recipients one subject suffered acute malaria with acute upper respiratory tract infection (0.1 mL dose) and one subject suffered cerebral malaria (0.25 mL dose). In the rabies control group three SAEs were reported: acute severe malaria with urinary tract infection and salmonella septicemia, bronchopneumonia with bronchial asthma and accidental death due to drowning. All SAEs were considered not to be related to study vaccines. Apart from the fatal SAE, all subjects made a full recovery and were not withdrawn from the study.
A total of 20 subjects out of 135 subjects enrolled in the Malaria-015 study were under 2 years of age. The safety and reactogenicity profiles of these subjects receiving the RTS,S/AS02A candidate vaccine were comparable to that seen in the older children. Table 8 details the antibody responses to anti-CS and Table 9 the antibody responses to anti-HBs for these two studies. All dose levels of RTS,S/AS02A (0.1 mL, 0.25 mL and 0.5 mL doses) were highly immunogenic for anti-CS and anti-HBs antibodies, irrespective of pre-vaccination HBsAg serostatus.
For both Malaria-015 and Malaria-020, all subjects receiving RTS,S/AS02A were seropositive for anti-CS antibodies post Dose 2. Post Dose 3 the lowest anti-CS antibody geometric mean titers (GMTs) occurred in the 0.1 mL RTS,S/AS02A dose group (Table  8 below). In the 0.25 mL and 0.5 mL RTS,S/AS02A dose groups, similar GMT values were recorded. All subjects in the RTS,S/AS02A groups were seroprotected for anti-HBs antibodies post Dose 2 in Malaria-015 and post Dose 3 (first post-vaccination assessment) in Malaria-020; the highest GMTs were observed in the 0.25 mL RTS,S/AS02A dose group in Malaria-015 and in the 0.5 mL RTS,S/AS02A dose group in Malaria-020 (Table 9   Grade 3  n/% = number/percentage of doses followed by a local/general symptom. *oral body temperature ≥ 37.5°C. Grade 3 limited arm motion; abduction at the shoulder < 30°. Grade 3 injection site pain; spontaneously painful. Grade 3 swelling; > 50 mm and persisting for more than 24 hours. Grade 3 fever; oral body temperature ≥ 39.0°C. For other symptoms; adverse event that prevents normal activity. Rel = related to study vaccine. Note: all local injection site symptoms were considered related to study vaccine.   n/% = number/percentage of doses followed by a local/general symptom. *oral body temperature ≥ 37.5°C. Grade 3 injection site pain; cries when limb is moved/spontaneously painful. Grade 3 swelling; > 20 mm. Grade 3 fever; oral body temperature ≥ 39.0°C. Grade 3 drowsiness; drowsiness that prevents normal activity. Grade 3 loss of appetite; not eating at all. Grade 3 irritability/fussiness; crying that cannot be comforted/prevents normal activity. Rel = related to study vaccine. Note: all local injection site symptoms were considered related to study vaccine.    The 0.25 mL dose RTS,S/AS02A was selected for future pediatric development, because it exhibited comparable immunogenicity to the 0.5 mL dose, with a tendency to a lower reactogenicity profile.

CONFIDENTIAL
Subsequent to these studies, RTS,S/AS02A (0.25 mL dose) was evaluated in a doubleblind, randomized, controlled Phase I study under a new schedule (0, 1, 2 month) in children aged 1 to 4 years in Mozambique (Malaria-025 [GSK data on file]). Subjects received RTS,S/AS02A (0.25 mL dose) or Engerix-B hepatitis B vaccine (GSK Biologicals, Rixensart, Belgium). The frequency of solicited symptoms per dose during the 4-day follow-up period for each vaccine group during the study is summarized in Table 10.
As previously observed in Malaria-020, local reactions at the site of injection were common. Unlike Malaria-020 where swelling at the injection site was the most frequently observed local reaction, pain was the most frequently observed reaction in Malaria-025. Grade 3 swelling was reported after 23% of doses of RTS,S/AS02A (0.25 mL dose), but did not occur following administration of Engerix-B. The incidence of swelling in the RTS,S/AS02A group decreased after Dose 2 of vaccine, but increased after Dose 3.
Few solicited general adverse events were reported. In the RTS,S/AS02A group the most frequently reported solicited adverse events related to vaccination were fever (9.5% after all doses) followed by loss of appetite (7.5% after all doses). In the Engerix-B comparator group, fever (1.2%) was the only solicited general adverse event related to vaccination. There was no trend in increase in solicited general adverse events related to vaccination with subsequent doses. Only one Grade 3 solicited general adverse event was reported in the study; one subject suffered fever in the RTS,S/AS02A group. All solicited general symptoms reported resolved within the 4 day follow-up period after vaccination. The frequency of solicited symptoms was similar to that previously observed in Malaria-020.
Unsolicited adverse events were recorded over a 30-day follow-up period after vaccination. Unsolicited symptoms were recorded with similar frequency in the study vaccine group compared to the control vaccine group. No unsolicited event was considered by the investigator to be related to vaccination. reflected the pattern of childhood morbidity expected in the population and unsolicited adverse events were balanced between treatment groups in terms of frequency and severity.  n/% = number/percentage of doses followed by a local/general symptom. *oral body temperature ≥ 37.5°C. Grade 3 injection site pain; cries when limb is moved/spontaneously painful. Grade 3 swelling; > 20 mm. Grade 3 fever; oral body temperature ≥ 39.0°C. Grade 3 drowsiness; drowsiness that prevents normal activity. Grade 3 loss of appetite; not eating at all. Grade 3 irritability/fussiness; crying that cannot be comforted/prevents normal activity. Rel = related to study vaccine. Note: all local injection site symptoms were considered related to study vaccine.
Two children experienced moderate anemia (one child with a hematocrit of 15%, the other with 24%) during the period to one month post Dose 3. One child who received RTS,S/AS02A had anemia associated with an acute case of malaria. In the control group the cause was not identified but the child recovered when administered a course of antibiotics and iron supplementation.
In total 7 subjects in the control group and 3 subjects in the RTS,S/AS02A group had an elevation in ALT during the period to one months post Dose 3 (reference range < 60 IU/L). One subject in the control group was observed to have an ALT level of 61 at a single time point. The subject was clinically well and, because the value was just outside the reference range, he was not investigated further. In the other 9 subjects the raised ALT was associated with a viral hepatitis. Five of these subjects had acute hepatitis A. One subject in each group was a chronic carrier of hepatitis B and 1 subject in the RTS,S/AS02A group was suffering from acute hepatitis B. One other subject who received Engerix-B was HBsAg positive and was a probable chronic carrier of hepatitis B; no confirmatory serology was performed.
No other clinically relevant abnormalities of hematological or biochemical laboratory parameters were observed.
A total of four serious adverse events were reported, two in each group, during the period to one month post Dose 3. One subject in the Engerix-B group suffered P. falciparum malaria; a second subject suffered glomerulonephritis secondary to skin lesions. In the RTS,S/AS02A (0.25 mL dose) group, one subject suffered a febrile convulsion 15 days after the second vaccination and another subject suffered from bronchopneumonia. All SAEs were considered not to be related to study vaccines. All subjects made a full recovery and were not withdrawn from the study.
All subjects receiving RTS,S/AS02A (0.25 mL dose) were seropositive (titers ≥ 1 µg/mL) for anti-CS antibodies 14 days after Dose 1 and titers increased with each subsequent injection. Post Dose 3 a GMC of 270.4 µg/mL (95% CI 183 to 400 µg/mL) was observed in the group receiving RTS,S/AS02A (0.25 mL dose), in comparison to the control group in which GMC did not change over time (Post Dose 3: GMC of 0.6 µg/mL, 95% CI 0.5 to 0.7 µg/mL) ( Table 8 above). Antibody concentrations in this study were higher than those observed previously in The Gambia.
Antibody GMTs to HBsAg after three doses of RTS,S/AS02A (0.25 mL dose) were significantly higher than those observed after 3 doses of Engerix-B: GMT 10 387 (95% CI 4872 to 22 142) compared to GMT 329 (95% CI 108 to 996). One month post Dose 3, seroprotection rates (anti-HBs titers ≥ 10 mIU/mL) for RTS,S/AS02A (0.25 mL dose) and Engerix-B were 96.3% and 95.2% respectively. There was 1 non-responder in each group (ATP cohort): in the RTS,S/AS02A (0.25 mL dose) group the subject was a chronic carrier of hepatitis B virus, in the Engerix-B group the subject was HBsAg negative.
The experience of the use of RTS,S/AS02A in children to date is that it has an acceptable reactogenicity and safety profile. The 0.25 mL dose is highly immunogenic for anti-CS and anti-HBs antibodies.

Phase IIb pediatric trial of RTS,S/AS02A (Malaria-026)
The trial Malaria-026 evaluated the efficacy of the 0.25 mL Dose of RTS,S/AS02A in children aged 1 to 4 years in Mozambique. It was a double-blind randomized design; 2022 children received either 3 doses of the candidate malaria vaccine, containing 25μg RTS,S and 0.25mL AS02A adjuvant, or 3 doses of a control vaccine using a 0, 1 and 2 month vaccination schedule. The control vaccination regime was 3 doses of hepatitis B vaccine (Engerix-B, GlaxoSmithKline Biologicals), administered to children of 24 months and older, or 2 doses of a 7-valent pneumococcal conjugate vaccine (Prevnar ® , Wyeth Lederle Vaccines) at Dose 1 and Dose 3 and 1 dose of Hemophilus influenzae type b vaccine (Hiberix ™ , GlaxoSmithKline Biologicals) at Dose 2 to children less than 24 months. Study subjects were recruited to two Cohorts for the study. Cohort 1 included children recruited from the town of Manhiça, Cohort 2 included children from the nearby 47 Modify Date 13/06/2006 Version : 5.1 town of Ilha Josina. Cohort 2 was used to examine vaccine efficacy (VE) against infection.

Primary Efficacy Endpoint, Malaria-026
The Primary Efficacy Endpoint of the trial was the determination of the VE against clinical malaria disease and was evaluated in Cohort 1. The malaria Case Definition for the Primary Efficacy Endpoint was 'the presence of P. falciparum asexual parasitemia above 2500 per µL on Giemsa stained thick blood films and the presence of fever (oral temperature ≥ 37.5°C) at the time of presentation and occurring in a child who is unwell and brought for treatment to a healthcare facility'.
In Cohort 1 and Cohort 2 the RTS,S/AS02A and control groups were well-balanced for age, gender, bednet usage, distance of residence from health facility and geographical area of residence. Malaria transmission intensity, as indicated by Hct, prevalence of splenomegaly and IFAT, was higher in Ilha Josina where Cohort 2 was evaluated than Manhiça where Cohort 1 was evaluated. Malaria transmission was not constant over the six-month period. Malaria disease rates in the control group were lower in the second half of the observation period.
VE was determined over a six-month surveillance period commencing from 14 days post Dose 3. As specified per RAP, the time at risk was adjusted for malaria drug usage, residence in the study, withdrawal and death and the estimate of effect was adjusted for the covariates of age of the subject, geographical area of the residence of the subject, bednet use, and distance of the subject's residence from the nearest health center. The estimate of VE determined as the time to the first clinical episode in the ATP Cohort for Efficacy (i.e. all children who received full vaccination courses and contributed to the time at risk) was 26.9% (95% CI 7.4% to42.2%) and after adjustment for covariates was 29.9% (95% CI 11.0% to 44.8%).
No waning of efficacy over the 6-month observation period was noted for the primary endpoint when analyzed by different methods (test for proportionality of hazards with Schoenfeld residuals, p=0.139). Consistent with these data, at the cross-sectional survey 6.5 months after Dose 3, prevalence of parasitemia in recipients of RTS,S/AS02A was 37% lower than in the recipients of control vaccines; 11.9% in RTS,S/AS02A vs 18.9%in controls, p=0.0003). Parasite densities in these children were similar between RTS,S/AS02A recipients and controls (geometric mean density 2271 vs 2513; p=0.699).

Secondary Efficacy Endpoints of clinical malaria disease, Malaria-026
The study evaluated three other Case Definitions of clinical malaria disease specified per protocol. These endpoints explored different parasite density threshold values and history of fever as opposed to documented fever. The point estimates were consistent for all the Secondary Case Definitions of malaria disease.
For the first episode meeting Secondary Case Definition 1 for malaria episodes assessed over 6 months of Dose 3 defined as: the time to the first clinical episode of symptomatic P. falciparum malaria meeting Secondary Case Definition 1 for malaria episodes (the presence of P. falciparum asexual parasitemia [any level of parasitemia] on Giemsa stained thick blood films and the presence of fever [oral temperature ≥ 37.5°C] at the time of presentation and occurring in a child who is unwell and brought for treatment detected by passive case detection) in children (1-4 years of age at first vaccination), determined over a six-month surveillance period post Dose 3; the VE was 28.6% (95% CI 10.4% to 43.1%; p=0.0036) First episode meeting Secondary Case Definition 2 for malaria episodes assessed over 6 months of Dose 3: the time to the first clinical episode of symptomatic P. falciparum malaria meeting Secondary Case Definition 2 for malaria episodes (the presence of P. falciparum asexual parasitemia [any level of parasitemia] on Giemsa stained thick blood films and a history of fever within 24 hours or documented fever [oral temperature ≥ 37.5°C] at the time of presentation and occurring in a child who is unwell and brought for treatment) detected by passive case detection in children (1-4 years of age at first vaccination), determined over a six-month surveillance period post Dose 3; the VE was 33.8% (95% CI 19.7% to 45.3%; p=<0.001) First episode meeting Secondary Case Definition 3 for malaria episodes assessed over 6 months of Dose 3: the time to the first clinical episode of symptomatic P. falciparum malaria meeting Secondary Case Definition 3 (the presence of P. falciparum asexual parasitemia above 15 000 per µL on Giemsa stained thick blood films and the presence of fever [oral temperature ≥ 37.5°C] at the time of presentation and occurring in a child who is unwell and brought for treatment to a healthcare facility) detected by passive case detection in children (1-4 years of age at first vaccination), determined over a six-month surveillance period post Dose 3; the VE was 31.7% (95% CI 11.5% to 47.2%; p=0.0039).

Incidence of moderate anemia, Malaria-026
All children presenting to a Health Center as outpatients who were evaluated for malaria also had a blood sample taken by fingerprick to determine their hematocrit (Hct). It should be noted that there was no requirement for concurrent malaria parasitemia in this case definition and this therefore measures all cause anemia, of which in the study area malaria is the most important cause. The adjusted estimate of VE against first incident case of moderate anemia (defined as Hct < 25%) was 28.2% (95% CI -19.6% to 56.9%; p=0.203) in the ATP Cohort for Efficacy.

All clinical episodes meeting Primary Case Definition for Malaria, Malaria-026
Relatively few children had second or third episodes of malaria that met the Primary Case Definition (see Table 11). No child experienced more than 3 episodes of clinical malaria during the course of the trial. The significance of the differences in the distribution of the number of episodes was p=0.0508 assessed using a Fisher exact test.  The malaria transmission season over which the trial was conducted was particularly intense. The increased number of cases (compared to number expected), particularly those at the severe end of the spectrum, allowed the inclusion of Exploratory Efficacy Endpoints to evaluate severe disease to the RAP. It should be emphasized that all case determination was done systematically according to predefined criteria and prior to unblinding.

Malaria requiring hospitalization
Malaria requiring hospitalization was defined as an admission where malaria was either the sole cause of illness or a significant contributing factor. It was analyzed as the proportion of children experiencing one or more episodes during the six-month surveillance period post Dose 3 in the ATP cohort. There were 42 affected children in recipients of RTS,S/AS02A compared to 62 in the controls. The unadjusted estimate of VE was 32.3% (95% CI 1.3% to 53.9%; p=0.053).

Severe malaria disease
The proportion of children affected by one or more episodes of severe malaria meeting the Case Definition was compared.

Total number of hospital admissions
The analysis of the total number of hospital admissions is restricted to those hospital admissions which occurred in the six-month surveillance period and in children belonging to the ATP Cohort for Efficacy, Cohort 1. In total there were 79 admissions in the RTS,S/AS02A group and 90 in the control Table 12. The Fisher exact test showed no statistical significance difference between the groups. Association of CS response with efficacy,  As specified in the RAP, two approaches to the analysis of the association of the CS response with hazard rate in the RTS,S/AS02A recipients were used. The association between CS and Hazard Rate by means of a comparison of hazard rates between the higher tertile (measured in the treated group) against the lower tertile of the anti-CS antibodies as well as the hazard rates per ten-fold increase in the value of anti-CS antibodies was calculated. Neither analysis detected an association reaching statistical significance (see Table 13) The first infection could be detected actively on a scheduled home visit or at the time a child presented at a health facility with clinical malaria. All children received sulfadoxine-pyrimethamine and amodiaquine 14 days prior to Dose 3 and only children with a negative blood slide for malaria parasites were included in the ATP Cohort for Efficacy. As specified in the RAP, the time at risk was adjusted for malaria drug usage, residence in the study, withdrawal and death. The unadjusted estimate of VE determined as the time to the first infection in the ATP Cohort for Efficacy (i.e. all children who received full vaccination courses and contributed to the time at risk) was 44.7% (95% CI 31.0% to 55.6%; p ≤ 0.001). After adjustment for the covariates of age of subject, bednet usage and distance of residence of subject from a health center, the VE was 45.0% (95% CI 31.4% to 55.9%; p ≤ 0.001).

Immunogenicity, Malaria-026
Full details of the CS and HBs responses for Malaria-026 can be found in Table 8 and Table 9 respectively.
A formal evaluation of non-inferiority of RTS,S/AS02A over Engerix-B in terms of anti-HBs response was completed in children 2 to 4 years of age in Malaria-026. Table 14 shows the outcome of the assessment of non-inferiority of RTS,S/AS02A compared to Engerix-B with respect to seroprotection rates in subjects HBsAg negative prevaccination. The lower limit of the 95% CI for the difference in anti-HBs seroprotection rates (≥10 mIU/mL) was 4.3%, hence greater than the a priori defined limit of -10%, thus demonstrating non-inferiority of RTS,S/AS02A compared to Engerix-B.   Table 15 shows the outcome of the assessment of non-inferiority of RTS,S/AS02A compared to Engerix-B with respect to the ratio of anti-HBs GMTs in HBsAg negative subjects at baseline. The upper limit of the 95% CI for the ratio of anti-HBs GMTs was 0.04, hence lower than the a priori defined limit of 2.0, thus demonstrating noninferiority of RTS,S/AS02A compared to Engerix-B. Solicited symptoms are shown in Table 16 and Table 17, corresponding to results from Cohort 1 and Cohort 2 respectively. As with previous studies with RTS,S/AS02A, local reactions at the site of injection were common. Pain was the most frequently observed local symptom. In the recipients of RTS,S/AS02A, the most commonly observed solicited general adverse event related to vaccination was fever (In Cohort 1, 11.1% after all doses in children < 24 months of age, 3.7% after all doses in children ≥ 24 months of age: in Cohort 2 related fever was observed after 4.9% of doses in children < 24 months, 1.3% of doses in children ≥ 24 months). In recipients of control vaccines in Cohort 1, fever was also the most commonly observed solicited adverse event related to vaccination (1.1% in children < 24 months of age [who received Prevnar and Hiberix] and 0.9% in children > 24 months of age [who received Engerix-B]). For recipients of RTS,S/AS02A < 24 months of age, a trend of increased local reactogenicity with sequential doses was observed. For recipients of RTS,S/AS02A ≥ 24 months of age, local reactogenicity was most frequent following Dose 3. The incidence of general symptoms of drowsiness, irritability, loss of appetite and fever (≥ 37.5°C) was higher in recipients of RTS,S/AS02A than recipients of control vaccine; Grade 3 events were infrequent. In children < 24 months, the most commonly reported AEs were Upper Respiratory Tract infection, Malaria and Diarrhea and in children ≥ 24 months Upper Respiratory Tract Infection (URTI), Malaria and Ascariasis; frequencies were similar in the RTS,S/AS02A and control groups.  R(1)<24; recipients of RTS,S/AS02A less than 24 months of age (Cohort 1) C(1)<24; recipients of control vaccines (Prevnar & Hiberix) less than 24 months of age (Cohort 1) R(1)>24; recipients of RTS,S/AS02A greater than or equal to 24 months of age (Cohort 1) C(1)>24; recipients of control vaccines (Engerix-B) greater than or equal to 24 months of age (Cohort 1) N = number of documented doses. n/% = number/percentage of doses followed by a local/general symptom. LL=95% lower limit UL=95% upper limit *oral body temperature ≥ 37.5°C. Grade 3 injection site pain; cries when limb is moved/spontaneously painful.   ; recipients of RTS,S/AS02A less than 24 months of age (Cohort 2) C(2)<24; recipients of control vaccines (Prevnar & Hiberix) less than 24 months of age (Cohort 2) R(2)>24; recipients of RTS,S/AS02A greater than or equal to 24 months of age (Cohort 2) C(2)>24; recipients of control vaccines (Engerix-B) greater than or equal to 24 months of age (Cohort 2) N = number of documented doses. n/% = number/percentage of doses followed by a local/general symptom. LL=95% lower limit UL=95% upper limit *oral body temperature ≥ 37.5°C. Grade 3 injection site pain; cries when limb is moved/spontaneously painful. Grade 3 swelling; > 20 mm. Grade 3 fever; oral body temperature ≥ 39.0°C. Grade 3 drowsiness; drowsiness that prevents normal activity. Grade 3 loss of appetite; not eating at all. Grade 3 irritability/fussiness; crying that cannot be comforted/prevents normal activity. Rel = related to study vaccine. Note: all local injection site symptoms were considered related to study vaccine.
Fifteen subjects died during the double-blind phase of the Malaria-026 study; 5 had received RTS,S/AS02A and 10 had received control vaccines. Four deaths of recipients of control vaccines had malaria as a sole or contributing factor; no deaths of recipients of RTS,S/AS02A had malaria as a contributing factor. None of the deaths were, in the judgment of the investigator, related to vaccination. Extrapolating from the mortality rate in the study population, approximately 17 deaths would be expected ( SAE; for recipients of RTS,S/AS02A ≥ 24 months of age, 14% of subjects suffered at least one SAE. This is compared to recipients of Prevnar and Hiberix (children < 24 months of age), 37% of whom suffered at least one SAE; for recipients of Engerix-B (those children ≥ 24 months of age) 20% of subjects suffered at least one SAE. It should be noted that apart from five deaths that occurred at home, the non-hospitalized SAEs were all children with episodes of acute malaria associated with a high parasite density (defined as 5+ parasitemia on quick read).
There were no SAEs judged related to vaccination.
In Cohort 1, SAE reports of malaria tended to be fewer in recipients of RTS,S/AS02A. In children <24 months who received RTS,S/AS02A 19% of children experienced a malaria episode compared to 33% in the control group. There was a similar trend in the children ≥ 24 months; in the recipients of RTS,S/AS02A 13% experienced a malaria episode compared to 16% in the recipients of control vaccines. In Cohort 2, where surveillance for malaria was intense, no difference was observed. For the other SAEs classified at the MedDRA preferred term level, there were no differences in the pattern of morbidity observed between recipients of RTS,S/AS02A and recipients of control vaccines. The pattern of morbidity and SAEs in the study participants was similar to that previously observed at the study site (personal communication, Dr. Pedro Alonso, October 2003) and that has been described in the region [Fidel, 2002;Iriso, 2000].

Phase II pediatric trial of RTS,S/AS02D; Bridging Study (Malaria-034)
Malaria-034 was a phase I/II randomized double-blind bridging study to evaluate the safety and immunogenicity of 3 doses of GlaxoSmithKline Biologicals' candidate vaccine RTS,S/AS02D (0.5 mL dose) in comparison to 3 doses of the existing formulation RTS,S/AS02A (0.25 mL dose) administered to children aged 3 to 5 years living in a malaria-endemic region of Mozambique. Both vaccines contain the same constituents but the final volume was adjusted to be compatible with existing EPI practices.
Two hundred children were enrolled into two groups of 100 subjects each, of which 189 (93 of whom received RTS,S/AS02D [D group] and 96 received RTS,S/AS02A [A group]) subjects were included in the ATP analysis of safety and reactogenicity and 143 (67 and 76 subjects respectively) subjects in the analysis of immunogenicity.
The safety profile of the two vaccine formulations was similar. Solicited symptoms were as shown in Table 18, with pain the more frequent local symptom and fever the most frequent general symptom reported. Symptoms were generally mild to moderate in intensity, and decreased in frequency with subsequent doses.
Unsolicited symptoms were reported following 69 (24.7%) doses in the RTS,S/AS02D group and following 56 (19.4%) in the RTS/SAS02A group. The most frequently reported symptoms were malaria (18% of the subjects in the D group and 25% in the A group) and upper respiratory tract infection (14% of the subjects in the D group and 12% in the A group). An unsolicited symptom, injection site erythema, deemed to be related to vaccination was reported for a single subject, following one dose in the D group. Three symptoms in the D group and 8 in the A group were graded 3 in intensity, none of which were related to vaccination.
One serious adverse event occurred in a subject in the A group. This subject had severe malaria which lasted 4 days and required hospitalization. The subject recovered and the event was deemed by the investigator to be unrelated to vaccination. Grade 3 0 0.0 0.0 3.9 0 0.0 0.0 3.8 For each dose and overall/subject: 95%CI = Exact 95% confidence interval N = number of subjects having received at least one dose n/% = number/percentage of subjects reporting a specified symptom LL=95% lower limit UL=95% upper limit Grade 3 injection site pain; cries when limb is moved/spontaneously painful. Grade 3 swelling; > 20 mm. Fever: oral body temperature ≥ 37.5°C. Grade 3 fever; oral body temperature ≥ 39.0°C. Grade 3 drowsiness; drowsiness that prevents normal activity. Grade 3 loss of appetite; not eating at all. Grade 3 irritability/fussiness; crying that cannot be comforted/prevents normal activity Non-inferiority of the RTS,S/AS02D vaccine formulation compared to the RTS,S/AS02A formulation was proven with respect to the immune response elicited. Table 19 shows the anti-CS seropositivity rates and GMTs one month following the third vaccine dose and Table 20 gives the ratios of post vaccination GMTs. All subjects in both groups were seropositive for anti-CS antibodies one month following the third vaccine dose. The RTS,S/AS02A:RTS,S/AS02D anti-CS GMT ratio was 0.9, with as upper limit of 95% CI: 1.3, hence below the a priori defined clinical limit of non-inferiority.   All subjects were seroprotected with respect to anti-HBs after vaccination, as shown in Table 21, and GMTs exceeded 17 000 mIU/mL in both groups. The RTS,S/AS02A: RTS,S/AS02D ratio of post-vaccination anti-HBs GMTs (Table 22) was 0.7 (with, as upper limit of 95% CI:1.1, which is below the a priori defined clinical limit of noninferiority, 3).

Table 21 Seroprotection rates and geometric mean titers (GMT) for anti-HBs antibody titers one month after dose 3 (ATP cohort for immunogenicity)
≥

Studies of RTS,S/AS02A in children; conclusions
Following demonstration of the safety and efficacy of RTS,S/AS02A in adults, assessment of the RTS,S/AS02A vaccine progressed into children. Studies commenced with dose selection studies in older children, before progressing to dose selection studies in younger children. The RTS,S/AS02A vaccine was shown to be safe and immunogenic in both age groups at all dose levels. Subsequent to the selection of a suitable dose (0.25 mL containing 0.25 μg of antigen) and schedule (0, 1 and 2 month) with these studies, assessment of safety and efficacy progressed further with a study in approximately 2000 1 to 4 year old volunteers. The vaccine was shown to be safe, immunogenic and efficacious.
Following this study, a bridging study to evaluate the safety and immunogenicity of a new formulation of the vaccine, RTS,S/AS02D (designed to be compatible with EPI vaccination) was carried out. The new formulation was found to have an acceptable safety and reactogenicity profile and to induce similar antibody titers for anti-CS and anti-HBs as RTS,S/AS02A.

Trials of RTS,S/AS01B in adults
The first clinical study of RTS,S adjuvanted with AS01B was initiated in healthy, malaria-naive adults, aged 18 to 45 years at WRAIR, Silver Spring, MD, USA. This study (Malaria-027) was a double-blind, randomized, controlled challenge, Phase I/IIa study involving two sequential cohorts, Cohort 1 (n=50) and Cohort 2 (n=50). Volunteers received 3 doses of either RTS,S/AS02A or RTS,S/AS01B vaccine at months 0, 1, 2, followed by a challenge with P. falciparum-infected mosquitoes 14 to 28 days post Dose 3 and rechallenge in protected subjects at approximately 6 months post Dose 3. Infectivity controls were enrolled for the challenge phases of the study. As of March 2005, Cohort 1 has completed all study procedures and Cohort 2 is ongoing.
To date, results are available for an interim analysis of safety (solicited and unsolicited events only), immunogenicity and efficacy data of Cohort 1 only. respectively). Grade 3 events tended to be more frequent in subjects receiving RTS,S/AS02A than those receiving RTS,S/AS01B (per dose: 28.4% and 17.6%; per subject: 48.0% and 30.8%, respectively).
The incidence of solicited local events recorded over the 6-day follow-up following each dose were slightly higher in the RTS,S/AS02A group compared to the RTS,S/AS01B group (Table 23). Grade 3 events of pain were observed to be more frequent in the RTS,S/AS02A group (after 14.9% of doses) than in the RTS,S/AS01B group (after 2.9% of doses). Grade 3 events of redness and swelling occurred with a similar and low frequency in both group.
The incidence of solicited general symptoms in the RTS,S/AS01B group was similar to that observed in the RTS,S/AS02A group (Table 23). Grade 3 events were infrequent, occurring in no more than 3 subjects in either group.
Unsolicited events occurred more frequently in the RTS,S/AS02A group (after 66.2% of doses) compared to the RTS,S/AS01B group. Grade 3 unsolicited events were rare, occurring after 6.8% of doses of RTS,S/AS02A and 4.4% of doses of RTS,S/AS01B. Two events in each vaccine group were graded 3 in intensity and were considered to be causally related to vaccine (RTS,S/AS02A: dizziness and insomnia; RTS,S/AS01B: chills and hyperhidrosis).
Two SAEs were reported by two subjects; the vaccine groups for both subjects have not been unblinded. One subject, a 42-year-old female developed gait disorder, vertigo, illdefined condition, progressive weakness and facial droop 99 days after Dose 3 for which she was subsequently hospitalized. A subcortical infarct within the left internal capsule with a non-embolic distribution was diagnosed. Following examination by a neurologist, the cerebral infarct was considered likely to be associated with the use of oral contraceptives. The subject recovered with sequelae. A second subject was hospitalized for a ruptured tendon which occurred 107 days after Dose 3. The second subject, Neither SAEs were considered to have a causal relationship to study drug.  At 28 days post dose 1, at least 95% of subjects were seropositive for anti-CS antibody titers in both the RTS,S/AS02A and RTS,S/AS01B vaccine groups (Table 24); all subjects in both groups were seropositive 56 days post dose 2 and remained so until 5 months post dose 3. GMTs for anti-CS antibodies tended to be higher at all time points in the RTS,S/AS01B group compared to the RTS,S/AS02A group.
At 28 days post dose 1, 68% of subjects in the RTS,S/AS02A group compared to 91% of subjects in the RTS,S/AS01B group had seroprotective anti-HBs antibody titers (Table  25); all subjects in both groups were seroprotected 56 days post dose 2 and remained so until 5 months post dose 3. GMTs for anti-HBs antibodies tended to be higher at all time points in the RTS,S/AS01B group compared to the RTS,S/AS02A group    Following challenge with P. falciparum 14 to 28 days after vaccination, 15/24 subjects (62.5%) in the RTS,S/AS02A group and 7/17 subjects (41.2%) in the RTS,S/AS01B group were infected (Table 26). All infectivity control subjects became infected.
Vaccine Efficacy (VE) compared to infectivity control was significant in both vaccine groups (P<0.001). The observed VE was higher in the RTS,S/AS01B group than in the RTS,S/AS02A group (

Concurrent protection against hepatitis B
The hepatitis B surface antigen (HBsAg) contained in the RTS,S candidate malaria and hepatitis B vaccine is encoded by the hepatitis B virus S protein gene that is identical to the gene used to express HBsAg in GSK Biologicals' Engerix-B vaccine against hepatitis B.
Non-inferiority of the anti-HBs response of RTS,S/AS02A compared to the licensed vaccine Engerix-B has been formally demonstrated in the study Malaria-026 (refer to Section 1.3.3.2.2).

Rationale for the study design
Preclinical studies and an ongoing Phase IIa study of RTS,S (Malaria-027) suggest that AS01B efficacy may be superior to that of AS02A. Efficacy of RTS,S/AS01B needs to be further evaluated as well as assurance of safety of the vaccine.
This study advances the clinical development of the RTS,S/AS01B vaccine to malaria endemic populations. As a primary objective it compares the safety and reactogenicity of RTS,S/AS01B to the RTS,S/AS02A vaccine which has been more extensively evaluated. As there is limited previous experience of the RTS,S/AS01B vaccine, the sample size of the study is limited for safety reasons.
As a secondary objective the study will establish proof of concept of efficacy of RTS,S/AS01B under conditions of natural exposure in an endemic country. Point estimates of efficacy will be determined for both RTS,S/AS01B and RTS,S/AS02A by comparing the time to first infection in the recipients of the experimental vaccines to controls. Under the anticipated malaria transmission intensity, the study has at least 80% power to detect a difference from control if the true vaccine efficacy is greater than or equal to 41%.

Safety monitoring plan
The safety monitoring plan for this trial is facilitated by the Local Safety Monitor (LSM); the role of the LSM is fully explained in 5.1.3.1. The LSM acts as an advocate for the study volunteers, advisor to the PI on clinical safety issues and a link between the study site, medical monitor and the Safety Monitoring Group (fully explained in 5.1.3.2). Furthermore, the LSM is empowered to temporarily suspend vaccination on the trial for major safety concerns, pending discussion with the SMG.
The SMG is empowered to temporarily suspend the trial on the basis of any safety concern or upon meeting predefined criteria for temporary suspension (Section 5.1.3.4). The SMG will review summarized safety and reactogenicity data at protocol-defined timepoints, so that the trial can be halted in a timely fashion if a safety issue is detected (refer to Section 5.1.3.2).
The full safety monitoring plan process is detailed in Section 5.1.3.

Assessment of proof-of-concept
Proof-of-concept will be assessed as time-delay to first infection with asexual forms of P. falciparum.
The time to infection was assessed in the challenge model conducted at the WRAIR; estimates of vaccine efficacy with RTS,S/AS02A were consistently approximately 40% [Kester, 2001;Stoute, 1997]. When the vaccine was evaluated under conditions of natural exposure in The Gambia [Malaria-005], the vaccine efficacy for the time to first infection in adults was 34% (CI 8.0% to 53.0%). The recently conducted Phase 2b trial in Mozambican children (Malaria-026) was the first study in which both clinical and parasitological endpoints were assessed. Measured concurrently in two groups of children, vaccine efficacy against first clinical episode was 30% (95% CI: 11.0% to 44.8%) and against first infection was 45% (95% CI: 31.4% to 55.9%).
Time to first infection will be used to assess proof-of-concept, because it has proven to be consistent when measured by different methodologies in different populations and because, in the recent Malaria-026 study, the point estimate of efficacy against infection was consistent with the estimate of prevention of clinical episodes.

Rationale for the use of Rabies vaccine as a control
Rabies vaccine has been chosen as the comparator because: 1) the vaccine can be administered on a 0, 1, 2 month schedule; 2) rabies vaccine has been used in previous studies of RTS,S-based vaccines in Africa; and 3) volunteers will benefit from receiving rabies vaccine as rabid animals occur in the study area.
Chiron's Rabipur ® vaccine will be used. When the Rabipur rabies vaccine is administered according to the recommended vaccination schedule (days 0, 7, 21), nearly 100% of subjects attain a protective titer. In two studies carried out in the US in 101 subjects, protective antibody titers >0.5 IU/mL were obtained by day 28 in all subjects. In studies carried out in Thailand in 22 subjects, and in Croatia in 25 subjects, antibody titers of >0.5 IU/mL were obtained by day 14 (injections on days 0, 7, 21) in all subjects [Dreesen, 1989;Nicholson, 1987;Vodopija, 1986;Wasi, 1986].
High antibody titers have also been demonstrated with off-label immunization with rabies vaccines. Among participants in England, Germany, France and Belgium who received two vaccinations one month apart, nearly 100% of the participants developed specific antibody and the geometric mean titer for the group was 10 IU [Ajjan, 1978;Costy-Berger, 1978;Cox, 1976;Kuwert, 1978]. The proposed vaccination schedule of 0, 1, and 2 months is therefore expected to be highly successful in conferring protective immunity against rabies among the control participants. However all subjects that are in contact with a potential rabid animal will be advised to seek medical attention immediately.

Rationale for testing cell-mediated immunity
RTS,S/AS02A has been shown to be a powerful inducer of antigen-specific humoral and CMI response in preclinical and clinical studies [GSK data on file, Malaria Investigator's Brochure 2003]. The RTS,S/AS02A and RTS,S/AS01B vaccines are believed to elicit a strong humoral immune response directed against surface-exposed sporozoite proteins, and elicit CMI responses characterized by predominantly CD4+-Th1 cells, that are hypothesized to either destroy infected hepatocytes and/or limit intracellular parasite development through appropriate cytokines. As CMI response is believed to be a key component in protecting vaccinees against the P. falciparum parasite it will be measured in this trial and exploratory analyses conducted.

To evaluate strain-specific vaccine effects in this trial of the RTS,S/AS02A and RTS,S/AS01B vaccines in Kenyan adults, DNA samples will be used for sequence analysis of the Th2R and Th3R sequences in the CSP gene of parasitemic individuals identified during follow-up by active detection of infections (ADI). Vaccine effects on multiplicity of infection (MOI) will also be investigated using polymorphic antigens loci, namely msp1 and msp2.
Amended 13 June 2006

HIV testing at screening
As part of this trial, no HIV testing is scheduled to be carried out at screening. It is a condition of the trial that all enrolled subjects are in good health (refer to Section 4.3), as confirmed by a range of clinical and blood tests at screening (refer to Section 4.4 for the exclusion criteria for this trial). Those volunteers who exhibit clinical features of immunosuppresion or immunodeficiency will not be enrolled (refer to Section 4.4). There is no reason to believe that any of the vaccines to be administered during this study may have any ill-effect on clinically asymptomatic volunteers infected with HIV.
In addition, HIV testing has been found to be culturally unacceptable at the site. The local community have hesitated to embrace HIV testing and local elders have voiced their concerns about mandatory HIV testing as part of any studies in this community.

Risks and benefits for study volunteers
1.8.1.

Risks of receipt of RTS,S/AS02A, RTS,S/AS01B or Rabies Vaccine and risk-management
Recipients of any of the investigational products in this study (RTS,S/AS02A, RTS,S/AS01B or Rabies Vaccine) may experience pain and/or swelling at the injection site, fever, headache, fatigue, nausea, vomiting and/or abdominal pain, joint pain and/or muscle aches. As with any vaccination, there is the potential for an anaphylactic reaction. Additionally, there may other reactions that at this time are not known.
To facilitate management of these potential problems, medical staff experienced in the management of anaphylactic reaction will observe patients for at least 30 minutes following each vaccination to ensure that should any anaphylactic reaction occur it is managed in a timely manner (refer to Section 5.3). Subjects will be followed up daily by study staff for 6 days following each dose of investigational product in order to evaluate and arrange treatment (where necessary) of any post-vaccination symptoms (refer to Section 5.4).

Risk of receipt of Malarone and risk management
Subjects who receive Malarone ® may experience stomach pain, headache, nausea, vomiting, loss of appetite, cough, rash, tiredness, mouth ulcers, hair loss or diarrhea.
Subjects will be seen by field workers on the day following Dose 1 and Dose 2 of Malarone ® , and by a study clinician one week post Dose 3 of Malarone ® . Each dose of Malarone ® will be given under the supervision of a fieldworker. Any subjects who are unwell at these visits will be referred to a study clinician for evaluation and treatment. Subjects who are unable to tolerate Malarone ® will be given Coartem ® (refer to Section 5.10).

Risk of receipt of Coartem and risk management
Subjects who receive Coartem ® may experience abdominal pain, anorexia, nausea, vomiting, diarrhea, headache and/or dizziness.
For those subjects who receive Coartem ® as an alternative clearance treatment, they will be seen by field workers on the day following the first two doses and the second two doses of Coartem, and by a study clinician one week post the final two doses of Coartem ® . Dose 1, Dose 3 and Dose 5 of Coartem ® will be given under the supervision of a fieldworker. Any subjects who are unwell at these visits will be referred to a study clinician for evaluation and treatment.

Risk of disclosure of volunteer's confidential/personal information to persons not involved in the study
In order to ensure that all information collected on study volunteers is kept confidential, the following safeguards will be applied: • Access to study files and personal information will be limited to only WRP and GlaxoSmithKline personnel with a need to access the information. • Study information will be kept in locked cabinets when not in use.
• All information or samples that leave WRP will be labeled in a way that it cannot be linked back to a specific study subject.

Benefits to study volunteers of taking part in the study
All volunteers for this study will receive the following benefits for their participation: • All volunteers will undergo a medical examination at screening free of charge. All volunteers, whether accepted for enrollment into the trial or not will benefit from this Where illnesses are newly-diagnosed, a referral to an appropriate specialist will be made for the volunteer. • For the duration of their participation in the trial, all randomized subjects will receive free health care from study medical personnel. • All enrolled subjects will be offered, free of charge, a full three dose course of vaccinations against rabies (except where the principal investigator deems that vaccination is unsuitable for safety concerns). Subjects who are randomized to receive the rabies control vaccine will receive these vaccinations during the course of the trial. Subjects who are randomized to receive RTS,S/AS01B or RTS,S/AS02A will be offered the vaccination course after they have completed all study procedures and returned for the final study visit.

Safety & Reactogenicity
• To compare the safety and reactogenicity of RTS,S/AS01B vs RTS,S/AS02A, when administered as 3 doses intramuscularly on a 0, 1 and 2 months schedule to adult volunteers living in a malaria endemic region Refer to Section 10.1 for definition of the primary endpoint.

Safety
• To describe the safety and reactogenicity of RTS,S/AS02A and RTS,S/AS01B, when administered as 3 doses intramuscularly on a 0, 1 and 2 months schedule to adult volunteers living in a malaria endemic region.

Immunogenicity
• To describe antibody responses to the circumsporozoite (anti-CS) antigen and hepatitis B surface antigen (anti-HBs) of RTS,S/AS02A and RTS,S/AS01B when administered as 3 doses intramuscularly on a 0, 1 and 2 months schedule to adult volunteers living in a malaria endemic region.

Proof-of-concept
• • To determine the prevalence of vaccine-like alleles of CSP in the study population. • Phase IIb, single-center, double blind (observer blind, participant blind), randomized controlled trial with three groups in one study site (randomization 1:1:1, RTS,S/AS01B: RTS,S/AS02A:rabies) • 255 adults will be enrolled. It is assumed that 90% will complete study procedures and be evaluable.

STUDY DESIGN OVERVIEW
• Prior to study start, a community information program will inform the local population of the study. Throughout the period of enrollment, study information will be presented at community centers. • Healthy male and female adults aged 18 to 35 years will be screened. Those determined to be eligible, based on the inclusion and exclusion criteria, will be enrolled in the study. • Route of administration: all vaccines will be administered by the intramuscular route to the deltoid of the non-dominant arm • Each subject will be observed for at least 30 minutes after vaccination to evaluate and treat any acute adverse events • After vaccination, the subjects will visit field workers at field stations for the detection of adverse events (AEs) occurring after vaccination for one week (days 1, 2, 3, 4, 5 and day 6 after each vaccination). Diary cards and thermometers will be provided for the field workers to record oral temperature and any local (at the injection site) or general adverse events. • One week prior to Dose 3, subjects will be treated with Malarone ® for presumptive clearance of malaria parasitemia (in the event that subjects are unable to tolerate Malarone ® , Coartem ® (artemether/lumefantrine, Novartis AG, Zurich, Switzerland) will be used as the alternative clearance treatment. • Surveillance for Active Detection of Infection (ADI) will begin 2 weeks after Dose 3. Subjects will visit field workers at field-stations near their homes weekly during the ADI period. The total period of surveillance for ADI is 14 weeks. Two Clinic Visits and 14 field-worker visits will contribute to the ADI. • All subjects will have a blood slide prepared and read to check for asexual P.
falciparum parasitemia one week post Dose 3. Any subject who tests positive will be treated with Coartem ® • At each contact for ADI, history of fever will be recorded and oral temperature taken; a smear will be taken for detection of malaria parasites. • Infection with malaria parasites is defined as P. falciparum asexual parasitemia > 0 • Subjects who are symptomatic at the time of ADI contact (i.e. history of fever within previous 24 hours or oral temperature >37.5°C) will have a blood slide read and treated within the same day if the blood slide is positive. • Subjects will be followed passively for the occurrence of clinical malaria from Day 0 until 16 weeks post Dose 3. Cases will be detected at Kombewa Clinic. If a subject feels ill they can present to a field station near their home and be transported to Kombewa Clinic for evaluation. • DNA will be extracted from EDTA blood of all subjects with patent asexual parasitemia. • Recording of serious adverse events will be throughout the study period. They will be captured through the morbidity surveillance system at Kombewa Clinic. • Anti-CS antibody titers will be determined at baseline, one month post Dose 2, 1 month post Dose 2, 1 month post Dose 3, 4 months post Dose 3 and 10 months post Dose 3. • Anti-HBs antibody titers will be determined at baseline, one month post Dose 3 and 10 months post Dose 3. • CS-specific CMI will be assessed at three four time points during the study; prevaccination, 1 month post Dose 3, and 4 months post Dose 3 and 9 months post Dose 3. Amended 13 June 2006. • Data collection will be by conventional CRF.
• Duration of the study will be approximately 12 months per subject.
• There will be a planned interim analysis on all safety humoral and immunogenicity data collected up to 30 days after Dose 3. • The final analysis of the study for all primary and secondary endpoints will take place on all data collected up to the end of the surveillance for ADI (16 weeks post Dose 3). • All subjects will be followed for safety for a period that extends to 10 months post Dose 3. Any data collected after the final analysis will be reported in an annex report. There are two distinct rainy seasons. The "long rainy season" of late March through May produces intense transmission from April through August and the "short rainy season" of October through December produces another intense transmission season from November through January.

Study site; malaria transmission
Malaria transmission occurs all year but is maximal during the two rainy seasons. The principal vector is Anopheles gambiae. P. falciparum parasitemia is present in over 90% of malaria cases. P. ovale and P. malariae, which together constitute less than 10% of cases, are usually present in mixed infections with P. falciparum.
The Malaria Infection Rate in adults has been estimated to be been roughly 95% during the long rains (April to August) and 72% during the short rains (November to January) (unpublished data, WRAIR). Approximately 25% of infected adults become symptomatic.
A longitudinal epidemiological study conducted in 2003/2004 in Kombewa indicated that monthly attack rates range from approximately 20% to 55% in children aged 1 to 3 years in the proposed study area (unpublished data WRAIR) ( Villages in this area are typically a loose conglomeration of family compounds near a family garden plot and grazing land. Houses are typically made of mud with thatched or corrugated roofs. In Kombewa Town, homes are often attached to family shops/businesses. Most homes do not have glass windows or screens. Water source is mainly from community wells and local streams though some households have their own wells. Most water sources are not chlorinated or covered.

Number of subjects
Subjects will be recruited by non-coercive methods according to existing policies. Eligible subjects will be male and female adults between the ages of 18 and 35 years.
The necessary number of volunteers will be screened in order to enroll approximately 255 eligible subjects, 85 to each of the RTS,S/AS02A, RTS,S/AS01B groups and Rabies Vaccine groups.

Inclusion criteria
All subjects must satisfy the following criteria at study entry: • A male or female of between 18 and 35 years of age at the time of first vaccination • Written informed consent obtained from the subject • Available to participate for the study duration (approximately 12 months) • Free of obvious health problems as established by medical history and clinical examination before entering into the study. • If the volunteer is female, she must be of non-childbearing potential, i.e. either surgically sterilized or one year post-menopausal; or, if of childbearing potential, she must be abstinent or have used adequate contraceptive precautions (i.e. natural family planning, intrauterine contraceptive device; oral contraceptives; diaphragm or condom in combination with contraceptive jelly, cream or foam; Norplant ® or DepoProvera ® ) for 30 days prior to vaccination, have a negative pregnancy test and must agree to continue such precautions for two months after completion of the vaccination series. • Negative pregnancy test

Exclusion criteria for enrollment
The following criteria should be checked at the time of study entry. If any apply, the subject must not be included in the study: • Planned administration/administration of a vaccine not foreseen by the study protocol within 30 days of the first dose of vaccine(s) with the exception of tetanus toxoid. • Any confirmed or suspected immunosuppressive or immunodeficient condition, including human immunodeficiency virus (HIV) infection. • A family history of congenital or hereditary immunodeficiency.
• History of allergic reactions significant IgE-mediated events or anaphylaxis to previous immunizations. • History of any neurologic disorders or seizures.
• Acute disease at the time of enrolment (acute disease is defined as the presence of a moderate or severe illness with or without fever). All vaccines can be administered to persons with a minor illness, such as diarrhoea or mild upper respiratory infection without fever, i.e. Oral temperature < 37.5°C. • Acute or chronic, clinically significant pulmonary, cardiovascular, hepatic or renal functional abnormality, as determined by physical examination or laboratory screening tests. • ALT above normal range as defined in Table 32 • Creatinine above normal range as defined in Table 32 CONFIDENTIAL • Hemoglobin below normal range as defined in Table 33 • Platelet count below normal range as defined in Table 33 • Absolute lymphocyte count below normal range as defined in Table 33 • Total white cell count below normal range as defined in Table 33 • HBsAg positive • Homozygous sickle cell disease (Hgb SS) • History of splenectomy.
• Administration of immunoglobulins, blood transfusions or other blood products within the three months preceding the first dose of study vaccine or planned administration during the study period. • Pregnant or lactating female (i.e. a positive pregnancy test).
• Female planning to become pregnant or planning to discontinue contraceptive precautions. • History of chronic alcohol consumption and/or drug abuse.
• Use of any investigational or non-registered drug or vaccine within 30 days preceding the first dose of study vaccine, or planned use during the study period. • Chronic administration (defined as more than 14 days) of immunosuppressants or other immune-modifying drugs within six months prior to the first vaccine dose (for corticosteroids, this will mean prednisone, or equivalent, ≥ 0.5 mg/kg/day. Inhaled and topical steroids are allowed). • Any chronic drug therapy to be continued during the study period.
• Major congenital defects or serious chronic illness.
• Previous participation in any other malaria vaccine trial.
• Simultaneous participation in any other clinical trial.
• Any other findings that the investigator feels would increase the risk of having an adverse outcome from participation in the trial.
As part of the screening procedure for this trial, no HIV testing will be performed.

Elimination criteria during the study
The following criteria should be checked at each visit subsequent to the first visit. If any become applicable during the study, it will not require withdrawal of the subject from the study but may determine a subject's evaluability in the according-to-protocol (ATP) analysis. See Section 10.6 for definition of study cohorts to be evaluated.
• Use of any investigational or non-registered product (drug or vaccine) other than the study vaccine(s) during the study period. • Chronic administration (defined as more than 14 days) of immunosuppressants or other immune-modifying drugs during the study period (for corticosteroids, this will mean prednisone, or equivalent, ≥ 0.5 mg/kg/day. Inhaled and topical steroids are allowed). • Administration of a vaccine (except tetanus toxoid) not foreseen by the study protocol during the period starting from 90 days before Dose 1 and ending 30 days after Dose 3. • Administration of immunoglobulins and/or any blood products during the study period. • Drug or alcohol abuse.

Absolute contraindications to further vaccination
The following adverse events (AEs) constitute absolute contraindications to further administration of RTS,S/AS02A, RTS,S/AS01B or Rabipur; if any of these AEs occur during the study, the subject must not receive additional doses of vaccine but may continue other study procedures at the discretion of the investigator (see Section 9). The subject must be followed until resolution of the event, as with any AE (see Section 8.7): • Acute allergic reaction, significant IgE-mediated event or anaphylactic shock following the administration of vaccine investigational product • Any confirmed or suspected immunosuppressive or immunodeficient condition, including human immunodeficiency virus (HIV) infection. • Pregnancy, i.e. a positive urine test (see Section 8.11).

Indications for deferral of vaccination
The following AEs constitute contraindications to administration of RTS,S/AS02A, RTS,S/AS01B or Rabipur at that point in time. If any one of these AEs occurs at the time scheduled for administration of investigational product, the investigational product may be administered to the subject at a later date but within the time window specified in the protocol (see Section 5.12), or withdrawn at the discretion of the investigator (see Section 9). The subject must be followed until resolution of the event, as with any AE (see Section 8.7).
• Acute disease at the time of administration of investigational product (acute disease is defined as the presence of a moderate or severe illness with or without fever). All vaccines can be administered to persons with a minor illness such as diarrhoea or mild upper respiratory infection without fever, i.e. Oral temperature < 37.5°C • Oral temperature of ≥ 37.5°C

5.
CONDUCT OF STUDY

Ethics and regulatory considerations
The study will be conducted according to Good Clinical Practice (GCP), the appended Declaration of Helsinki (Protocol Appendix A), and local rules and regulations of the country.
The following two sections provide guidance to the study investigator of the minimal ethical and regulatory requirements required by GSK but the final responsibility for the interactions between the Institutional Review Boards (IRBs) and Independent Ethics Committees (IECs) and for informed consent remain with the Principal Investigator and may be subject to local rules and regulations.

Institutional Review Board/Independent Ethics Committee (IRB/IEC)
The IRB/IEC must be constituted according to the local laws/customs of each participating country. The ICH Harmonized Tripartite Guideline for Good Clinical Practice recommends that the IRB/IEC should include: (a) At least five members.
(b) At least one member whose primary area of interest is in a non-scientific area.
(c) At least one member who is independent of the institution/study site.
Only those IRB/IEC members who are independent of the investigator and the study sponsor (USAMMDA), GSK Biologicals or WRAIR should vote/provide opinion on a study-related matter.
This protocol and any other documents that the IRB/IEC may need to fulfill its responsibilities, including subject recruitment procedures and information about payments and compensation available to subjects, will be submitted to the IRB/IEC by the principal investigator. Written unconditional approval of the IRB/IEC must be in the possession of the investigator and GSK Biologicals and USAMMDA before commencement of the study. This approval must refer to the study by exact protocol title and number, and should identify the documents reviewed and state the date of review. Relevant GSK Biologicals' data will be supplied by the principal investigator to the independent IRB/IEC for review and approval of the protocol. Verification of IRB/IEC unconditional approval of the protocol and the written informed consent statement will be transmitted by the principal investigator to GSK Biologicals' Study Monitor, using the standard notification form, prior to shipment of vaccine supplies and CRFs to the site. • all subsequent protocol amendments, informed consent changes or revisions of other documents originally submitted for review; • serious and/or unexpected adverse events occurring during the study, where required, • all subsequent protocol modifications (for information); • new information that may affect adversely the safety of the subjects or the conduct of the study; • an annual update and/or request for re-approval, where required; • when the study has been completed, where required.
If a trial is prematurely terminated or suspended for reasons including, but not limited to, safety or ethical issues or severe non-compliance, the sponsor, USAMMDA will promptly inform the regulatory authorities of the termination or suspension and the reason(s) for the termination or suspension. If required by applicable regulations, the investigator must inform the IEC/IRB promptly and provide the reason for the suspension or termination (see Section 5.1.3 and Appendix B for further details).

Informed consent
The details of the informed consent process are provided in Appendix C. The following principles will also apply.
In obtaining and documenting informed consent, the investigator should comply with the applicable regulatory requirement(s), and should adhere to GCP and to the ethical principles that have their origin in the appended Declaration of Helsinki. Prior to the beginning of the trial, the investigator should have the IRB/IEC's written approval/favorable opinion of the written informed consent form and any other written information to be provided to the subjects.
Information should be given in both oral and written form whenever possible and as deemed appropriate by the IRB/IEC.
An investigator or designate will describe the protocol to potential subjects face to face. The Subject Information and Consent Form may be read to the subjects, but, in any event, the investigator or designate shall give the subjects ample opportunity to inquire about details of the study and ask any questions before dating and signing the Consent Form.
Subject information and consent forms must be in a language fully comprehensible to the prospective subjects. Informed consent shall be documented by the use of a written consent form approved by the IRB/IEC and signed and dated by the subjects and by the person who conducted the informed consent discussion. The signature confirms the consent is based on information that has been understood. All illiterate individuals will have the study, the Subject Information and Consent Form explained to them point by point by the interviewer in the presence of an impartial witness. The subjects will thumbprint or sign the consent form. The witness will also sign and date the consent form. Oral witnessed consent will replace written consent only in countries where the local custom is contrary or if the subjects incapacity precludes this and provided that the local legal obligations are fulfilled.
Each subject's signed informed consent form must be kept on file by the investigator for possible inspection by Regulatory Authorities and/or GSK Biologicals' professional and Regulatory Compliance persons. Only the signed Informed Consent Agreement pages of each volunteer will be kept on file at the investigator's site. In addition one entire representative copy of the informed consent documentation will be kept on file by the investigator for reference. The subjects should receive a copy of the signed and dated written informed consent form and any other written information provided to the subjects and should receive copies of any signed and dated consent form updates. Any amendments to the written information will be provided to subjects.
Both the informed consent discussion and the written informed consent form and any other written information to be provided to the subjects should include explanations of the following: (a) That the trial involves research.
(b) The purpose of the trial.
(c) The trial treatment(s) and the probability for random assignment to each treatment.
(d) The trial procedures to be followed, including all invasive procedures.
(f) Those aspects of the trial that are experimental.
(g) The reasonably foreseeable risks or inconveniences to the subjects and, when applicable, to an embryo, fetus or nursing infant.
(h) The reasonable expected benefits. When there is no intended clinical benefit to subjects, the subjects should be made aware of this.
(i) The alternative procedure(s) or course(s) of treatment/methods of prevention that may be available to subjects, and their important potential benefits and risks.
(j) The compensation and/or treatment available to subjects in the event of trial-related injury.
(k) The anticipated prorated payment, if any, to subjects for participating in the trial.
(l) The anticipated expenses, if any, to subjects for participating in the trial. (m) That the subjects' participation in the trial is voluntary and subjects may refuse to participate or withdraw from the trial, at any time, without penalty or loss of benefits to which subjects are otherwise entitled.
(n) That the monitor(s), the auditor(s), the IRB/IEC, and the regulatory authority(ies) will be granted direct access to the subject's original medical records for verification of clinical trial procedures and/or data, without violating the confidentiality of subjects, to the extent permitted by the applicable laws and regulations and that, by signing a written informed consent, the subject's is authorizing such access.
(o) That records identifying subjects will be kept confidential and, to the extent permitted by the applicable laws and/or regulations, will not be made publicly available. If the results of the trial are published, subjects' identity will remain confidential.
(p) That the subjects will be informed in a timely manner if information becomes available that may be relevant to the subjects willingness for continued participation in the trial.
(q) The person(s) to contact for further information regarding the trial and the rights of trial subjects, and who to contact in the event of trial-related injury.
(r) The foreseeable circumstances and/or reasons under which a subject's participation in the trial may be terminated.
(s) The expected duration of a subject's participation in the trial.
(t) The approximate number of subjects involved in the trial.
GSK Biologicals will prepare a model Informed Consent Form which will embody all the elements described above. While it is strongly recommended that this model document be followed as closely as possible, the informed consent requirements given in this document are not intended to pre-empt any local regulations which require additional information to be disclosed for informed consent to be legally effective. Clinical judgment, local regulations and requirements should guide the final structure and content of the document.
The investigator has the final responsibility for the final presentation of Informed Consent Form, respecting the mandatory requirements of local regulations. The consent form generated by the investigator with the assistance of the sponsor's representative, must be approved (along with the protocol, and any other necessary documentation) by the IRB/IEC and be acceptable to GSK Biologicals.

Safety monitoring plan
A local safety monitor (LSM) will be in place for the trial.
A Safety Monitoring Group (SMG) comprising medical representation from KEMRI, the sponsor's representative (USAMMDA), GSK Biologicals and WRAIR will review data. The PI, LSM and SMG are empowered to suspend the trial for any safety concern. To supplement this, the protocol defines criteria for both the immediate suspension of all vaccination and also for the progression to the next sequential dose. Blinded safety reports will be produced by the Principal Investigator for the SMG on the first 60 subjects to receive Dose 1 and Dose 2 (5.1.3.3).
The decision making process for resuming vaccination or permanently stopping the trial is described in Section 5.1.3.4.

Local Safety Monitor (LSM)
The overall role of the LSM, who is an experienced clinician based in-country, will be to support the PI and to act as a link between the PI and the SMG (refer to Section 5.1.3.2).
The LSM's role will include: • Acting as the study volunteer's advocate; • Promptly communicating relevant safety information to the medical monitor and the SMG; • Providing advice to the PI on whether a set of clinical circumstances in a study warrants formal notification to the medical monitor and the SMG; • Unblinding a subject if deemed necessary to allow for adequate treatment; • Suspension of vaccination for a major safety concern pending discussion with the SMG (see Section 5.1.3.4.1 for full details); A second, qualifed, experienced physician based in-country will serves as the alternate LSM, in the event the LSM is out of the country or unavailable to fulfill time-sensitive duties.
The LSM is required to review all unanticipated problems (refer to Section 8.3) involving risk to subjects or others, serious adverse events and all subject deaths associated with the protocol and provide an unbiased written report of the event. At a minimum, the LSM should comment on the outcomes of the event or problem, and in the case of a serious adverse event or death, comment on the relationship to participation in the study. The LSM should also indicate whether he concurs with the details of the report provided by the study investigator. Reports for events determined by either the investigator or LSM to be possibly or definitely related to participation and reports of events resulting in death should be promptly forwarded to the HSRRB and GSK Biologicals thorough evaluation of the safety reports after Dose 1 and Dose 2. The members do not form a part of the investigator groups; i.e. the investigator site, the GSK Biologicals malaria vaccine clinical project team or the investigator group at WRAIR. They will be one of the parties responsible for deciding whether to progress to the next dose and whether to resume immunizations if the trial has been suspended. The SMG may, if deemed necessary, convene a meeting with, or request further information from the PI or LSM.
A charter will be agreed upon prior to initiation of this study that will further define the role of the SMG.

Safety Reports
Safety reports will be written on the first 60 subjects to receive Dose 1 and Dose 2. These reports will be blinded to vaccine allocation and will contain: • All SAEs and any relationship to vaccines to date; • For each dose, all Grade 3 solicited and unsolicited symptoms by relationship to vaccine; − Should the PI judge a Grade 3 case to be unrelated to vaccination, an alternative explanation for the cause of the symptom will be provided; • All indicators of hepatic and renal function (as per Table 32) above the normal range (i.e. Gradings 1 to 3); − For all subjects above normal range, clinical details will be provided; • Parameters of complete blood count below normal range (as per Table 33) post Dose 1; − For all subjects below normal range, clinical details will be provided; • All withdrawals of study subjects by the PI due to adverse events; − For all subjects withdrawn clinical details will be provided.

Indications for immediate suspension of all vaccination
The PI will suspend all vaccination for any of the following SAEs; • Death or life-threatening SAE which is judged to be related to the study vaccine; • Anaphylactic shock reaction or significant IgE-mediated event in an enrolled subject following vaccination.
The LSM, sponsor (USAMMDA) or GSK Biologicals may suspend all vaccination for any one SAE or pattern of SAEs.

Indications for suspension of progression to next sequential dose
If the blinded safety report shows ≥ 35% of subjects have a Grade 3 systemic reaction (solicited or unsolicited) If the blinded safety report shows ≥ 5% of subjects have a Grade 3 laboratory abnormality of any one indicator of hepatic or renal function as defined in Table 32.
If the blinded safety report shows ≥ 5% of subjects are withdrawn by the PI or withdraw themselves due to local or systemic reactogenicity.

Process if the trial is suspended
The trial may be temporarily suspended by the LSM, PI or the SMG. The SMG will review all available safety data which may include a full description of reactogenicity. If the SMG judge it to be necessary, the safety data will be unblinded. However the final responsibility to recommend whether or not the trial should be stopped permanently rests with the sponsor (USAMMDA) in consultation with GSK Biologicals, after having considered all the safety information available.
In the event that the trial is temporarily suspended, the FDA will be informed by the sponsor (USAMMDA). If the SMG's recommendation is to continue, then a report will be submitted to the FDA detailing the rationale used in reaching this decision. The agreement of the FDA will be obtained prior to restarting the trial.

Exposure to rabies
All subjects will be reminded at each Clinic Visit that should they come into contact with an animal who they suspect to have rabies, they must immediately consult a physician for treatment.

Cross over immunization with rabies vaccine
After completion of the study (12 months after Dose 1), rabies vaccination will be offered to all subjects that did not receive it during the study (i.e. those subjects that received RTS,S/AS02A or RTS,S/AS01B)

Community information
The community in which the study will take place will be informed about the nature and design of the study. Community leaders (assistant chiefs and local village elders) will be formally briefed in their own language on the nature and purpose of the study. They will have the opportunity to ask questions of the PI or his designees.

Recruitment
Adults age 18 to 35 years will be recruited from the villages in Kombewa District around the Kombewa Clinic. Non-coercive means of recruitment will be used according to existing U.S. Army regulations (viz., AR 70-25 and AR 40-38). Prospective subjects will view a videotaped briefing on the nature and purpose of the study and will then receive both oral and written explanations of the study. Afterwards, written informed consent will be obtained from each person who wants to participate in the study. No question relating to the potential study subject's health will be asked prior to consenting. So that language and illiteracy will not be impediments to informed consent, all briefings and explanations will be in Dholuo, the local language. All screening tests and procedures (refer to 5.2.3 and Appendix C) will be performed during a separate visit. It is expected that at least 500 adult volunteers will have to be recruited to achieve the final enrolment number. Refer to Appendix C for an overview of the recruitment plan of the study.

Screening of volunteers
Screening will be done on a separate visit from recruiting. Only adults aged 18 to 35 years at the scheduled time of first vaccination with a written Informed Consent, signed/thumb printed and dated will be eligible to be screened for the trial.
Volunteers will provide a medical history through a one-on-one interview with a clinical officer. Volunteers will also undergo physical examination and standard laboratory screening tests, which include complete blood count (CBC), creatinine, and ALT. Blood to screen for hemoglobinopathies (sickle cell disease/trait, G6PD deficiency, and alphathalassemia) will also be drawn. Volunteers may be screened one additional time if rescreening is determined to be necessary (e.g. after treatment for malaria). Volunteers will be excluded from participation if they meet any of the exclusion criteria. Volunteers excluded from this study because of significant abnormalities will be referred to a named specialist at the New Nyanza Hospital for evaluation and treatment as described in Section 5.7.3). All screening tests will be completed within 42 days prior to entry into the study. Information gathered during screening (medical history, physical examination, and laboratory analysis) will be recorded in the Source Documents and the GSK Biologicals' CRFs.
A photograph will be taken of each volunteer who is screened and stored securely in a computer at KC. When screening and review of inclusion/exclusion criteria are complete, a picture ID card of each eligible volunteer will be created using the stored photo. In addition to the subject's photo, name, subject number and the study number and name, the card will contain contact information for KC. This will ensure that the subject can contact an investigator, and the clinic can be contacted if medical care is received outside the study health facilities. Copies of these pictures will be kept in the subjects' records to aid the study staff in confirmation of the volunteers identification for future visits. The photos of subjects who are determined ineligible to participate will be deleted from the computer.
At the screening visit each subject will be given a Subject Number and each will have a clinic record prepared. Subject Numbers will be issued consecutively in the order in which the subjects arrive at the health center. This number will be used throughout the study to identify every document and blood sample associated with the volunteer. The clinic records will contain the Subject Number, the subject's date of birth, medical history, findings of the physical examination, the date of screening visit, whether the subject was enrolled, and (where applicable) reasons for exclusion from the study. The name of the study will be written on both the ID card and clinic records of enrolled volunteers.
All screening laboratory studies will be performed at WRP laboratories. After reviewing the medical history, physical examination and laboratory results, any reasons for noneligibility will be documented on the screening list and in the GSK Biologicals Screening CRF.
Field workers will notify each volunteer of their eligibility to enroll in the study. It is expected that at least 500 volunteers will have to be screened to meet the required number for enrolment. Any volunteers who are found to be ineligible for the trial due to illness will be referred for specialist medical care as detailed in Section 5.7.3.

Vaccination process
After screening, all eligible volunteers will be asked to return for vaccination. On the day of Dose 1, randomization will occur on a first come, first served basis until the target number for the study has been reached. The RTS,S/AS02A, RTS,S/AS01B and Rabies vaccines will be packaged in identical boxes and will be identified by a Treatment Number. The unique Treatment Number will identify each dose of the vaccine administered to each subject. After randomization the Treatment Number will assign the subject to one group or another in a blinded way. The Treatment Number will be issued consecutively at Clinic Visit 2. Each subject will retain the same Treatment Number for their subsequent vaccine doses. The Treatment Number will be recorded on the subject's Clinic Record and in the GSK Biologicals CRF after the vaccine has been administered. The Clinic Record and GSK Biologicals CRF link the Subject Number and the Treatment Number.
Vaccinations will take place at KC. On each vaccination day, criteria for continued eligibility will be reviewed and verified. A history-directed physical examination will be done and oral temperature, blood pressure and baseline general symptom history will be recorded. Volunteers will then have venous blood collected for laboratory analysis as detailed in Section 5.3. The volunteer will then be placed in one of the individual vaccination rooms adjacent to the pharmacy.
After the identity of the subject is again checked (by comparing the Subject Number and photograph in the subject's record with the subject's photo ID), the photo ID and the Clinic Record will be given to the pharmacy through a hatch connecting the vaccination room with the pharmacy. The pharmacy will then prepare the appropriate vaccine following a consecutive treatment number assignment and return it to the vaccination room (with the Clinic Record) through the same hatch, for administration.
Since the vaccines used in this study are of distinct appearance, the Pharmacist and the Vaccinator are not blinded and perform no other function in the study (refer to Section 6.4). The Pharmacist will select the sealed box labeled with the subject's Treatment Number, containing the vials numbered with the Treatment Number, remove the vaccine vials and fill a syringe according to this study protocol (refer to Section 6.2). The Pharmacist will then place a numbered opaque label with the subject's Treatment Number on the syringe. The purpose of masking the syringe is to blind the subject. After administering the vaccination to the subject, the Vaccinator will enter the Treatment Number administered to the subject on the clinic form. Subjects will be vaccinated by deltoid intramuscular injection into the non-dominant arm by a qualified nurse. The volunteer will not know what injection was given. A staff member experienced in resuscitation will be available at all vaccination sessions. Facilities and equipment will be available to give emergency treatment in the case of an anaphylactic reaction following administration of vaccines.
Immediately after vaccination, each subject will proceed to the next station for assessment of local and systemic reactions. Each subject will be observed for at least 30 minutes post-vaccination. Appropriate medical treatment will be readily available in the unlikely case of an anaphylactic reaction following the administration of the vaccine.
Subjects who cannot be vaccinated on the originally scheduled date (due to acute illness, local lesion at the injection site or scheduling conflicts) may be vaccinated if eligible within the next 5 days. In the particular case of any subject found to be febrile (oral temperature ≥ 37.5°C), a malaria blood film (MBF) will be taken to investigate for malaria. Subjects will be treated as required for their condition and will be followed up until resolution of any symptoms and be vaccinated if their clinical symptoms resolve within 5 days.
Those who cannot be re-vaccinated within 5 days of their scheduled date will continue all study procedures apart from receiving further study vaccinations.

Home follow-up visits for assessment of reactogenicity (7day follow-up period)
Subjects will be evaluated daily for seven days after each vaccination (day of vaccination plus six more days). Evaluation will be conducted at designated field stations by trained field workers under the supervision of the PI. Subjects will be evaluated by soliciting post-vaccination signs and symptoms from the volunteer and by examination of the injection site. Results of each visit will be recorded by the field worker on the diary card for that subject.
In the event that a field worker finds any Grade 3 solicited or unsolicited sign/symptom, the subject will be brought to KC for examination by a study clinician. The evaluation and any further clinical data, including treatment provided, will be written on the diary card as well as clinic forms and transcribed onto the CRF. If the clinician determines that the subject has experienced a serious adverse event (SAE), appropriate measures will be taken to notify the Local Safety Monitor (LSM), the SMG, the IRBs, the sponsor, USAMMDA, GSK Biologicals and WRAIR.
Every effort will be made to ensure that each subject completes every visit. If a subject does not appear for a scheduled clinic visit, field workers will be dispatched to their home that same day to attempt to contact them. Regular attempts to contact the subject will continue until the study ends or a reason is found for missing the visit (e.g. the subject moved away from the study area). When the subject is contacted the field worker will measure vital signs and collect information on any solicited or unsolicited symptoms since the last scheduled visit.
After each follow-up period, diary cards will be checked and verified by the PI or designate prior to transcription onto CRFs. The PI has a primary responsibility for the data transcribed onto the CRFs. Unresolved AEs will be followed-up by field workers until resolution under the supervision of the Principal Investigator and data will be entered onto the CRF. The procedures and frequency of visits will be outlined in a SOP at the investigator's site.

Monitoring of hematological and biochemical laboratory parameters
Results of hematological and biochemical laboratory tests will be reviewed as soon as possible after they are generated. Any value outside the normal range will be managed as appropriate by a medically qualified individual under the supervision of the Principal Investigator. The hematological and biochemical parameters will be documented on the CRF. For all values outside the normal range, the reason and/or clinical condition will be documented. Guidance on when to report abnormalities as SAEs is given in Section 8.3.

Surveillance for SAEs (all subjects)
All study subjects will have access to free health care at Kombewa Clinic 24 hours/day through one of the field stations located within one mile of their residence. Study subjects will be encouraged to present to the clinic or field station for any medical issue that may arise during the study. When a subject presents to a field station, transportation will be provided to KC where medical staff is available 24 hours/day. This will be the primary means of SAE surveillance.
Subjects are also given contact numbers for the clinical staff at KC on their study ID card. Should a study subject have to present to some other medical facility they are instructed to present the card to the health care provider who can then contact KC and arrange for transportation, treatment or payment of treatment for the subject. If a subject is treated at a medical facility other than KC, an attempt will be made to obtain a copy of the treatment record for review by the PI.
Additionally, volunteers will be asked if they have been seen or treated at an outside medical facility since their last visit to KC. If so, the PI will request a copy of the record from the relevant medical facility The record of every acute clinic visit (at KC or otherwise) will be reviewed by the PI or designate for SAEs. The capabilities of the clinic and continuous staffing have been developed to ensure any acute illness or outpatient medical need of anyone involved in a study can be addressed 24 hours a day. The KC is staffed at all times (24 hours a day) during a study with at least a clinical officer, community nurse and a driver with an evacuation vehicle. During working hours, Monday though Friday, the basic staffing also includes a medical officer (physician), pharmacist, lab technician, and microscopist.
All outpatient services for study participants will be provided by the staff at KC. Transportation to the clinic and medical staff will be available 24 hours a day.

New Nyanza Provincial General Hospital
New Nyanza Provincial General Hospital (NNPGH) in Kisumu is the primary referral site for study participants requiring inpatient care or blood transfusion. If either is required, KC personnel will transport the subject to NNPGH. This hospital includes two adult wards with 30 beds each, oxygen supplementation capabilities, 24-hour nursing and physician coverage, radiology, laboratory, pathology, surgical suites, and screened blood supply. The subject will be admitted to the care of Kenyan physicians on staff. Subjects who are admitted will be followed daily by clinical personnel from KC as well. Specialty care will be coordinated for by KC personnel as required. The medical record from NNPGH will be the SAE source document.

Referring of Excluded Volunteers to Medical Care
If a volunteer is found to have a medical condition that excludes them from the trial they will be informed at a private appointment with a member of the clinical staff. The clinical staff member will take as much time as required to explain the medical condition including its severity, potential causes, long-term complications (if there are any), impact on current lifestyle and evaluation and treatment options. Once it is clear the volunteer understands the medical condition, the clinical staff member will develop an evaluation and treatment plan with the volunteer and ensure that the volunteer understands their options.
WRP has identified specialty and sub-specialty physicians in the local area. If a volunteer is excluded from the study for a medical condition or laboratory abnormality not previously known to the volunteer they will be referred for care to one of these physicians according to their illness. WRP personnel will coordinate with the relevant physician and the volunteer to make an appointment and provide transportation to the appointment if necessary. The history, physical exam, and laboratory findings that lead to exclusion and referral for evaluation will be provided to the subject and the referral physician. WRP will not pay for the referral but will ensure that the appointment is coordinated and transportation is provided.

Management and treatment of malaria in all subjects
Infection with malaria parasites is defined as P. falciparum asexual parasitemia > 0. If a subject is found to have a positive malaria smear (with or without symptoms) the subject will be treated with a regimen of artemether/lumefantrine (Coartem ® , Novartis AG, Zurich, Switzerland In the event of treatment failure or inability to tolerate artemether/lumefantrine, Malarone ® (atovaquone and proguanil hydrochloride, GlaxoSmithKline, Uxbridge, UK) or quinine will be used as the alternative outpatient treatment. The dosing regimen of quinine for adults with acute malaria is 600 mg orally every 8 hours for seven days.
Subjects with vomiting or manifestations of severe malaria (including mental status changes, convulsions, renal failure) will be admitted to NNPGH for treatment with parenteral antimalarials. Quinine will be used for severe malaria. A member of the KC clinical team will review the subject admitted to NNPGH daily until discharge from hospital.
All study subjects treated for malaria will be followed (at the clinic or by field worker home visit) until resolution of symptoms. All treatment courses will be administered under the direction of the attending clinician and recorded in the subject's record. Subjects found to have clinical malaria will be treated before any further vaccination. Subjects will be given the next dose of vaccine if their clinical symptoms resolve within the vaccination window. If the clinical symptoms do not resolve within that period, the subject will not be vaccinated; however, the subject will be followed for collection of all study data points.

Passive case detection of clinical cases of malaria
In the event that a subject presents to one of the field stations with fever (≥ 37.5ºC) or history of fever during the past 24 hours they will be transported to KC and evaluated by a clinician. An MBF will be taken and read immediately and complete blood count (CBC) evaluated. All subjects with asexual parasitemia will be treated. The procedures for MBFs are outlined in site SOPs.
Passive case detection of malaria will begin with screening and continue until 16 weeks post Dose 3 (end of the double-blind period).

Active detection of infection
The ADI period for malaria infection will begin at Day 74 (Field Worker Visit 40), 14 days after Dose 3. ADI visits will occur weekly for 14 weeks. Blood samples will be taken for examination of malaria parasitemia at each ADI visit. ADI visits will continue through to Month 6½ (Clinic Visit 8).
Three weeks prior to the start of surveillance for malaria infection (i.e. 1 week prior to Dose 3), all subjects will be presumptively treated for aymptomatic parasitemia with Malarone ® (atovaquone and proguanil hydrochloride, GlaxoSmithKline, Uxbridge, UK).
(in the event that subjects are unable to tolerate Malarone ® , Coartem ® (artemether/lumefantrine, Novartis AG, Zurich, Switzerland) will be used as the alternative clearance treatment. The subjects will visit field workers for three consecutive days. At each of these visits, the field worker will give the subject four tablets of Malarone ® (containing 250 mg atovaquone and 100 mg proguanil hydrochloride), and observe the subject taking the tablets. The first dose of medication will be administered at the field stations on Study Day 53 (Field Worker Visit 32). The second dose will be given under observation the next morning by the field worker team (Study Day 54; Field Worker Visit 33). The third dose (the final one) will be given by the field worker team the following morning (Study Day 55; Field Worker Visit 34).
The absence of parasitemia prior to the start of the ADI period will be checked by MBF one week post Dose 3 (Study Day 66; Clinic Visit 6). Any subject with any level of parasitemia at this point will be treated with the second-line treatment for malaria (Coartem ® ).
For the ADI period, a field worker visit will consist of the subject presenting to a field station or the KC and completing a brief surveillance for infection morbidity questionnaire, which will include the reporting of malaria symptoms and a record of oral temperature. If the subject is well and afebrile, a blood-sample will be taken and examined for malaria parasitemia and evaluated for CBC. Blood slides from the sample will be Giemsa-stained and read at KC to determine the presence of parasites. Treatment for asymptomatic subjects with positive MBF will be sent by a field worker within 72 hours of the when the MBF was taken.
In the event that a subject reports a history of fever within the preceding 24 hours or has a documented oral temperature of ≥ 37.5°C, no blood slides will be taken by the field worker but transport by a project vehicle for the subject to KC will be arranged. The surveillance for infection morbidity questionnaire will record that the subject was transferred to KC for further evaluation. At KC the subject will be evaluated by a clinician and a venous blood sample will be taken for CBC, determination of parasitemia. Subjects with a positive MBF will be managed according to the procedures detailed above (see Section 5.8). Subjects who are diagnosed in such a manner will be classified as diagnosed by ADI.
At first detection of malaria infection with asexual forms of P. falciparum, either by ADI surveillance or on clinical presentation with malaria disease, the subject will be considered to be infected and no further visits for surveillance of infection will be undertaken. The subject will still be under morbidity surveillance and encouraged to seek care at the KC.

Subject identification
Subject Numbers will be issued consecutively to all volunteers on the day of screening.
To identify the volunteer at subsequent contacts, they will be issued a photo ID that includes the volunteer's subject number.

Recording of medication
Administer drug for clearance of parasitemia ADI; Active Detection of Infection X; Drug for the presumptive clearance of Plasmodium parasitemia will be given during these visits a all subjects will be required to attend these cross-sectional visits, regardless of whether or not they have previously reported malaria during the ADI period b Includes analysis of hemoglobin, total white cell count absolute lymphocyte count and platelets. Done with safety labs and with MBF to determine parasite count c If a subject presenting to a health center or field site reports fever within the preceding 24 hours or has a documented fever (oral temperature ≥ 37.5°C) then blood will be taken for parasitemia determination and hemoglobin d any subjects who test positive for malaria parasitemia on this visit will be administered Coartem ® to reclear them e these blood samples are carried out on subjects for as long as they take part in the ADI Phase; if a subject has a positive MBF they no longer partake in this sampling f this visit serves as both the final visit for soliciting symptoms at Day 6 post Dose 3 and the first visit for ADI g all medications to be recorded at these visits h record administration of immunosuppresants or other immune-modifying drugs during this period (for corticosteroids this means prednisone or equivalent, ≥ 0.5 mg/kg/day. Inhaled or topical steroids are allowed and should not be recorded. ALSO all immunoglobulins and blood products should be recorded during this period. ALSO any antimalarials. I record administration of immunosuppresants or other immune-modifying drugs during this period (for corticosteroids this means prednisone or equivalent, ≥ 0.5 mg/kg/day. Inhaled or topical steroids are allowed and should not be recorded. ALSO all immunoglobulins and blood products should be recorded during this period. j SAEs related to study procedures will be collected • is used to indicate a study procedure that requires documentation in the indivudual CRF and ○ is used to indicate a study procedure that does not require documentation in the individual CRF. *SAEs that are related to study participation or are related to a concurrent medication will be collected and recorded from the time the subject consents to participate in the study until they are discharged Amended 13 June 2006

Detailed description of study visits
When materials are provided by GSK Biologicals, it is MANDATORY that all clinical samples (including serum samples) will be collected and stored using exclusively those materials in the appropriate manner. The use of other materials could result in the exclusion of the subject from the ATP analysis (see Section 10.6 for definition of study cohorts to be evaluated). The investigator must ensure that his/her personnel and the laboratory(ies) under his/her supervision comply with this requirement. However, when GSK Biologicals does not provide material for collecting and storing clinical samples, then appropriate materials from the investigator's site are to be used. Refer to Appendix D and Appendix E.
The subjects will be instructed to contact the investigator immediately should the subject manifest any signs or symptoms they perceive as serious.
The following schedule reflects the visits for a single subject.  • Randomize subject to a vaccination group • Confirm that the subject's randomization number and Subject Number agree with label on syringe • Administer study vaccine Dose 1; record site, date of injection.

After vaccination:
• Observe each vaccinee for at least 30 minutes.
• Record blood pressure, oral temperature at the end of post-vaccination observation period • Examine site of injection

• Record solicited and unsolicited AEs
• Record any post-vaccination SAEs • Instruct subject to return to KC immediately should subject manifest any sign or symptom which they perceive as serious.

Field Worker Visit Code Numbers 21 to 25: Daily Post-vaccination follow up visits Day 1 to Day 5
• Examine site of injection • Confirm that the subject's randomization number and Study Number agree with label on syringe • Administer study vaccine Dose 3; record site, date of injection.

After vaccination:
• Observe vaccinee for at least 30 minutes.
• Record blood pressure, oral temperature at the end of post-vaccination observation period • Examine site of injection

• Record any post-vaccination SAEs
• Instruct subject to return to KC immediately should subject manifest any sign or symptom which they perceive as serious. All subjects who are positive for malaria parasites should be treated with the secondary clearance drug, Coartem ® as described in Section 5.8. These subjects are to be included in ADI surveillance

Field Worker Visit Code Number 40 and 41: ADI visits #1 and 2 Weekly Visits
• Record oral temperature, solicit history of fever in the last 24 hours • Transport any subject with a fever or history of fever in the past 24 hours to KC for evaluation, including MBF and CBC and malaria parasite genotyping • Draw 2 mL of blood from asymptomatic patients for:

All subjects who received Dose 3 should be invited to this visit regardless of whether they have had a positive MBF or reported having had malaria during the preceding ADI visits
• Record blood pressure • Record oral temperatures, solicit history of fever in the last 24 hours • Record any subject complaints, interim history, history-directed physical examination and examination of the vaccination site for any abnormalities • Transport any subject with a fever or history of fever in the past 24 hours to KC for evaluation, including MBF and CBC and malaria parasite genotyping  Separation of serum from the blood samples will be performed at the investigator's center.
Hematology and biochemistry determination will be performed at the KEMRI laboratories. The acceptable reference ranges will be specified in an SOP at the investigator site and in the GSK central study file.
CS-specific CMI will be determined at KEMRI and at GSK Biologicals by intra-cellular cytokine staining (CD4, CD8, CD3, IFN-gamma and activation markers) and measured as frequency (percent) of cytokine positive cells.

DNA extraction, PCR and determination of P. falciparum genotypes (MOI) will be performed at the Walter Reed laboratory, Kisumu, Kenya. The determination of CSP polymorphic sequences will be done at HPA Malaria Reference Laboratory, LSHTM, UK. Amended 13 June 2006
Serological responses will be primarily measured by evaluating antibody responses to CS (anti-CS also referred to as anti-R32LR antibodies). Antibody levels will be measured by standard Enzyme Linked Immunosorbent Assay (ELISA) methodologies using R32LR antigen as coating antigen and a standard reference in order to assess the relative titer of each test sample. Results will be reported in EU/mL. Anti-HBs antibody levels will be measured by ELISA with a commercial AUSAB EIA kit from Abbott or equivalent.
Serum for antibody determination will be collected by venous blood sample (as specified at each visit in Sections 5.4 and 5.5). Samples for safety will be analyzed at the time they are collected.
Serum samples from each subject will be shipped to GSK Biologicals, Rixensart, Belgium. Any serum not immediately used in antibody assays will be stored at -20°C or less and would only be used to assess the immune response to vaccination or to assess any potential toxicity of the vaccine (Assay details are provided in Appendix F).

Investigational products
The RTS,S/AS02A, RTS,S/AS01B vaccines to be used have been developed and manufactured by GSK Biologicals.
The Quality Control Standards and Requirements for the candidate vaccines are described in separate release protocols and the required approvals have been obtained.
Commercial vaccines are assumed to comply with the specifications given in the manufacturer's Summary of Product Characteristics.
Refer to Appendix G for details of vaccine supplies.

RTS,S antigen presentation:
• The lyophilized antigen pellet contains 62.5 μg of RTS,S with 25.2 mg of sucrose as cryoprotectant per 3 mL monodose vial. The pellet is reconstituted with adjuvant in liquid form and 0.5 mL of reconstituted vaccine contains 50 µg RTS,S.
A dose of 0.5 mL will be delivered. The presentation of the reconstituted RTS,S/AS02A candidate malaria vaccine is an opaque milky liquid.

RTS,S antigen presentation:
• The lyophilized antigen pellet contains 62.5 μg of RTS,S with 25.2 mg of sucrose as cryoprotectant per 3 mL monodose vial. The pellet is reconstituted with adjuvant in liquid form and 0.5 mL of reconstituted vaccine contains 50 µg RTS,S.

AS01B adjuvant:
• AS01B contains 50 μg of MPL ® , 50 μg QS21 (QS21 is a triterpene glycoside purified from the bark of Quillaja saponaria) in a suspension of liposomes in phosphate buffered saline per 0.5 mL and is presented in 3 mL monodose vials.
A dose of 0.5 mL will be delivered. The presentation of the reconstituted RTS,S/AS01B candidate malaria vaccine is an opalescent liquid.

Rabipur Rabies Vaccine
Rabipur, is a sterile freeze-dried vaccine obtained by growing the fixed-virus strain Flury LEP in primary cultures of chicken fibroblasts.
The potency of one dose (1.0 mL) Rabipur is at least 2.5 IU of rabies antigen. Rabipur is a white, freeze-dried vaccine for reconstitution with the diluent prior to use; the reconstituted vaccine is a clear to slightly opaque, colorless suspension.
A dose of 1.0 mL will be delivered. The presentation of the reconstituted vaccine is as a clear or slightly opaque suspension. RTS,S/AS02A will be supplied such that the reconstituted vaccine volume will provide a 0.5 mL adult dose. One 0.5 mL dose will be aspirated from each vial and used.
Disinfect top of vaccine vial (pellet) with alcohol swabs and let dry. Inject complete contents of one PFS of diluent into vial of lyophilized vaccine. Remove and discard the syringe and needle under appropriate safety precautions. The pellet is then dissolved by gently shaking the vial. Wait for 1 minute to ensure complete dissolution of vial contents before withdrawing a sufficient volume to provide a 0.5 mL dose (volume required for RTS,S/AS02A) of the reconstituted vaccine solution using a fresh needle and syringe for injection. The reconstituted vaccine should be administered by slow IM injection, using a 25G needle with length of 1 inch (25 mm), in the deltoid of the arm within 4 hours of reconstitution (storage at 2°C to 8°C).

RTS,S/AS01B (0.5 mL dose)
RTS,S/AS01B will be supplied such that the reconstituted vaccine volume will provide a 0.5 mL adult dose. One 0.5 mL dose will be aspirated from each vial and used.

Disinfect top of vaccine vial (pellet) and adjuvant vial with alcohol swabs and let dry.
Aspirate the contents of the adjuvant vial in a syringe and inject adjuvant into the vial of lyophilized antigen. Remove and discard the syringe and needle under appropriate safety precautions. The pellet is then dissolved by gently shaking the vial. Wait for 1 minute to ensure complete dissolution of vial contents before withdrawing a sufficient volume to provide a 0.5 mL dose (volume required for RTS,S/AS01B) of the reconstituted vaccine solution using a fresh needle and syringe for injection. The reconstituted vaccine should be administered by slow IM injection, using a 25G needle with length of 1 inch (25 mm), in the deltoid of the arm within 4 hours of reconstitution (storage at 2°C to 8°C).

Rabipur rabies vaccine (1.0 mL dose)
Disinfect top of vaccine vial with alcohol swabs and let dry. Inject the entire contents of the diluent ampoule into the vaccine vial. Keeping the syringe and needle in place, the freeze-dried vaccine is dissolved by gently shaking the vial. Wait for 1 minute to ensure complete dissolution of vial contents before withdrawing a sufficient volume to provide a 1.0 mL dose still using the original needle and original syringe. The original needle should then be replaced with a fresh needle for IM injection. The reconstituted vaccine should be used immediately. All vaccine/adjuvant/water for injection vials will be stored in a safe and locked place with no access for unauthorized personnel. Storage temperature will be monitored daily, according to SOPs at the investigator's site. An alarm system and a back-up refrigerator will be available in case of power failure/breakdown.

ALL VACCINE VIALS/PRE-FILLED SYRINGES (RTS,S, and Rabies antigen), AND ADJUVANTS (AS02A and AS01B water for injection) MUST BE STORED IN THE REFRIGERATOR (+2°C to +8°C) AND MUST NOT BE FROZEN.
The study monitor must be contacted if the cold chain is broken (e.g. vaccines become frozen or refrigeration fails).
Storage conditions for transport of vaccines from country medical department or dispatch center to study sites or between sites are described in Appendix D.

Treatment allocation and randomization
6.4.1.

Randomization of supplies
A randomization list will be generated at GSK Biologicals, Rixensart, using a standard SAS ® (Statistical Analysis System) program and will be used to assign the vaccines to treatment numbers. A 1:1:1 randomization of RTS,S/AS02A : RTS,S/AS01B : Rabipur will be used throughout the study.

Randomization of subjects
Subjects will be allocated sequentially to treatment numbers in the order that they present for vaccination.

Method of blinding and breaking the study blind
Data pertaining to the investigational products will be collected in a double blinded (observer blinded) manner. 'Double blinded (observer blinded)' means that the recipient of the investigational product as well as those responsible for the evaluation of safety, immunogenicity and efficacy endpoints will all be unaware which treatment (RTS,S/AS02A, RTS,S/AS01B or Rabipur) was administered to a particular subject. The only study staff aware of the vaccine assignment for RTS,S/AS02A, RTS,S/AS01B or Rabipur will be those responsible for the preparation and administration of investigational product; these staff will play no other role in the study.
Code break envelopes, for each study enrolled subject and associating each treatment number with a specific vaccine, will be kept in a safe and locked place with no access for unauthorized personnel by the Local Safety Monitor in Kenya as well as by Central Safety at GlaxoSmithKline Biologicals, Rixensart.
If deemed necessary for reasons such as safety, the Local Safety Monitor in Kenya as well as GSK Biologicals Central Safety will unblind the specific enrolled subject without revealing the study blind to the investigators.
One interim analysis will be conducted during the study: an analysis of Safety and Immunogenicity post Clinic Visit 7. To maintain the blind the interim analysis will be performed by a statistician independent of the GSK Biologicals and WRAIR clinical teams.
A formal reporting and analysis plan (RAP) will be developed and agreed upon by the PI, the Sponsor (USAMMDA) and GSK Biologicals prior to unblinding. Once the study is completed and the GSK Biologicals reference database locked, GSK Biologicals will be responsible for initiating the execution of the statistical analysis plan in collaboration with WRAIR and breaking the blind.
GSK Biologicals' policy (incorporating ICH E2A guidance, EU Clinical Trial Directive and Federal Regulations) is to unblind any serious adverse event (SAE) report associated with the use of the investigational product, which is unexpected and attributable/suspected, prior to regulatory reporting. The Clinical Safety physician is responsible for unblinding the treatment assignment in accordance with specified time frames for expedited reporting of SAEs (Refer to Section 8.8).

Replacement of unusable vaccine doses
Additional vaccine doses will be provided to replace those that are unusable (see Appendix G for details of supplies).
In addition to the vaccine doses provided for the planned number of enrolled subjects, 3% additional doses will be supplied. In case a vaccine dose is broken or unusable, the investigator should replace it with a replacement vaccine dose. If a vaccine dose needs replacement, the envelope with the corresponding treatment number will designate the replacement without unblinding the study using a coded letter system. Although the sponsor, USAMMDA, and GSK Biologicals need not be notified immediately in these cases, documentation of the use of the replacement vaccine must be recorded by the investigator on the vaccine administration page of the CRF and on the vaccine accountability form.

Packaging
See Appendix G.

Vaccine accountability
See Appendix G.

Concomitant medication/treatment
At each study visit/contact, the investigator should question the enrolled subject about any medication(s) taken.
All antipyretic, analgesic, and antibiotic and administered at ANY time during the period starting with administration of each dose and ending 30 days after each dose are to be recorded with generic name of the medication (trade names are allowed for combination drugs, i.e., multi-component drugs), medical indication, total daily dose, route of administration, start and end dates of treatment.
All antimalarial drugs administered from the beginning of the study until the end of the ADI period (i.e. 4 months post Dose 3) are to be recorded with generic name of the medication (trade names are allowed for combination drugs, i.e., multi-component drugs), medical indication, total daily dose, route of administration, start and end dates of treatment.
Any treatments and/or medications which are listed as elimination criteria in Section 4.5, e.g., any immunoglobulins, other blood products and any immune modifying drugs administered within three months preceding the first dose or at any time during the study period are to be recorded with generic name of the medication (trade names are allowed for combination drugs only), medical indication, total daily dose, route of administration, start and end dates of treatment. Refer to Sections 4.4 and 4.5.
Any vaccine not foreseen in the study protocol administered in the period beginning 30 days preceding each dose and ending 30 days after each dose is to be recorded with trade name, route of administration and date(s) of administration. Refer to Sections 4.4 and 4.5.
Any concomitant medication administered prophylactically in anticipation of reaction to the vaccination must be recorded in the CRF with generic name of the medication (trade names are allowed for combination drugs only), total daily dose, route of administration, start and end dates of treatment and coded as 'Prophylactic'.

ADVERSE EVENTS AND SERIOUS ADVERSE EVENTS
The investigator is responsible for the detection and documentation of events meeting the criteria and definition of an adverse event (AE) or serious adverse event (SAE) as provided in this protocol. During the study, when there is a safety evaluation, the investigator or site staff will be responsible for detecting AEs and SAEs, as detailed in this section of the protocol. In addition, for this study the investigator is responsible for the timely reporting of unanticipated problems (refer to Section 8.3).
Each subject will be instructed to contact the investigator immediately should they manifest any signs or symptoms they perceive as serious.

Definition of an adverse event
An AE is any untoward medical occurrence in a clinical investigation subject, temporally associated with the use of a medicinal product, whether or not considered related to the medicinal product.
An AE can therefore be any unfavorable and unintended sign (including an abnormal laboratory finding), symptom, or disease (new or exacerbated) temporally associated with the use of a medicinal product.
Examples of an AE include: • Exacerbation of a chronic or intermittent pre-existing condition including either an increase in frequency and/or intensity of the condition. • New conditions detected or diagnosed after investigational product administration even though it may have been present prior to the start of the study. • Signs, symptoms, or the clinical sequelae of a suspected overdose of either investigational product or a concurrent medication (overdose per se should not be reported as an AE/SAE). • Signs, symptoms temporally associated with vaccine administration.
AEs may include pre-or post-treatment events that occur as a result of protocolmandated procedures (i.e., invasive procedures, modification of subject's previous therapeutic regimen).
N.B. AEs to be recorded as endpoints (solicited events) are described in Section 8.4.1.
All other AEs will be recorded as UNSOLICITED AEs.
Example of events to be recorded in the medical history section of the CRF: Pre-existing conditions or signs and/or symptoms present in a subject prior to the start of the study (i.e. prior to the first study procedure) should be recorded in the medical history section of the subject's CRF. e. is a congenital anomaly/birth defect in the offspring of a study subject.

Definition of a serious adverse event
f. Medical or scientific judgment should be exercised in deciding whether reporting is appropriate in other situations, such as important medical events that may not be immediately life-threatening or result in death or hospitalization but may jeopardize the subject or may require medical or surgical intervention to prevent one of the other outcomes listed in the above definition. These should also be considered serious. Examples of such events are invasive or malignant cancers, intensive treatment in an emergency room or at home for allergic bronchospasm, blood dyscrasias or convulsions that do not result in hospitalization.

Definition of an Unanticipated Problem
Unanticipated problems are those problems that are not specifically described in the protocol or other study documents. They are problems that involve risks to subjects and others, although the risk may not result in bodily harm. For example, misplacing a subject's study records containing identifiable private information results in the risk of breach of confidentiality. Confidentiality may or may not be breached, but either way this would be a reportable event. Another example would be administering the wrong agent to a subject at one time point in a series of vaccinations. Risks to others must also be reported. For example, an inadvertent exposure of a household contact in a smallpox vaccine trial would be a reportable event. Problems resulting in risks to members of the research team are also reportable. Reporting will be carried out with forms supplied by the HSRRB. Reports by the investigator should be promptly sent to the US Army Medical Research and Materiel Command's Human Subjects Research Review Board and to the medical monitor at GSK Biologicals; details of the addresses to which reports should be sent are given in Section 8.8.3. Appropriate supporting documents, such as laboratory reports, pathology reports, and discharge summaries should be submitted with the unanticipated problem report. The medical monitor should indicate whether she concurs with the details provided in the investigator's report. A medical monitor report that comments on the outcomes of the event and the relationship of the event to participation in the study must be submitted to the HSRRB within ten calendar days. Follow-up reports should be submitted until resolution of the unanticipated problem.

Clinical laboratory parameters and other abnormal assessments qualifying as adverse events and serious adverse events
Abnormal laboratory findings (e.g., clinical chemistry, hematology, urinalysis) or other abnormal assessments (e.g. blood film) that are judged by the investigator to be clinically significant will be recorded as AEs or SAEs if they meet the definition of an AE, as defined in Section 8.1 or SAE, as defined in Section 8.2. Clinically significant abnormal laboratory findings or other abnormal assessments that are detected during the study or are present at baseline and significantly worsen following the start of the study will be reported as AEs or SAEs. However, clinically significant abnormal laboratory findings or other abnormal assessments that are associated with the disease being studied, unless judged by the investigator as more severe than expected for the subject's condition, or that are present or detected at the start of the study and do not worsen, will not be reported as AEs or SAEs.
The investigator will exercise his or her medical and scientific judgment in deciding whether an abnormal laboratory finding or other abnormal assessment is clinically significant.

Time period, frequency, and method of detecting adverse events and serious adverse events
All AEs occurring within 30 days following administration of each dose of vaccine must be recorded on the Adverse Event form in the subject's CRF, irrespective of severity or whether or not they are considered vaccination-related.
The standard time period for collecting and recording SAEs will begin at randomization or the first receipt of vaccine and will end 30 days following administration of the last dose of study vaccine for each subject. See Section 8.8 for instructions for reporting and recording SAEs.
Additionally, in order to fulfil international reporting obligations, SAEs that are related to study participation (e.g. procedures, invasive tests, a change from existing therapy) or are related to a concurrent medication will be collected and recorded from the time the subject consents to participate in the study until she/he is discharged.
All AEs either observed by the investigator or one of his clinical collaborators or reported by the subject spontaneously or in response to a direct question will be evaluated by the investigator. AEs not previously documented in the study will be recorded in the Adverse Event form within the subject's CRF. The nature of each event, date and time (where appropriate) of onset, outcome, intensity and relationship to vaccination should be established. Details of any corrective treatment should be recorded on the appropriate page of the CRF. Refer to Section 6.9.
As a consistent method of soliciting AEs, the subject should be asked a non-leading question such as: "Have you felt different in any way since receiving the vaccine or since the previous visit?" N.B. The investigator should record only those AEs having occurred within the time frame defined above.
AEs already documented in the CRF, i.e. at a previous assessment, and designated as "not recovered/not resolved" or "recovering/resolving" should be reviewed at subsequent visits, as necessary. If these have resolved, the documentation in the CRF should be completed.
N.B. If an AE changes in frequency or intensity during the specified reporting period, a new record of the event will be entered.
When an AE/SAE occurs, it is the responsibility of the investigator to review all documentation (e.g., hospital progress notes, laboratory, and diagnostics reports) relative to the event. The investigator will then record all relevant information regarding an AE/SAE on the CRF or SAE Report Form as applicable. It is not acceptable for the investigator to send photocopies of the subject's medical records to GSK Biologicals in lieu of the appropriate completed AE/SAE pages. However, there may be instances when copies of medical records for certain cases are requested by GSK Biologicals. In this instance, all subject identifiers will be blinded on the copies of the medical records prior to submission to GSK Biologicals.
The investigator will attempt to establish a diagnosis of the event based on signs, symptoms, and/or other clinical information. In such cases, the diagnosis should be documented as the AE/SAE and not the individual signs/symptoms. The visiting field worker will record these adverse events according to detailed SOPs available on study site during the field worker visits.
N.B. Temperature will be recorded on days 1 to 6 by the field worker or Principal Investigator. Should additional temperature measurements be performed at other times of day, the highest temperature will be recorded.

8.6.
Evaluating adverse events and serious adverse events 8.6.1.

Assessment of intensity
Intensity of the following AEs will be assessed as described: Muscle ache that interferes with normal activity 3 Muscle ache that prevents normal activity *Fever is defined as oral temperature ≥37.5°C The maximum intensity of local injection site swelling will be scored at GSK Biologicals as follows: The maximum intensity of fever will be scored at GSK Biologicals as follows:  Hemoglobin <11 g/dL (males) or < 9.5 g/dL (females) Platelet count < 77 000 per mm 3 Absolute lymphocyte count < 1 x10 3 /μL Total white cell count < 3 x10 3 /μL (males) or < 2.5 x 10 3 /μL (females) The investigator will make an assessment of intensity for all other AEs, i.e. unsolicited symptoms, including SAEs reported during the study. The assessment will be based on the investigator's clinical judgment. The intensity of each AE and SAE recorded in the CRF or SAE Report Form, as applicable, should be assigned to one of the following categories: 1 (mild) = An AE which is easily tolerated by the subject, causing minimal discomfort and not interfering with everyday activities.
2 (moderate) = An AE which is sufficiently discomforting to interfere with normal everyday activities.
3 (severe) = An AE which prevents normal, everyday activities (in adults, such an AE would, for example, prevent attendance at work and would necessitate the administration of corrective therapy).
An AE that is assessed as Grade 3 (severe) should not be confused with a SAE. Grade 3 is a category utilized for rating the intensity of an event; and both AEs and SAEs can be assessed as Grade 3. An event is defined as 'serious' when it meets one of the pre-defined outcomes as described in Section 8.2.

Assessment of causality
The investigator is obligated to assess the relationship between investigational product and the occurrence of each AE/SAE. The investigator will use clinical judgment to determine the relationship. Alternative causes, such as natural history of the underlying diseases, concomitant therapy, other risk factors and the temporal relationship of the event to the investigational product will be considered and investigated. The investigator will also consult the Investigator Brochure and/or Product Information, for marketed products, in the determination of his/her assessment.
There may be situations when a SAE has occurred and the investigator has minimal information to include in the initial report to the study contacts for reporting of a serious adverse event (refer to Section 8.8.2). However, it is very important that the investigator always makes an assessment of causality for every event prior to transmission of the SAE Report Form to the study contacts for reporting of a serious adverse event. The investigator may change his/her opinion of causality in light of follow-up information, amending the SAE Report Form accordingly. The causality assessment is one of the criteria used when determining regulatory reporting requirements.
In case of concomitant administration of multiple vaccines, it may not be possible to determine the causal relationship of general AEs to the individual vaccines administered. The investigator should, therefore, assess whether the AE could be causally related to vaccination rather than to the individual vaccines.
All solicited local (injection site) reactions will be considered causally related to vaccination. Causality of all other AEs should be assessed by the investigator using the following question: Is there a reasonable possibility that the AE may have been caused by the investigational product ?
NO : The AE is not causally related to administration of the study vaccines.
There are other, more likely causes and administration of the study vaccines is not suspected to have contributed to the AE.

YES
: There is a reasonable possibility that the vaccines contributed to the AE.
Non-serious and serious AEs will be evaluated as two distinct events. If an event meets the criteria to be determined "serious" (see Section 8.2 for definition of serious adverse event), it will be examined by the investigator to the extent to be able to determine ALL contributing factors applicable to each serious adverse event.

Follow-up of adverse events and serious adverse events and assessment of outcome
After the initial AE/SAE report, the investigator is required to proactively follow each subject and provide further information to study contacts for reporting of a serious adverse event (refer to Section 8.8.2) on the subject's condition.
All AEs and SAEs documented at a previous visit/contact and designated as not recovered/not resolved or recovering/resolving will be reviewed at subsequent visits/contacts.
Investigators will follow-up subjects: • with SAEs or subjects withdrawn from the study as a result of an AE, until the event has resolved, subsided, stabilized, disappeared, the event is otherwise explained, or the subject is lost to follow-up; • or, in the case of other non-serious AEs, until they complete the study or they are lost to follow-up.
Clinically significant laboratory abnormalities will be followed up until they have returned to normal, or a satisfactory explanation has been provided. Additional information (including but not limited to laboratory results) relative to the subsequent course of such an abnormality noted for any subject must be made available to the Study Monitor.
The Sponsor, USAMMDA, Kenya National Ethical Review Committee and GSK Biologicals may request that the investigator perform or arrange for the conduct of supplemental measurements and/or evaluations to elucidate as fully as possible the nature and/or causality of the AE or SAE. The investigator is obliged to assist. If a subject dies during participation in the study or during a recognized follow-up period, study contacts for reporting of a serious adverse event will be provided with a copy of any available post-mortem findings, including histopathology.
New or updated information will be recorded on the originally completed SAE Report Form, with all changes signed and dated by the investigator. SAEs will be reported promptly to the study contacts for reporting of a serious adverse event (refer to Section 8.8.2) once the investigator determines that the event meets the protocol definition of an SAE. The investigator or designate will fax the SAE reports to the study contacts for reporting of a serious adverse event WITHIN 24 HOURS OF HIS/HER BECOMING AWARE OF THESE EVENTS. Additional or follow-up information relating to the initial SAE report is also to be reported to the study contacts for reporting of a serious adverse event within 24 hours of receipt of such information.

Completion and transmission of serious adverse event reports to the study contacts for reporting of a serious adverse event
Once an investigator becomes aware that a SAE has occurred in a study subject, she/he will report the information to study contacts for reporting of a serious adverse event (refer to Section 8.8.2) within 24 hours as outlined in Section 8.8.1. The SAE Report Form will always be completed as thoroughly as possible with all available details of the event, signed by the investigator (or designee), and forwarded to study contacts for reporting of a serious adverse event within the designated time frames. If the investigator does not have all information regarding an SAE, he/she will not wait to receive additional information before notifying study contacts for reporting of a serious adverse event of the event and completing the form. The form will be updated when additional information is received and forwarded to study contacts for reporting of a serious adverse event WITHIN 24 HOURS as outlined in Section 8.8.1.
The investigator will always provide an assessment of causality at the time of the initial report as described in Section 8.6.2.
Facsimile (Fax) transmission of the SAE Report Form is the preferred method to transmit this information to the Study Contacts for Reporting SAEs. In rare circumstances and in the absence of facsimile equipment, notification by telephone is acceptable, with a copy of the SAE Report Form sent by overnight mail. Initial notification via the telephone does not replace the need for the investigator to complete and sign the SAE Report Form within 24 hours as outlined in Section 8.8.1.
In the event of a death determined by the investigator to be related to vaccination, sending of the fax must be accompanied by telephone call to the Study Contacts for Reporting SAEs. . A complete written report should follow the initial notification. In addition to the methods above, the complete report can be sent to the addresses given below.

Regulatory reporting requirements for serious adverse events
The investigator will promptly report all SAEs to study contacts for reporting of a serious adverse event (refer to Section 8.8.2) in accordance with the procedures detailed in Section 8.8. The Sponsor, USAMMDA has a legal responsibility to promptly notify, as appropriate, both the local regulatory authority and other regulatory agencies about the safety of a product under clinical investigation. Prompt notification of SAEs by the investigator to the Study Contacts for Reporting SAEs is essential so that legal obligations and ethical responsibilities towards the safety of other subjects are met. The investigator, or responsible person according to local requirements, will comply with the applicable local regulatory requirements related to the reporting of SAEs to the IRB/IEC and, if required, to the applicable government authority.
Investigator safety reports are prepared according to FDA requirements by the Sponsor (USAMMDA) and are forwarded to investigators as necessary. An investigator safety report is prepared for a SAE(s) that is both attributable to investigational product and unexpected. The purpose of the report is to fulfill specific regulatory and Good Clinical Practice (GCP) requirements, regarding the product under investigation.
An investigator who receives an investigator safety report describing a SAE(s) or other specific safety information (e.g., summary or listing of SAEs) from GSK Biologicals and/or USAMMDA will file it with the Investigator Brochure or other appropriate study documentation and will notify the IRB or IEC, if appropriate according to local requirements.

Post study adverse events and serious adverse events
A post-study AE/SAE is defined as any event that occurs outside of the AE/SAE detection period defined in Section 8.4. Investigators are not obligated to actively seek AEs or SAEs in former study participants.
However, if the investigator learns of any SAE, including a death, at any time after a subject has been discharged from the study, and he/she considers the event reasonably related to the investigational product, the investigator will promptly notify the Study Contacts for Reporting SAEs.

Pregnancy
Subjects who become pregnant during the study (up to 30 days after receiving the last vaccine dose) must not receive additional doses of study vaccine investigative product but may continue other study procedures at the discretion of the investigator.
The investigator, or his designee, will collect pregnancy information on any subject who becomes pregnant while participating in this study. The investigator, or his designee, will record pregnancy information on the Pregnancy Report Form and submit it to study contacts for reporting of a serious adverse event (refer to Section 8.8.2) within 24 hours of learning of a subject's pregnancy. The subject will be followed to determine the outcome of the pregnancy. At the end of the pregnancy, whether that be full-term or prematurely, information on the status of the mother and child will be forwarded to study contacts for reporting of a serious adverse event. Generally, follow-up will be no longer than six to eight weeks following the estimated delivery date.
While pregnancy itself is not considered an AE or SAE, any pregnancy complication or elective termination of a pregnancy for medical reasons will be recorded as an AE or a SAE, as described in Section 8.1 and 8.2, and will be followed as described in Section 8.7.
A spontaneous abortion is always considered to be a SAE and will be reported as described in Section 8.8. Furthermore, any SAE occurring as a result of a post-study pregnancy AND considered reasonably related in time to receipt of the investigational product by the investigator, will be reported to study contacts for reporting of a serious adverse event as described in Section 8.10. While the investigator is not obligated to actively seek this information from former study participants, he may learn of a pregnancy through spontaneous reporting.
Information on pregnancies identified during the screening phase/prior to vaccine administration does not need to be collected; this information need not be communicated to safety.

Treatment of adverse events
Treatment of any adverse event is at the sole discretion of the investigator and according to current good medical practice. Any medication administered for the treatment of an AE should be recorded in the subject's CRF. Refer to Section 6.9.

Subject completion
A subject who returns for the concluding visit/is available for the concluding contact foreseen in the protocol is considered to have completed the study.

Subject withdrawal
Subjects who are withdrawn for AEs must be clearly distinguished from subjects who are withdrawn for other reasons. Investigators will follow subjects who are withdrawn as result of a SAE/AE until resolution of the event (see Section 8.7). Withdrawals will not be replaced.

Subject withdrawal from the study
From an analysis perspective, a 'withdrawal' from the study is any subject who did not come back for the concluding visit.
A subject qualifies as a 'withdrawal' from the study when no study procedure has occurred, no follow-up has been performed and no further information has been collected for this subject from the date of withdrawal/last contact.
Investigators will make an attempt to contact those subjects who do not return for scheduled visits or follow-up.
Information relative to the withdrawal will be documented on the Study Conclusion page of the CRF. The investigator will document whether the decision to withdraw from the study was made by the subject or the investigator and which of the following possible reasons was responsible for withdrawal: • serious adverse event • non-serious adverse event • protocol violation (specify) • consent withdrawal, not due to an adverse event • moved from the study area • lost to follow-up • other (specify).

Subject withdrawal from investigational product
A 'withdrawal' from the investigational product is any subject who does not receive the complete treatment, i.e. when no further planned dose is administered from the date of withdrawal. A subject withdrawn from the investigational product may not necessarily be withdrawn from the study as further study procedures or follow-up may be performed (safety or immunogenicity) if planned in the study protocol.
Information relative to premature discontinuation of the investigational product will be documented on the Vaccine Administration page of the CRF. The investigator will document whether the decision to discontinue further vaccination / treatment was made by the subject or the investigator and which of the following possible reasons was responsible for withdrawal: • serious adverse event, • non-serious adverse event, • other (specify).

Safety & Reactogenicity
• Occurrence of Grade 3 solicited or unsolicited general reactions after each vaccination during a seven day follow-up period (day of vaccination and 6 subsequent days)

Safety
• Occurrence of SAEs until 4 months post Dose 3.
• Occurrence of unsolicited AEs after Dose 1, 2 and 3 of vaccine over a 30 day followup period (day of vaccination and 29 subsequent days) • Occurrence of solicited general and local reactions over a 7 day follow-up period (day of vaccination and 6 subsequent days) after Dose 1, 2 and 3 of vaccine. • Occurrence of parameters of hematological monitoring below normal range.
• Occurrence of parameters of biochemical monitoring above normal range

Immunogenicity
Endpoints assessed prior to vaccination, and 1 month post Dose 3:

• Anti HBs antibody titers
Endpoints assessed prior to vaccination,, 1 month post Dose 2, 1 month post Dose 3 and 4 months post Dose 3: • Anti-CS antibody titers

Efficacy
• The time to first malaria infection (first recording of infection of asexual stage falciparum parasites detected by the active case detection or passive case detection) over a period starting 14 days after Dose 3 and extending for 14 weeks. • The asexual P. falciparum parasitemia (prevalence and density) at 16 weeks post Dose 3

Safety
• The occurrence of SAEs from 4 months post Dose 3 until study conclusion

Cell Mediated Immunity
Endpoint assessed prior to vaccination, 1 month post Dose 3 and 4 months post Dose 3.
• For each subject, the frequency of CS-specific CD4+ and CD8+ T-cells, as measured by intracellular cytokine staining.

Efficacy
• The percentage change in hemoglobin value between baseline and 16 weeks post Dose 3.

Sample size for the Primary Objective
The primary objective will be assessed by comparing the proportion of subjects experiencing a Grade 3 solicited or unsolicited general reaction after each vaccination during a seven day follow-up period (day of vaccination and six subsequent days). In a previous study with RTS,S/AS02A in Africa (Malaria-008) the proportion affected was 10%. The proportion affected was higher in a recent study conducted in non-immune adults at WRAIR (Malaria-027); 39%. Therefore Table 34, below, has been generated for a wide range of hypothetical proportions of Grade 3 general symptoms.

Sample Size for the Secondary Objective for Safety
The data set will be examined, comparing the rates of SAEs at the Medical Dictionary for Regulatory Activities (MedDRA) preferred term level. SAEs are expected to occur infrequently and the study has therefore the power to detect only large differences in the frequencies of SAEs compared to control. Refer to Table 35.

Sample Size for the Secondary Objective for Proof-of-Concept
The minimum vaccine efficacy that a study of this size has the power to detect (i.e. vaccine vs. control) is shown in Table 36. This assumes an attack rate of 72%.   10.6. Study cohorts to be evaluated 10.6.1.

Total Vaccinated cohort
The Total Vaccinated Cohort will include all vaccinated subjects for whom data are available. Thus, the total analysis of safety will include all subjects with at least one vaccine administration documented and the total analysis of immunogenicity/efficacy will include vaccinated subjects for whom data concerning immunogenicity/efficacy endpoint measures are available. The Total Vaccinated Cohort analysis will be performed per treatment actually administered.

According-To-Protocol (ATP) cohort for analysis of safety
The ATP cohort for analysis of safety will include all evaluable subjects; • who have received at least one dose of study vaccine according to their random assignment • have sufficient data to perform an analysis of safety (at least one vaccine dose with safety follow-up) • for whom administration site of study vaccine is per protocol • who have not received a vaccine not specified or forbidden in the protocol and for whom elimination criteria were not applied • for whom the randomization code has not been broken except for when unblinding has been carried out by the SMG for Safety Analysis. • who meet all eligibility criteria.

According To Protocol (ATP) cohort for analysis of immunogenicity
The ATP cohort for analysis of immunogenicity will include all evaluable subjects (i.e. those meeting all eligibility criteria, complying with the procedures defined in the protocol, with no elimination criteria during the study) for whom data concerning immunogenicity endpoint measures are available.

According To Protocol (ATP) cohort for analysis of proof-ofconcept
The ATP cohort for analysis of proof-of-concept will include all evaluable subjects (i.e. those meeting all eligibility criteria, complying with the procedures defined in the protocol, with no elimination criteria during the study) for whom data concerning efficacy endpoint measures are available.

Derived and transformed data
• A subject seropositive for anti-CS antibody is a subject whose antibody titer is greater than or equal to the cut-off value (anti-CS ≥ 0.5 EU/mL). • A subject seropositive for anti-HBs antibody is a subject whose antibody titer is greater than or equal to the cut-off value (anti-HBs ≥ 3.3 mIU/mL).
• A subject seroprotected for anti-HBs antibody is a subject whose antibody titer is greater than or equal to the seroprotective level (anti-HBs ≥ 10 mIU/mL).
• The Geometric Mean Titers (GMTs) calculations are performed by taking the antilog of the mean of the log10 titer transformations. Antibody titers below the cut-off of the assay will be given an arbitrary value of half the cut-off for the purpose of GMT calculation.
For a given subject and a given immunogenicity measurement, missing or non-evaluable measurements will not be replaced. Therefore, an analysis will exclude subjects with missing or non-evaluable measurements.
For the analysis of solicited local/general symptoms, only subjects for which information on the solicited local/general symptom sheet is available for the considered study dose will be included in the analysis.

Final analyses
The final analysis will take place after 4 months post Dose 3 of vaccine (Month 6½). It will complement the interim analysis which takes place 1 month post Dose 3 (Day 90) to provide complete analysis on all primary and secondary endpoints. Section 10.8 describes the analyses to be carried out for the annex report (data up until 12 months post Dose 1).
For the safety primary objective, the occurrence of Grade 3 solicited or unsolicited general reactions will be determined on the Total Vaccinated Cohort. The proportion of subjects with a Grade 3 solicited or unsolicted reaction, reported from study start until study conclusion (end of the double-blind phase) will be tabulated with exact 95% CI.
Comparisons between groups will be done using Fisher's Exact Test.

Secondary objectives
The occurrence of SAEs will be determined on the Total Vaccinated Cohort. The proportion of subjects with an SAE, classified by the MedDRA preferred term level, reported from study start until study conclusion (end of the double-blind phase) will be tabulated with exact 95% CI. Comparisons between groups will be done using Fisher's Exact Test for each preferred term.
The occurrence of adverse events will be determined on the Total Vaccinated Cohort. The proportion of subjects with an AE, classified by the MedDRA preferred term level, reported from study start until study conclusion (end of the double-blind phase) will be tabulated with exact 95% CI. Comparisons between groups will be done using Fisher's Exact Test.
The percentage of subjects with at least one local adverse event (solicited and unsolicited), with at least one general adverse event (solicited and unsolicited) and with any adverse event during the solicited follow-up period will be tabulated with exact 95% CI after each vaccine dose and overall. The percentage of doses followed by at least one local adverse event (solicited and unsolicited), by at least one general adverse event (solicited and unsolicited) and by any adverse event will be tabulated, overall vaccination course, with exact 95% CI. Comparisons between groups will be done using Fisher's Exact Test. Similar tables will be generated for Grade 3 events, the relationship of the event to vaccination and for fever, temperature (in 0.5°C increments).
The percentage of subjects reporting each individual solicited local and general adverse event during the solicited follow-up period will be tabulated with exact 95% CI. The percentage of doses followed by each individual solicited local and general adverse event will be tabulated, overall vaccination course, with exact 95% CI. Comparisons between groups will be done using Fisher's Exact Test. Similar tables will be generated for Grade 3 events, the relationship of the event to vaccination and for fever, temperature (in 0.5°C increments).
For each sampling timepoint, the biochemical parameters (ALT, bilirubin, creatinine) above normal range and hematological parameters (hemoglobin, total WBC, platelets and absolute lymphocyte count) below normal range will be described. Similar tables will be generated for Grade 3 parameters (as determined from Table 32).

Analyses of immunogenicity
The primary analysis will be based on the ATP cohort for analysis of immunogenicity. If the percent of enrolled subjects excluded from this ATP cohort is more than 5%, a second analysis based on the Total Vaccinated cohort will be performed to complement the ATP analysis.

Anti-CS antibodies
The percentage of subjects with sero-positive levels of anti-CS (proportion of subjects with anti-CS antibody titers greater than or equal to 0.5 EU/mL) with 95% CI will be determined prior to vaccination, at 1 month post Dose 2, 1 Month post Dose 3 and 4 months post Dose 3. Antibody titers will be summarized by GMT with 95%CI. Antibody titers after the third dose will also be investigated using reverse cumulative curves.

Anti-HBs antibodies
The percentage of subjects with sero-positive levels of anti-HBs (proportion of subjects with anti-HBs antibody titers greater than or equal to 3.3 mIU/mL), the percentage of subjects with seroprotective levels of anti-HBs (≥10 mIU/mL) with 95% CI will be determined prior to vaccination at 1 Month post Dose 3. Antibody titers will be summarized by GMT with 95%CI. Antibody titers after the third dose will also be investigated using reverse cumulative curves.

Case Definitions
Infection is defined as P. falciparum asexual parasitemia > 0 detected on a scheduled ADI visit or by passive case detection.

Calculation of the time at risk
For the efficacy endpoint (time to first malaria infection), the time at risk will be counted in days, and expressed as person years at risk (days/365.25).
For each subject in the Total Cohort, time at risk begins 14 days after Dose 3 for those that received Dose 3 or 2½ months after Dose 1 (Day 75) otherwise. For the According to Protocol Cohort, time at risk begins 14 days after Dose 3.
The time at risk will end whenever one of the following conditions happen first: fits the case definition for malaria infection, loss of follow up, emigration from the study area, withdrawal, death, end of follow-up period (4 months post Dose 3).
The time at risk will take into account absences from the study area and anti-malaria drug therapy. Absences from the study area of 1 weeks or more will be recorded in multiples of 1 week. The date of departure will be documented. Similarly, if treatment for malaria is administered, the subject will not be considered susceptible to malaria infection for the longest duration of the combination of drugs the subject could receive for this episode as follows: 28 days if received Coartem ® , 7 days days if having received Malarone ® , 7 days if having received Fansidar (sulfadoxine-pyrimethamine) and 7 days if having received quinine alone.
If an episode is detected during a period of time not counting for the time at risk it will not be included in the analysis, however a table will be presented showing the number of such episodes not included in the analysis.
Episodes will be included in the analysis only if the subject is identified properly showing his/her ID Card at the contact with the health facility and the study number is written in the OPD form. Only blood slides that complete the reading process according to SOP will be considered in the establishment of a malaria infection.

Vaccine efficacy compared to Rabies control vaccine
Time to first infection of P. falciparum will be examined using Kaplan-Meier curves for all groups. The distribution of both malaria vaccine groups will be compared with the Wilcoxon test (if efficacy varies with time) or the Log-rank test (if it does not). Vaccine Efficacy will be assessed using Cox regression models. Vaccine efficacy is defined as 1 minus R where R is the hazard ratio of the RTS,S/AS02A or RTS,S/AS01B group versus the Rabies Vaccine group (with 95% CI). Crude and adjusted estimates for covariates factors (see Section 10.6.3.3) will be presented.
Cox regression assumes proportional hazards throughout the follow-up period. This assumption will be checked by plotting by group the log of the cumulative hazard against the log of time. Under the assumption of proportionality of the hazard, both curves should be parallel. A test based on the Schoenfield residuals will be performed.
If there is strong evidence that the hazard is not constant over the surveillance period, then alternative approaches to analyze the data will be examined according to the following: • Cox regression models with time varying covariates, using fractional polynomials to identify the function of the time that best fits. The following functions of the time will be explored: -2, -1, -½, log, identity, ½, 2, 3. The Akaike's criterion and the Schwarz Bayesian criterion will be explored. • Piecewise Cox regression model, partitioning the surveillance period in three periods based on the equal number of events For the assessment of the parasite density at Month 5½, a 2x2 table presenting the frequency of positive and negative will be presented. The effect of the group will be evaluated using the Fisher exact test. The geometric mean of those positive in each group will be calculated. Difference in geometric mean and 95% CI will be estimated. The effect of the group will be evaluated using the t-test. In case the assumptions of the t-test are not satisfied (normality of residuals and equal variance) a non-parametric Wilcoxon test will be used instead.

Vaccine efficacy of RTS,S/AS02A against RTS,S/AS01B
Time to first infection of P. falciparum will be examined using Kaplan-Meier curves for both groups. The distribution will be compared with the Wilcoxon test (if efficacy varies with time) or the Log-rank test (if it does not). Vaccine Efficacy will be assessed using Cox regression models. Vaccine efficacy is defined as 1 minus R where R is the hazard ratio of the RTS,S/AS02A group versus the RTS,S/AS01B (with 95% CI). Crude and adjusted estimates for covariates factors (see Section 10.8.4.4) will be presented.

Adjustment for covariates
The risk for malaria infection and development of clinical disease depends on numerous factors related to the parasite, host and vector biology. Estimates of Vaccine Efficacy will be made for the following covariates: • age: continuous variable An exploratory examination of the marginal effect each of the above covariates have on the treatment estimate may also be undertaken.

Hemoglobin values
The percentage change in hemoglobin values will be calculated as 100 x (14 weeks post Dose 3 minus baseline) / baseline. Descriptive statistics (mean, standard deviation, median, minimum and maximum) will be tabulated by group (RTS,S/AS02A RTS,S/AS01B and Rabies vaccine recipients). Comparisons between groups will be perfomred by Wilcoxon Rank Sum test (two sided, alpha = 0.05).

Between group comparison of the multiplicity of infection of emergent infections will be performed as described in the analyses of previous vaccine trials [Alloueche 2003; Haywood 1999; Enosse, 2006]. Briefly, to determine whether the vaccine reduces the number of P. falciparum genotypes, the number of genotypes in each isolate will be taken as the number of alleles of msp1 or msp2, whichever is the greater. The differences between mean MOI in the vaccinated vs. control groups will be compared using the Wilcoxon test. Regression analysis will be used to assess the effect of the vaccine on multiplicity of infection adjusted for geometric mean parasite density and age.
Amended 13 June 2006

Planned Interim Analysis
One interim analysis is planned at one month post Dose 3, Clinic Visit 7. The interim analysis will be performed by GSK Biologicals. In order to maintain blinding, the analyses will be performed by a statistician independent of the GSK Biologicals clinical teams. The interim analysis will be done on cleaned data and will contain analyses of reactogenicity, safety and immunogenicity.
This interim analysis will contain the final analyses for the reactogenicity (local and general solicited symptoms within 7 days of vaccination and unsolicited symptoms within 14 or 30 days of vaccination) and immunogenicity on Clinic Visit 2, 3 and 7. 10.9.1.

Analysis of demographics/baseline characteristics
Demographic characteristics (age, gender) of each study cohort will be tabulated.
The distribution of subjects enrolled will be tabulated as a whole and per group.

Analyses of safety
The analysis for safety will be conducted on the Total Cohort for Safety.
For the safety primary objective, the occurrence of Grade 3 solicited or unsolicited general reactions will be determined on the Total Vaccinated Cohort. The proportion of subjects with a Grade 3 solicited or unsolicted reaction, reported from study start until study Clinic Visit 7 will be tabulated with exact 95% CI. Comparisons between groups will be done using Fisher's Exact Test for each preferred term.
The occurrence of SAEs will be evaluated on the Total Vaccinated Cohort. The proportion of subjects with an SAE, classified by the MedDRA preferred term level, reported from study start until study Clinic Visit 7 will be tabulated with exact 95% CI.
Comparisons between groups will be done using Fisher's Exact Test.
The occurrence of adverse events will be evaluated on the Total Vaccinated Cohort. The proportion of subjects with an AE, classified by the MedDRA preferred term level, reported from study start until Clinic Visit 7 will be tabulated with exact 95% CI. Comparisons between groups will be done using Fisher's Exact Test. Similar tables will be generated for Grade 3 events and the relationship of the event to vaccination.
For each sampling timepoint, the biochemical parameters (ALT, bilirubin, creatinine) above normal range and hematological parameters (hemoglobin, total WBC, platelets and absolute lymphocyte count) below normal range will be described. Similar tables will be generated for Grade 3 parameters (as determined from Table 32).

Analyses of immunogenicity
The primary analysis will be based on the ATP cohort for analysis of immunogenicity. If the percent of enrolled subjects excluded from this ATP cohort is more than 5%, a second analysis based on the Total Vaccinated cohort will be performed to complement the ATP analysis.

Anti-CS antibodies
The percentage of subjects with sero-positive levels of anti-CS (proportion of subjects with anti-CS antibody titers greater than or equal to 0.5 EU/mL) with 95% CI will be determined prior to vaccination, at 1 month post Dose 2, 1 Month post Dose 3 and 4 months post Dose 3. Antibody titers will be summarized by GMT with 95%CI. Antibody titers after the third dose will also be investigated using reverse cumulative curves.

Anti-HBs antibodies
The percentage of subjects with sero-positive levels of anti-HBs (proportion of subjects with anti-HBs antibody titers greater than or equal to 3.3 mIU/mL), the percentage of subjects with seroprotective levels of anti-HBs (≥10 mIU/mL) with 95% CI will be determined prior to vaccination, at 1 Month post Dose 3. Antibody titers will be summarized by GMT with 95%CI. Antibody titers after the third dose will also be investigated using reverse cumulative curves.

ADMINISTRATIVE MATTERS
To comply with Good Clinical Practice important administrative obligations relating to investigator responsibilities, monitoring, archiving data, audits, confidentiality and publications must be fulfilled. See Appendix B for details.

INTRODUCTION
It is the mission of the physician to safeguard the health of the people. His or her knowledge and conscience are dedicated to the fulfilment of this mission.
The Declaration of Geneva of the World Medical Association binds the physician with the words, "The health of my patient will be my first consideration," and the International Code of Medical Ethics declares that, "A physician shall act only in the patient's interest when providing medical care which might have the effect of weakening the physical and mental condition of the patient." The purpose of biomedical research involving human subjects must be to improve diagnostic, therapeutic and prophylactic procedures and the understanding of the etiology and pathogenesis of disease.
In current medical practice most diagnostic, therapeutic or prophylactic procedures involve hazards. This applies especially to biomedical research.
Medical progress is based on research which ultimately must rest in part on experimentation involving human subjects.
In the field of biomedical research a fundamental distinction must be recognized between medical research in which the aim is essentially diagnostic or therapeutic for a patient, and medical research, the essential object of which is purely scientific and without implying direct diagnostic or therapeutic value to the person subjected to the research.
Special caution must be exercised in the conduct of research which may affect the environment, and the welfare of animals used for research must be respected.
Because it is essential that the results of laboratory experiments be applied to human beings to further scientific knowledge and to help suffering humanity, the World Medical Association has prepared the following recommendations as a guide to every physician in biomedical research involving human subjects. They should be kept under review in the future. It must be stressed that the standards as drafted are only a guide to physicians all over the world. Physicians are not relieved from criminal, civil and ethical responsibilities under the laws of their own countries.

I. BASIC PRINCIPLES
1. Biomedical research involving human subjects must conform to generally accepted scientific principles and should be based on adequately performed laboratory and animal experimentation and on a thorough knowledge of the scientific literature.
2. The design and performance of each experimental procedure involving human subjects should be clearly formulated in an experimental protocol which should be transmitted for consideration, comment and guidance to a specially appointed committee independent of the investigator and the sponsor provided that this independent committee is in conformity with the laws and regulations of the country in which the research experiment is performed.
3. Biomedical research involving human subjects should be conducted only by scientifically qualified persons and under the supervision of a clinically competent medical person. The responsibility for the human subject must always rest with a medically qualified person and never rest on the subject of research, even though the subject has given his or her consent.
4. Biomedical research involving human subjects cannot legitimately be carried out unless the importance of the objective is in proportion to the inherent risk to the subject.
5. Every biomedical research project involving human subjects should be preceded by careful assessment of predictable risks in comparison with foreseeable benefits to the subject or to others. Concern for the interests of the subject must always prevail over the interests of science and society.
6. The right of the research subject to safeguard his or her integrity must always be respected. Every precaution should be taken to respect the privacy of the subject and to minimize the impact of the study on the subject's physical and mental integrity and on the personality of the subject.
7. Physicians should abstain from engaging in research projects involving human subjects unless they are satisfied that the hazards involved are believed to be predictable. Physicians should cease any investigation if the hazards are found to outweigh the potential benefits.
8. In publication of the results of his or her research, the physician is obliged to preserve the accuracy of the results. Reports of experimentation not in accordance with the principles laid down in this Declaration should not be accepted for publication.
9. In any research on human beings, each potential subject must be adequately informed of the aims, methods, anticipated benefits and potential hazards of the study and the discomfort it may entail. He or she should be informed that he or she is at liberty to abstain from participation in the study and that he or she is free to withdraw his or her consent to participation at any time. The physician should then obtain the subject's freely-given informed consent, preferably in writing.
10. When obtaining informed consent for the research project the physician should be particularly cautious if the subject is in a dependent relationship to him or her or may consent under duress. In that case the informed consent should be obtained by a physician who is not engaged in the investigation and who is completely independent of this official relationship.
11. In case of legal incompetence, informed consent should be obtained from the legal guardian in accordance with national legislation. Where physical or mental incapacity makes it impossible to obtain informed consent, or when the subject is a minor, permission from the responsible relative replaces that of the subject in accordance with national legislation. Whenever the minor child is in fact able to give a consent, the minor's consent must be obtained in addition to the consent of the minor's legal guardian.
12. The research protocol should always contain a statement of the ethical considerations involved and should indicate that the principles enunciated in the present Declaration are complied with.

II. MEDICAL RESEARCH COMBINED WITH PROFESSIONAL CARE (Clinical research)
1. In the treatment of the sick person, the physician must be free to use a new diagnostic and therapeutic measure, if in his or her judgement it offers hope of saving life, re-establishing health or alleviating suffering.
2. The potential benefits, hazards and discomfort of a new method should be weighed against the advantages of the best current diagnostic and therapeutic methods.
3. In any medical study, every patient -including those of a control group, if anyshould be assured of the best proven diagnostic and therapeutic method. This does not exclude the use of inert placebo in studies where no proven diagnostic or therapeutic method exists.
4. The refusal of the patient to participate in a study must never interfere with the physician-patient relationship.
5. If the physician considers it essential not to obtain informed consent, the specific reasons for this proposal should be stated in the experimental protocol for transmission to the independent committee (I, 2).
6. The Physician can combine medical research with professional care, the objective being the acquisition of new medical knowledge, only to the extent that medical research is justified by its potential diagnostic or therapeutic value for the patient.

III. NON-THERAPEUTIC BIOMEDICAL RESEARCH INVOLVING HUMAN SUBJECTS (Non-clinical biomedical research)
1. In the purely scientific application of medical research carried out on a human being, it is the duty of the physician to remain the protector of the life and health of that person on whom biomedical research is being carried out.
USAMMDA will promptly inform, via written communication, all investigators and/or institutions conducting the study, if the study is suspended or terminated for safety reasons, and will also inform the regulatory authorities of the suspension or termination of the study and the reason(s) for the action. If required by applicable regulations, the investigator must inform the IEC/IRB promptly and provide the reason for the suspension or termination.
If the study is prematurely discontinued, all study data must be returned to GSK Biologicals. In addition, arrangements will be made for all unused investigational product(s) in accordance with the applicable GSK procedures for the study. Financial compensation to investigators and/or institutions will be in accordance with the agreement established between the investigator and/or institutions and GSK.

IV. Case Report Form Instructions
Prior to screening the first potential participant, the investigator will provide the Site Monitor with a list (Site Staff Signature Sheet) showing the signature and hand-written initials of all individuals authorized to make or change entries on CRFs (already defined).
If the authorized individuals should change during the study, the investigator is to inform GSK Biologicals of the specific change(s).
CRFs (and subject diary cards, if applicable), will be supplied by GSK Biologicals for recording all data. It is the responsibility of the investigator or co-investigator to ensure that CRFs (and subject diary cards) are legible and completely filled in with a black ink fountain or ballpoint pen.
Errors must be corrected by drawing a single line through the incorrect entry and writing in the new value/data positioned as close to the original as possible. The correction must then be initialled, dated and justified, where necessary, by the authorized individual making the change. The original entry must not be obliterated, overwritten or erased when a correction is made.
When a subject completes a visit, it is anticipated that relevant sections of the CRF will be completed by the investigator (or designated staff as documented in the Site Staff Signature Sheet) as soon as possible after the last data becoming available. Similarly, when a subject completes a study, it is anticipated that all relevant CRF pages will be completed promptly after the last data becoming available. This also applies to forms for potential study participants who were screened but not randomized to a study group.
As soon as the subject has completed/withdrawn from the study and the CRF is completed, the principal investigator or designated physician(s) under his/her supervision will sign the study conclusion pages of the CRF to confirm that they have reviewed the data and that the data are complete and accurate. In all cases the investigator remains accountable for the study data collected.
An original (top copy) CRF or log sheets must be submitted for all subjects who have undergone protocol specific procedures, whether or not the subject completed the study.
While completed CRFs are reviewed by a GSK Biologicals professional monitor at the study site, errors detected by subsequent in-house CRF review may necessitate clarification or correction of errors with documentation and approval by the investigator or appropriately qualified staff as documented on the Site Staff Signature Sheet. In all cases, the investigator remains accountable for the study data. Wherever possible the investigator should assist in the clarification or correction of errors detected after study finalization promptly after being brought to the attention of the investigator (preferably within 48 hours).
Any questions or comments related to the CRF should be directed to the assigned Site Monitor.

V. Monitoring by GSK Biologicals
Monitoring visits by a professional representative of GSK Biologicals will be scheduled to take place as close as possible to entry of the first subject, during the study at appropriate intervals and after the last subject has completed the study. It is anticipated that monitoring visits will occur at a frequency defined before study start.
These visits are for the purpose of confirming that GSK Biologicals' studies are being conducted in compliance with the relevant Good Clinical Practice regulations/ guidelines, verifying adherence to the protocol and the completeness and accuracy of data entered on the CRF pages/RDE screens and Vaccine Inventory Forms. The monitor will verify CRF/RDE entries by comparing them with the source data/documents that will be made available by the investigator for this purpose. Data to be recorded directly into the CRF pages/RDE screens will be specified in writing preferably in the source documentation agreement form that is contained in both the monitor's and investigator's study file. For RDE, the monitor will mark completed and approved screens at each visit. The investigator must ensure provision of reasonable time, space and adequate qualified personnel for monitoring visits.

VI. Archiving of Data
Following closure of the study, the investigator must maintain all site study records in a safe and secure location. The records must be maintained to allow easy and timely retrieval, when needed (e.g., audit or inspection), and, whenever feasible, to allow any subsequent review of data in conjunction with assessment of the facility, supporting systems, and staff. Where permitted by applicable laws/regulations or institutional policy, some or all of these records can be maintained in a validated format other than hard copy (e.g., microfiche, scanned, electronic for studies with an eCRF, for example); however, caution needs to be exercised before such action is taken. The investigator must assure that all reproductions are legible and are a true and accurate copy of the original, and meet accessibility and retrieval standards, including re-generating a hard copy, if required. Furthermore, the investigator must ensure there is an acceptable back-up of these reproductions and that an acceptable quality control process exists for making these reproductions.
GSK will inform the investigator/ institution of the time period for retaining these records to comply with all applicable regulatory requirements. However, the investigator/ institution should seek the written approval of the sponsor, USAMMDA, GSK Biologicals and WRAIR before proceeding with the disposal of these records. The minimum retention time will meet the strictest standard applicable to that site for the study, as dictated by ICH GCP E6 Section 4.9, any institutional requirements or applicable laws or regulations, or GSK standards/procedures; otherwise, the minimum retention period will default to 15 years.
The investigator/ institution must notify GSK of any changes in the archival arrangements, including, but not limited to, the following: archival at an off-site facility, transfer of ownership of the records in the event the investigator leaves the site.

VII. Audits
For the purpose of compliance with Good Clinical Practice and Regulatory Agency Guidelines it may be necessary for GSK Biologicals or a Drug Regulatory Agency to conduct a site audit. This may occur at any time from start to after conclusion of the study.
When an investigator signs the protocol, he agrees to permit drug regulatory agencies and GSK Biologicals audits, providing direct access to source data/ documents. Furthermore, if an investigator refuses an inspection, his data will not be accepted in support of a New Drug Registration and/or Application, Biologics Licensing Application.
GSK Biologicals has a substantial investment in clinical studies. Having the highest quality data and studies are essential aspects of vaccine development. GSK Biologicals has a Regulatory Compliance staff who audit investigational sites. Regulatory Compliance assesses the quality of data with regard to accuracy, adequacy and consistency. In addition, Regulatory Compliance assures that GSK Biologicals studies are in accordance with GCP and that relevant regulations/guidelines are being followed.
To accomplish these functions, Regulatory Compliance selects investigational sites to audit. These audits usually take 1 to 2 days. The GSK Biologicals' audits entail review of source documents supporting the adequacy and accuracy of CRFs, review of documentation required to be maintained, and checks on vaccine accountability. The GSK Biologicals' audit therefore helps prepare an investigator for a possible regulatory agency inspection as well as assuring GSK Biologicals of the validity of the database across investigational sites.
The Inspector will be especially interested in the following items: •

Confidentiality:
All information provided by GSK and all data and information generated by the site as part of the study (other than a subject's medical records) will be kept confidential by the investigator and other site staff. This information and data will not be used by the investigator or other site personnel for any purpose other than conducting the study. These restrictions do not apply to: (1) information which becomes publicly available through no fault of the investigator or site staff; (2) information which it is necessary to disclose in confidence to an IEC or IRB solely for the evaluation of the study; (3) information which it is necessary to disclose in order to provide appropriate medical care to a study subject; or (4) study results which may be published as described in the next paragraph. If a written contract for the conduct of the study which includes confidentiality provisions inconsistent with this statement is executed, that contract's confidentiality provisions shall apply rather than this statement.

Publication:
For multicentre studies, the first publication or disclosure of study results shall be a complete, joint multicentre publication or disclosure coordinated by GSK. Thereafter, any secondary publications will reference the original publication(s).
Prior to submitting for publication, presentation, use for instructional purposes, or otherwise disclosing the study results generated by the site (collectively, a "Publication"), the investigator shall provide GSK with a copy of the proposed Publication and allow GSK a period of at least thirty (30) days [or, for abstracts, at least five (5) working days] to review the proposed Publication. Proposed Publications shall not include either GSK confidential information other than the study results or personal data on any subject, such as name or initials.
At GSK's request, the submission or other disclosure of a proposed Publication will be delayed a sufficient time to allow GSK to seek patent or similar protection of any inventions, know-how or other intellectual or industrial property rights disclosed in the proposed Publication.
If a written contract for the conduct of the study, which includes publication provisions inconsistent with this statement is executed, that contract's publication provisions shall apply rather than this statement.

Appendix C Overview of the Recruitment Plan
Prior to enrollment, a specific information campaign towards the targeted population will be implemented. This will start with the administrative and senior leaders of the villages in the recruitment area around KC and expand to the barazas (town meetings) that everyone in the village can attend.
The sessions will explain the problem of malaria in this community, the current strategies for its control, as well as the limitations of these strategies. The need and the difficulties of developing a vaccine against malaria will be discussed, as well as an outline of the proposed trial, including the rationale, the background data available and the study objectives. Particular attention will be paid to study procedures including screening of subjects for hepatitis B, immunization and blood collection. In that respect a full discussion on the purpose of blood collection and the associated risks will be carried out.
Next, Field Workers will be available in villages in the study area to answer any additional questions on a one-on-one basis, schedule volunteers for a briefing and coordinate transportation to the briefing. On the day of the scheduled briefing Field Workers with transportation will escort them to KC.
At KC, volunteers will view a video in their native language that explains the nature and purpose of the study and reviews the entire informed consent document. There will be a period for asking questions afterwards. Volunteers will then receive both oral and written explanations of the study one-on-one with a study coordinator or clinician and again allowed to ask questions. Afterwards, written informed consent will be obtained from each person who wants to participate in the study. In addition, all volunteers who sign an informed consent form will be required to fill in a form that will be entered into the US Army Medical Research and Materiel Command Volunteer Registry Data Base. This form requires details of the volunteer's sex, date of birth, permanent home address, permanent home phone number, as well as their local address and phone number if it is different from their permanent details. In the instructions to the volunteer, this form states that the disclosure of each volunteer's Social Security Number (SSN) is mandatory; however, as SSNs are not issued in Kenya, no SSN will be recorded on this form. The intent of the data base is to readily answer questions concerning an individual's participation in research sponsored by USAMRMC and to ensure that the USAMRC can exercise its obligation to ensure research volunteers are adequately warned of risks and to provide them with new information as it becomes available. The information will be stored at USAMRMC for a minimum of 75 years. So that language and illiteracy will not be impediments to informed consent, all briefings and explanations will be in Dholuo, the local language.
Screening may be done on a separate visit from recruiting/consenting. Only adults aged 18 to 35 years that have previously given consent will be eligible to be screened for the trial.
Volunteers will provide a medical history through a one-on-one interview with a clinical officer and undergo physical examination and standard laboratory screening tests, which include complete blood count (CBC), creatinine, and ALT. Blood to screen for hemoglobinopathies (sickle cell disease/trait, G6PD deficiency, and alpha-thalassemia) will also be drawn. Subjects may be screened one additional time if re-screening is determined to be necessary (e.g., after treatment for malaria). Field Workers will notify each volunteer of their eligibility to enroll in the study. Subjects will be excluded from participation if they meet any of the exclusion criteria. Subjects excluded from this study because of significant abnormalities will have follow up evaluation and treatment coordinated for them by the KC personnel (refer to Section 5.7.3 of the Study Protocol). • The label should be attached to the tube as follows (see diagram): − first attach the paper part of the label to the tube − then wrap the label around the tube so that the transparent, plastic part of the label overlaps with the label text and bar code and shields them.
This will ensure optimal label attachment. • If necessary, any hand-written additions to the labels should be made using indelible ink.

Serology testing
Serological responses will be measured principally by evaluating antibody responses to HBs and to CSP repeats (anti R32LR). Other serological testing may be done that are not currently planned. This is included in the informed consent. If a study not specifically detailed in this protocol is proposed it must first be evaluated and approved by the Kenyan Ethical Review Committee. Serum for antibody determination will be collected at the time points defined in the flowchart in Sections 5.4.
Antibody levels against CS will be measured at GSK Biologicals (or a designated laboratory) by standard ELISA methodology using plate adsorbed R32LR antigen with a standard reference antibody as a control according to SOPs from the laboratory. Results will be reported in EU/mL.
Antibody to hepatitis B surface antigen will be measured at GSK Biologicals using a commercially available ELISA immunoassay (AUSAB EIA test kit from Abbott) or equivalent according to the assay instructions. Results will be reported in mIU/mL.

Determination of parasitemia
Estimates of asexual P. falciparum parasite density will be made in the microscopy lab at Kombewa Cinic according to laboratory SOPs. Two slides will be air dried, stained with Giemsa and read on a light microscope with a 100x oil immersion lens and 10x eyepieces. Parasite density will be assessed by counting the number of asexual stage parasites per 200 leukocytes. Slides will be declared negative only after 100 high power fields have been read. Parasite numbers will be converted to a parasite density (count/µL) based on the WBC from a recent CBC. All slides will be read twice independently. If there is a discrepancy between the two slide readings it will be read a third time by one of the senior (R3) readers. A discrepancy occurs if the first two readers disagree on presence of malaria or malaria species, or there is a 1.5 fold difference or greater in calculated parasite densities. If there are no discrepancies, as defined above, between the first two readers, the definitive result will be the average of the two densities. If there is a discrepancy, the final result will be the results from the senior (R3) reader. Differences in parasite density are not calculated if both readers have densities less than 10. The final result is the average of the two densities.

Cell-mediated immunity Methodology
T-cell mediated immunity (CMI) will be investigated in this study. CMI will be assessed at three four time points during the study, corresponding to prevaccination, 1 month post Dose 3, and 4 months post Dose 3 and 9 months post Dose 3. A sample volume of 9 mL will be taken at each of these timepoints. Amended 13 June 2006 Three CMI laboratories will be involved in the assessment of the CMI response:

Role and Methodology; CMI Laboratory Kisumu Kenya
Whole blood (9 mL) will be incubated at 37°C for two hours with the relevant antigens. Then, an inhibitor of secretion (brefeldin A) will be added for an additional 4 hours incubation. This step allows the cytokine/activation markers to accumulate within the stimulated cells. After washing and fixing steps, an aliquot of the cells (6 mL) will be frozen at minus 70 to minus 80°C and transferred to Rixensart for analysis. The remaining 3 mL of cells will be analyzed at Kisumu.
Flow-cytometry characterization will be used on cells. For this analysis, the cell samples will be processed with classical intracellular cytokine stainings and analyses designed to identify the frequency of antigen specific CD4/CD8 T-Cells. T-cell immunity will be explored in a short term antigen-stimulation (i.e., PfCS protein peptides) of whole blood by intracellular cytokine staining and determination of cell surface activation markers using flow cytometry (Beckton Dickenson Fast Immune™ kits will be used; these contain all the necessary reagents including the fixation and freezing medium).

Role and Methodology; CMI Laboratory GSK Biologicals, Rixensart
Flow-cytometry characterization will be used on cells transferred from Kisumu, Kenya. For this analysis, the cell samples will be thawed before being processed with classical intracellular cytokine staining and analyses designed to identify the frequency of antigen specific CD4/CD8 T-Cells.

Role and Methodology; CMI Laboratory WRAIR, Washington
The senior staff at this laboratory will support the CMI Laboratory in Kisumu, Kenya, but no samples would be transferred to this laboratory.

Biochemical and hematological analyses
Hematological and biochemical testing will be done at KEMRI in Kenya, following laboratory SOPs.  of 94ºC (1 min), 60ºC (1 min) and 72ºC (1 min) and final extension at 72ºC for 10 min. 5 μL 2nd round PCR products are monitored on a 2.0% agarose gel in 1xTAE buffer to check the quality, size and yield of the PCR products before proceeding to product purification and sequencing. The following samples will also be included in every run: Positive control = P. falciparum culture DNA; Negative control 1 = uninfected host genomic DNA; Negative control 2 = no temperate control.

Determination of CSP polymorphic sequences
The distribution of polymorphic variants within the Th2R and Th3R regions of P falciparum CSP will be determined using high-throughput direct sequencing of the PCR products. The sequencing strategy will be validated in a pilot study of 50 PCR samples randomly selected from pre-intervention blood samples from study participants. This will also provide important information regarding the pattern of CSP polymorphism in the study area, and an estimate of the expected data loss due to ambiguous sequence reads from mixed alleles. The sequencing using CSPf2 and

Genotyping of msp-1, msp-2 and detection of multiplicity of infection (MOI)
Allele specific, nested PCR of 3 polymorphic regions of P. falciparum genes, namely msp1 block 2 and msp2 will be used to detect the genetic structure of the parasite populations, essentially as described by Snounou, 1999

Amended 13 June 2006
• tubes with screw caps for serum samples, • racks and cardboard boxes for the tubes of serum.
The investigator or pharmacist must sign a statement that he/she has received the clinical supplies for the study.
It is NOT permitted to use any of the supplies provided by GSK Biologicals for purposes other than those specified in the protocol.

Vaccine packaging
The vaccines will be packed in labeled boxes. The box label will contain, as a minimum, the following information: study number, treatment number, lot number (or numbers, when double-blind), instructions for vaccine administration and any other relevant regulatory requirements.

Vaccine shipment from GSK Biologicals Rixensart to local country medical department, dispatching centre or investigational site
On arrival of vaccine shipment, the freeze watch should be removed from the vaccine boxes and checked after 10 minutes at room temperature. The temperature recording chart (chart from Cox recorder or print-out data of the electronic device) should be obtained from the temperature recording device.
The following documents should be completed and returned to GSK Biologicals on reception of vaccine shipment: Investigational supply receipt Notification of vaccine delivery/temperature control Temperature recording (chart).
In case of any temperature deviation, the official approval for the use of vaccine must be obtained from GSK Biologicals.

Vaccine accountability
At all times the figures on supplied, used and remaining vaccine doses should match. At the end of the study, it must be possible to reconcile delivery records with those of used and unused stocks. An explanation must be given of any discrepancies.
After approval from GSK Biologicals and in accordance with GSK SOP NPD-112, used and unused vaccine vials/syringes/containers should be destroyed at the study site using locally approved biosafety procedures and documentation unless otherwise described in the protocol. If no adequate biosafety procedures are available at the study site, the used and unused vaccine vials/syringes/containers are to be returned to an appropriate GSK Biologicals site for destruction, also in accordance with current GSK SOP NPD-112.

Transfers of clinical vaccines or products from country medical department or dispatch centre to study sites or between sites
Storage temperatures must be maintained during transport and deviations must be reported to Clinical Operations Logistics for guidance. All transfers of clinical vaccines or products must be documented using the Clinical Supply Transfer Form.
All packaging and shipment procedures for transfer of clinical vaccines or products must follow procedures approved by GSK Biologicals.
Clinical vaccines or products should always be sent by contract courier designated by GSK Biologicals, unless otherwise requested by GSK Biologicals.
Alternative local validated procedures may be followed after the documentation for these procedures has been sent to Clinical Operations Logistics and approval has then been obtained from the qualified person (or designee) in GSK Biologicals, Rixensart, before any shipment of vaccines.

A number of errors/omissions in the original version of the document have been corrected in this version.
These include the following: Two additional names have been added to the USAMRU-K team and the spelling of one investigator's name corrected.

The fax number of the GSK Medical Monitor has been corrected
The text relating to the submission of protocol modifications to the IRBs/IECs has been corrected both in the main protocol text and in the relevant Appendix. Previously it was stated that protocol modifications are submitted to the IRB/IEC for information only. The IRB/IEC must receive any protocol amendments/modifications and express a favorable opinion of it before it is applied.
It is intended that for all study visits that are not carried out at the clinic, the subjects will be seen by a field worker or some other qualified study personnel. The fact that these visits may not necessarily be carried out solely by field workers has been clarified throughout the protocol.
An inclusion criterion that all volunteers should be healthy (as established by clinical history and medical examination) in order to be enrolled in the trial was inadvertently left out of the protocol. This has been added.
Text has been added to clarify that only those pages of the informed consent documentation that are signed by each volunteer will be filed at the investigator's site.
In order to ensure that the Safety monitoring plan for the trial is practical a number of clarifications have been added: • While the members of the SMG are representatives of WRAIR, KEMRI and GSK Biologicals, clarification has been added that the members of the SMG are not members of the investigational teams at those institutions.

•
It is not intended to present data relating to the duration of symptoms in the safety reports to be written on the first 60 subjects to receive Dose 1 and Dose 2.

•
Text detailing that the SMG may request Study Reports to be unblinded if they deem it to be necessary has been added.

•
Previously it was stated that the SMG may stop the trial permanently. It was intended that the decision to stop the trial permanently should rest with the sponsor, USAMMDA (in consultation with GSK Biologicals) The drug regimen for the clearance of malaria parasites from all subjects one week prior to Dose 3 of vaccines has been corrected. Previously some sections of the protocol stated that one dose of Malarone ® consisted of one tablet. It is intended that each dose of Malarone ® should consist of four tablets. This has been corrected throughout the documentation.
It is intended that subjects who have a positive MBF during the ADI portion of the study should receive treatment for asymptomatic malaria within 72 hours of the MBF being taken, not 36 hours as stated in the protocol.
Corrections have been made to the 'list of study procedures' The text relating to the detailed description of study visits has been corrected to ensure that it is consistent with the outline of study procedures (Table 27). In addition a number of procedures, namely recording of pulse and time of vaccination have been removed from this section. These procedures may be carried out by the investigator, but are not recorded on the CRF.
The text relating to the dosage and administration of Rabipur rabies vaccine has been corrected. This is to reflect the fact that for this trial, 'long' and 'short' needles will not be supplied with this vaccine in order to facilitate blinding of subjects. In addition the Rabipur diluent will be supplied in ampoules for this trial.
There was an error in the text relating to the preparation of Investigator safety reports. The text has been corrected to ensure that Investigator safety reports are prepared by the Sponsor (USAMMDA) in accordance with FDA policy (not GSK policy as previously stated) In order to ensure that toxicity grading is in line with recent draft guidance from the FDA, the intensity scales for the monitoring of hepatic and renal function have been amended.
Instructions for handling cells for cell-mediated immunity assay have been added to the Appendices A number of typographical errors have been corrected Amended text has been included in bold italics in the following sections:

of this trial, no HIV testing is scheduled to be carried out at screening. It is a condition of the trial that all enrolled subjects are in good health (refer to Section 4.3), as confirmed by a range of clinical and blood tests at screening (refer to Section 4.4 for the exclusion criteria for this trial). Those volunteers who exhibit clinical features of immunosuppresion or immunodeficiency will not be enrolled (refer to Section 4.4). There is no reason to believe that any of the vaccines to be administered during this study may have any ill-effect on clinically asymptomatic volunteers infected with HIV. In addition, HIV testing has been found to be culturally unacceptable at the site. The local community have hesitated to embrace HIV testing and local elders have voiced their concerns about mandatory HIV testing as part of any studies in this community.
Section 1.7: Risks and benefits for study volunteers Section 1.7.1:

For those subjects who receive Coartem ® as an alternative clearance treatment, they will be seen by field workers on the day following the first two doses and the second two doses of Coartem, and by a study clinician one week post the final two doses of Coartem ® . Dose 1, Dose 3 and Dose 5 of Coartem ® will be
given under the supervision of a fieldworker. Any subjects who are unwell at these visits will be referred to a study clinician for evaluation and treatment. Section 1.7.4: Risk of disclosure of volunteer's confidential/personal information to persons not involved in the study In order to ensure that all information collected on study volunteers is kept confidential, the following safeguards will be applied: • Access to study files and personal information will be limited to only WRP and GlaxoSmithKline personnel with a need to access the information. • Study information will be kept in locked cabinets when not in use.

•
All information or samples that leave WRP will be labeled in a way that it cannot be linked back to a specific study subject. Section 1.7.5: Benefits to study volunteers of taking part in the study All volunteers for this study will receive the following benefits for their participation: •

All volunteers will undergo a medical examination at screening free of charge. All volunteers, whether accepted for enrollment into the trial or not will benefit from this free health check-up. The results of all tests will be communicated to all volunteers. Where illnesses are newlydiagnosed, a referral to an appropriate specialist will be made for the volunteer. •
For the duration of their participation in the trial, all randomized subjects will receive free health care from study medical personnel.

• All enrolled subjects will be offered, free of charge, a full three dose course of vaccinations against rabies (except where the principal investigator deems that vaccination is unsuitable for safety concerns). Subjects who are randomized to receive the rabies control vaccine will receive these vaccinations during the course of the trial. Subjects who are randomized to receive RTS,S/AS01B or RTS,S/AS02A will be offered the vaccination course after they have completed all study procedures and returned for the final study visit. Section 3: •
One week prior to Dose 3, subjects will be treated with Malarone ® for presumptive clearance of malaria parasitemia (in the event that subjects are unable to tolerate Malarone ® , Coartem ® (artemether/lumefantrine, Novartis AG, Zurich, Switzerland) will be used as the alternative clearance treatment Section 4.3: •

Free of obvious health problems as established by medical history and clinical examination before entering into the study. •
If the volunteer is female, she must be of non-childbearing potential, i.e. either surgically sterilized or one year post-menopausal; or, if of childbearing potential, she must be abstinent or have used adequate contraceptive precautions (i.e. natural family planning, intrauterine contraceptive device; oral contraceptives; diaphragm or condom in combination with contraceptive jelly, cream or foam; Norplant ® or DepoProvera ® ) for 30 days prior to vaccination, have a negative pregnancy test and must agree to continue such precautions for two months after completion of the vaccination series.

Section 4.4:
• History of allergic reactions significant IgE-mediated events or anaphylaxis to previous immunizations. Section 4.6.1: The following adverse events (AEs) constitute absolute contraindications to further administration of RTS,S/AS02A, RTS,S/AS01B or Rabies Vaccine Rabipur; if any of these AEs occur during the study, the subject must not receive additional doses of vaccine but may continue other study procedures at the discretion of the investigator (see Section 9). The subject must be followed until resolution of the event, as with any AE (see Section 8.7):

CONFIDENTIAL
• Anaphylactic reaction Acute allergic reaction, significant IgE-mediated event or anaphylactic shock following the administration of vaccine investigational product (Amended 24 June 2005).
Section 5.1.1: No deviations from, or changes to, the protocol should be initiated without prior written consent of the sponsor, USAMMDA, or GSK Biologicals and IRB/IEC favorable opinion of an appropriate amendment or administrative change except when necessary to eliminate immediate hazards to the subjects or when the change(s) involves only logistical or administrative aspects of the study (e.g., change of monitor[s], telephone number[s]). Modifications are submitted to the IRB/IEC for information only. However, written verification that the modification was submitted should be obtained. Approvals/verifications must be transmitted in writing to GSK Biologicals' Study Monitor by the principal investigator Section 5.1.2: Each subject's signed informed consent form must be kept on file by the investigator for possible inspection by Regulatory Authorities and/or GSK Biologicals' professional and Regulatory Compliance persons. Only the signed Informed Consent Agreement pages of each volunteer will be kept on file at the investigator's site. In addition one entire representative copy of the informed consent documentation will be kept on file by the investigator for reference. The subjects should receive a copy of the signed and dated written informed consent form and any other written information provided to the subjects and should receive copies of any signed and dated consent form updates. Any amendments to the written information will be provided to subjects.

Section 5.1.3.1: The LSM is required to review all unanticipated problems (refer to Section 8.3) involving risk to subjects or others, serious adverse events and all subject deaths associated with the protocol and provide an unbiased written report of the event. At a minimum, the LSM should comment on the outcomes of the event or problem, and in the case of a serious adverse event or death, comment on the relationship to participation in the study. The LSM should also indicate whether he concurs with the details of the report provided by the study investigator. Reports for events determined by either the investigator or LSM to be possibly or definitely related to participation and reports of events resulting in death should be promptly forwarded to the HSRRB and GSK Biologicals.
Section 5.1.3.2: The SMG is not a an independent Data Safety Monitoring Board. It is a group of experienced clinicians, from the organizations involved in the study, that is charged with assisting the PI and LSM with timely and thorough evaluation of the safety reports after Dose 1 and Dose 2. The members do not form a part of the investigator groups; i.e. the investigator site, the GSK Biologicals malaria vaccine clinical project team or the investigator group at WRAIR. They will be one of the parties responsible for deciding whether to progress to the next dose and whether to resume immunizations if the trial has been suspended. The SMG may, if deemed necessary, convene a meeting with, or request further information from the PI or LSM Section 5.1.3.3: Safety reports will be written on the first 60 subjects to receive Dose 1 and Dose 2. These reports will be blinded to vaccine allocation and will contain: • All SAEs and any relationship to vaccines to date; • For each dose, all Grade 3 solicited and unsolicited symptoms by relationship to vaccine and duration of symptom; Section 5.1.3.4.1: The PI will suspend all vaccination for any of the following SAEs; • Death or life-threatening SAE which is judged to be related to the study vaccine; • Anaphylactic shock reaction or significant IgE-mediated event in an enrolled subject following vaccination.

169
Modify Date 13/06/2006 Version : 5.1 Section 5.1.3.4.3: The trial may be temporarily suspended by the LSM, PI or the SMG. The SMG will review all available safety data which may include a full description of reactogenicity. If the SMG judge it to be necessary, the safety data will be unblinded. However the final responsibility to recommend whether or not the trial should be stopped permanently rests with the SMG the sponsor (USAMMDA) in consultation with GSK Biologicals, after having considered all the safety information available.
Section 5.2.3: Screening will be done on a separate visit from recruiting. Only adults aged 18 to 35 years at the scheduled time of first vaccination with a written Informed Consent, signed/thumb printed and dated will be eligible to be screened for the trial… At the screening visit each subject will be given a Subject Number and each will have a clinic record prepared. Subject Numbers will be issued consecutively in the order in which the subjects arrive at the health center. This number will be used throughout the study to identify every document and blood sample associated with the volunteer. The clinic records will contain the Subject Number, the subject's date of birth, medical history, findings of the physical examination, the date of screening visit, whether the subject was enrolled, and (where applicable) reasons for exclusion from the study. The name of the study will be written on both the ID card and clinic records of enrolled volunteers.
Section 5.7.3: If a volunteer is excluded from the study for a medical condition or laboratory abnormality not previously known to the subject volunteer they will be referred for care to one of these physicians according to their illness.
Section 5.10: Three weeks prior to the start of surveillance for malaria infection (i.e. 1 week prior to Dose 3), all subjects will be presumptively treated for aymptomatic parasitemia with Malarone ® (atovaquone and proguanil hydrochloride, GlaxoSmithKline, Uxbridge, UK). (in the event that subjects are unable to tolerate Malarone ® , Coartem ® (artemether/lumefantrine, Novartis AG, Zurich, Switzerland) will be used as the alternative clearance treatment. The subjects will visit field workers for three consecutive days. At each of these visits, the field worker will give the subject one four tablets of Malarone ® (containing 250 mg atovaquone and 100 mg proguanil hydrochloride), and observe them the subject taking the tablets it. The first dose of medication will be administered at the field stations on Study Day 53 (Field Worker Visit 32). The second dose will be given under observation the next morning by the field worker team (Study Day 54; Field Worker Visit 33). The third dose (the final one) will be given by the field worker team the following morning (Study Day 55; Field Worker Visit 34). … For the ADI period, a field worker visit will consist of the subject presenting to a field station or the KC and completing a brief surveillance for infection morbidity questionnaire, which will include the reporting of malaria symptoms and a record of oral temperature. If the subject is well and afebrile, a blood-sample will be taken and examined for malaria parasitemia and evaluated for CBC. Blood slides from the sample will be Giemsa-stained and read at KC to determine the presence of parasites. Treatment for asymptomatic subjects with positive MBF will be sent by a field worker within 36 72 hours of the when the MBF was taken.

Section 5.12 (footnotes):
Shaded areas indicate visits pertaining to ADI b Includes analysis of hemoglobin, total white cell count absolute lymphocyte count and platelets. Done with safety labs and with MBF to determine parasite count j SAEs related to study procedures will be collected Section 5.13: • Record oral temperature, blood pressure, pulse • Record SAEs experienced by the vaccinee since the last visit Section 6.2.3: Disinfect top of diluent vaccine vial with alcohol swabs and let dry. Using the longer of the 2 needles supplied, inject the entire contents of the diluent vial ampoule into the vaccine vial. Keeping the syringe and needle in place, the freeze-dried vaccine is dissolved by gently shaking the vial. Wait for 1 minute to ensure complete dissolution of vial contents before withdrawing a sufficient volume to provide a 1.0 mL dose still using the long original needle and original syringe. The long original needle should then be replaced with the shorter a fresh needle for IM injection. The reconstituted vaccine should be used immediately. Section 6.6: In addition to the vaccine doses provided for the planned number of enrolled subjects, 5 3% additional doses will be supplied. In case a vaccine dose is broken or unusable, the investigator should replace it with a replacement vaccine dose. If a vaccine dose needs replacement, the envelope with the corresponding treatment number will designate the replacement without unblinding the study using a coded letter system. Although the sponsor, USAMMDA, and GSK Biologicals need not be notified immediately in these cases, documentation of the use of the replacement vaccine must be recorded by the investigator on the vaccine administration page of the CRF and on the vaccine accountability form. Section 6.9: All antimalarial drugs administered from the beginning of the study until the end of the ADI period (i.e. 6½ 4 months post Dose 3) are to be recorded with generic name of the medication (trade names are allowed for combination drugs, i.e., multi-component drugs), medical indication, total daily dose, route of administration, start and end dates of treatment. Section 8: The investigator is responsible for the detection and documentation of events meeting the criteria and definition of an adverse event (AE) or serious adverse event (SAE) as provided in this protocol. During the study, when there is a safety evaluation, the investigator or site staff will be responsible for detecting AEs and SAEs, as detailed in this section of the protocol. In addition, for this study the investigator is responsible for the timely reporting of unanticipated problems (refer to Section 8.3).

BB-IND 11220
Protocol title: A Phase IIb randomized, double-blind, controlled study of the safety, immunogenicity and proof-of-concept of RTS,S/AS02A, and RTS,S/AS01B, two candidate malaria vaccines in malaria-experienced adults living in Western Kenya.

Amendment 2
Amendment date:

Rationale/background for changes:
For logistic reasons, the screening interval for the trial is being extended from 4 weeks (28 days) to 6 weeks (42 days) Amended text has been included in bold italics in the following sections:

Exploratory: Parasite Genotyping • To determine the prevalence of vaccine-like alleles of CSP in the study population. • To investigate whether vaccination with RTS,S/AS0A2 and RTS,S/AS01B modifies the distribution of amino acid substitutions in the Th2R and Th3R sequence regions of the CSP gene in subsequent P. falciparum infections. • To investigate whether vaccination with RTS,S/AS0A2 and RTS,S/AS01B modifies the number of P. falciparum genotypes (multiplicity of infection) as determined by analysis of msp-1 and msp-2 genes.
Study design DNA will be extracted from EDTA blood of all subjects with patent asexual parasitemia.
CS-specific CMI will be assessed at three four time points during the study; prevaccination, 1 month post Dose 3, and 4 months post Dose 3 and 9 months post Dose 3.

Exploratory endpoints: Cell Mediated Immunity
Endpoint assessed prior to vaccination, 1 month post Dose 3, and 4 months post Dose 3 and 9 months post Dose 3.

The CSP is the predominant protein found in the surface of the sporozoite. Studies of the genetic diversity of the gene encoding the CSP of P. falciparum have demonstrated the existence of high levels of genetic polymorphisms within the region known to contain T-cell epitopes in isolates from different areas in Africa [Escalante, 2002], which is therefore potentially important for generation of immunity. This has raised the concern whether a vaccine containing a sequence of a selected strain of P. falciparum would confer protection against other variant parasites. In the Gambia, the RTS,S/AS02A
vaccine was demonstrated to induce protection against first P. falciparum infections that was not strainspecific. The frequency of the vaccine allele type, which was derived from the sequence of the CSP allele in laboratory clone 3D7, was similar to that of all other CSP alleles studied in the vaccine vs control group [Alloueche, 2003]. The key polymorphic sites in the CSP gene which are encompassed by the RTS,S antigen are the T-cell epitopes at the carboxy-terminus of the protein, designated Th2R and Th3R. Polymorphisms in these epitopes were the focus of the evaluation of strain-specificity of vaccine efficacy in the Gambian trial [Alloueche, 2003]. Evidence was also found in that study that RTS,S modified the average number of clones (multiplicity) carried during post-vaccination parasitemia. Modification of multiplicity of P. falciparum infections was also found after vaccination with the chimaeric peptide multimer vaccine SPf66 [Haywood, 1999]. These findings were confirmed by recent analysis of CSP sequences and clone multiplicity in 521 parasite isolates from Mozambiquan children who participated in a large Phase II study of the efficacy of RTS,S/AS02A [Alonso, 2004;Enosse, 2006].

Refer to Section 10.4 for definition of the exploratory endpoints.
Section 3. STUDY DESIGN OVERVIEW DNA will be extracted from EDTA blood of all subjects with patent asexual parasitemia. CS-specific CMI will be assessed at three four time points during the study; prevaccination, 1 month post Dose 3, and 4 months post Dose 3 and 9 months post dose 3. Section 5.12. Outline of study procedures

EDTA blood will be collected during ADI from individuals with microscopically confirmed asexual
parasitemia. DNA will be extracted from 200 μL of the EDTA blood using QIAmp DNA blood mini kit as described by the manufacturer (QIAGEN Inc., CA). DNA samples will be stored at -80°C until use.

Polymerase chain reaction
Primers that bind to conserved sequences flanking the Th2R and Th3R region of P. falciparum CSP gene will be used in a nested PCR to amplify a 381 base pair fragment covering nucleotide 936 to 1317 of the CSP gene. For the first round PCR, the following primers will be used: First round PCR:

Reverse primer (CSPr2) -5'-tctaattaaggaacaagaaggat -3'
The composition of 1st PCR reaction mixture is shown below: products are monitored on a 2.0% agarose gel in 1xTAE buffer to check the quality, size and yield of the PCR products before proceeding to product purification and sequencing. The following samples will also be included in every run: Positive control = P. falciparum culture DNA; Negative control 1 = uninfected host genomic DNA; Negative control 2 = no temperate control.

QA/QC:
For QA/QC reasons, if the positive control sample doesn't amplify or the negative control samples amplify, the whole run will be repeated.

The distribution of polymorphic variants within the Th2R and Th3R regions of P falciparum CSP will be determined using high-throughput direct sequencing of the PCR products. The sequencing strategy will be validated in a pilot study of 50 PCR samples randomly selected from pre-intervention blood samples from study participants. This will also provide important information regarding the pattern of CSP polymorphism in the study area, and an estimate of the expected data loss due to ambiguous sequence reads from mixed alleles. The sequencing using CSPf2 and CSPr2 primers will be performed in an ABI 3730 capillary sequencer (provided by the GMP). The sequence of each PCR product will be manually checked by at least two investigators and a consensus reached or the assay repeated. Data will be collated from all isolates with a single CSP allele, or where a clear majority allele can be unambiguously identified. For those isolates in which a clear majority allele is not determined from the sequence electropherogram, the majority allele will be determined by cloning of PCR products and sequencing.
Modify Date 13/06/2006 Version : 5.1

Genotyping of msp-1, msp-2 and detection of multiplicity of infection (MOI)
Allele specific, nested PCR of 3 polymorphic regions of P. falciparum genes, namely msp1 block 2 and msp2 will be used to detect the genetic structure of the parasite populations, essentially as described by Snounou, 1999. Following electrophoresis, staining with ethidium bromide and observation under ultra-violet illumination, bands corresponding to different parasite allelic forms will be distinguished and counted and the number of genotypes for msp1 and msp2 loci determined. Gels will be doublescored independently by 2 investigators, and either consensus reached or the assay repeated. To gain a preliminary estimate of MOI in the study population, this genotyping will be performed on the 50 samples used in the sequencing pilot.