Protocol. This trial protocol has been provided by the authors to give readers additional information about their work.

Transcription

1 Protocol This trial protocol has been provided by the authors to give readers additional information about their work. Protocol for: Olotu A, Fegan G, Wambua J, et al. Seven-year efficacy of RTS,S/AS01 malaria vaccine among young African children. N Engl J Med 2016;374: DOI: /NEJMoa

2 This supplement contains the following items: 1. Original protocol, final protocol, summary of changes. 2. Original statistical analysis plan, final statistical analysis plan, summary of changes

3 1) Title of project Extended follow up of a Phase IIb vaccine trial with RTS,S in Kilifi District, Kenya 2) Investigators Ally Olotu Patricia Njuguna Philip Bejon Edna Ogada Margaret Mackinnon Britta Urban Pete Bull Trudie Lang Norbert Peshu Kevin Marsh Faith Osier Juliana Wambua (Kenya Medical Research Institute, Centre for Geographic Medicine Research Coast) 3) Abstract The RTS,S/AS01E candidate malaria vaccine is being developed for the routine immunization of infants and children living in malaria-endemic areas as part of the Expanded Program of Immunization (EPI). 30% efficacy against clinical malaria and 58% efficacy against severe malaria disease was seen with a related vaccine, RTS,S/AS02A, in children aged 1 to 4 years (Malaria-026) in Mozambique. The efficacy against clinical malaria and infection was sustained beyond 18 months. The RTS,S/AS01 vaccines have been developed in parallel with the RTS,S/AS02 vaccines, and differ in the adjuvant formulation. A recent challenge study with RTS,S/AS01B in malaria-naïve adults has shown higher humoral immunogenicity, a favourable Th1 cell response and a tendency to higher vaccine efficacy. This was confirmed in a study in Kenyan adults. RTS,S/AS01E (0.5 ml dose) is composed of the same active constituents as 0.25 mls of RTS,S/AS01B, but as appropriate for paediatric doses. The clinical development plan for the candidate vaccine RTS,S is designed to lead to a rapid licensure, and does not provide for long term follow up. The utility of a partially effective vaccination depends heavily on the overall effect of malaria incidence during a child s acquisition of natural immunity. However, other cohorts vaccinated in Phase II studies have already discontinued follow up for episodes of malaria, and the planned Phase III studies will run for at most 30 months post vaccination. The established immunology programme in Kilifi, and the infrastructure for monitoring and quality assuring clinical trials offer an opportunity to obtain much longer term data on the protective effect of vaccination. This will inform the design of Phase 4 studies and may prove critical in informing public health policy once the vaccine is licensed. In Malaria-049, the efficacy of a three dose regimen of RTS,S/AS01E against clinical malaria disease due to infection with P. falciparum is being determined in 5-17mth old children in Kilifi, Kenya (SSC 1132) and Korogwe, Tanzania. 12 to 18 months follow up is KEMRI protocol. Extended follow up of a Phase IIb vaccine trial with RTS,S in Kilifi District, Kenya. Version 1 26 th July

4 underway. We propose an additional 4 years follow up to describe the overall impact on a child s lifetime incidence of malaria, and the impact of the vaccine on natural acquisition of immunity to malaria. We will continue to monitor the incidence of febrile malaria episodes by active surveillance, and measure immunological markers of naturally acquired immunity in the laboratory. 4) Introduction The worldwide mortality and morbidity from Plasmodium falciparum malaria is high 1-3. There is evidence that Insecticide Treated Nets 4 and newer anti-malarials 5 are reducing this mortality, but there are still barriers to implementation 6. Nevertheless, there is recent evidence of falling malaria incidence in some areas 7, and this has renewed interest in the prospects for malaria control and even eradication in previously endemic areas 8. However, it is unclear what the next step towards the goal of eradication is, and it has been argued that a malaria vaccine is required in order to bring about a lasting improvement in public health 9. The RTS,S Vaccine RTS,S consists of the carboxyl terminal (a.a ) of the 3D7 circumsporozoite protein fused to the hepatitis B surface antigen, co-expressed in yeast with the non-fused hepatitis B surface antigen. The vaccine is in clinical trials in combination with two different adjuvant systems; AS01 and AS02. The proprietary adjuvant AS02 was the first to be deployed in the field, and is composed of an oil-in-water emulsion and the two immunostimulants QS21 and MPL. The AS01 system is composed of the same immunostimulants (QS21 and MPL) but is formulated as liposomes rather than as an emulsion. There is preliminary evidence that this system is more immunogenic, and non-significantly higher efficacy in experimental challenge has been seen. Studies of efficacy to date Clinical development of the RTS,S vaccine began with experimental challenge of malaria naïve volunteers 10,11, using the AS02 adjuvant system. An overall delay to parasitaemia and complete protection in 30-40% of volunteers was seen. Field trials in adults in The Gambia demonstrated 70% efficacy against parasitisation during the first 2 months, and none thereafter, with an overall 34% efficacy 12. RTS,S with AS02 was then given to children in Mozambique, demonstrating 30% efficacy in time to first clinical episode over the initial 6 months follow up post vaccination 13 and 30% efficacy overall during 18 months follow up 14. A further 9 months follow up data is available, but monitoring has now been discontinued. The same vaccine showed 65% efficacy against acquisition of parasitaemia in infants over 3 months follow up 15. All of the available efficacy data to date is given in terms of delay to first episode of parasitaemia, with the exception of episodes of febrile malaria in Mozambique 13,14, where multiple episodes are also reported. However, multiple episodes are not reported for the full 18 months follow up, but are given separately according to the original 6 months follow up and the subsequent 12 months follow up. Hence, there were relatively few children (<2%) with more than 2 episodes of malaria reported. KEMRI protocol. Extended follow up of a Phase IIb vaccine trial with RTS,S in Kilifi District, Kenya. Version 1 26 th July

5 Subsequent studies suggest that the AS01 system may be more immunogenic and efficacious. Sporozoite challenge studies of malaria naïve volunteers show significantly greater immunogenicity and non-significantly greater protection against sporozoite challenge (data presented at MIM meeting, Cameroon, 2006). Greater immunogenicity was also found in adult cohorts in Kenya, and a greater delay in time to parasitaemia. Based on these unpublished data, the clinical development plan for RTS,S favours a phase 3 trial with AS01 to lead to a licensure application. A series of phase 2 trials in Sub Saharan Africa are therefore underway to establish safety, immunogenicity and preliminary efficacy data before a larger phase 3 trial can be justified with the AS01 adjuvant. Efficacy is being studied in a two-centre study of 5-17 month old children in Kilifi (Kenya) and Korogwe (Tanzania). The study is sponsored by GSK/MVI, and is planned to continue for months follow up per subject. A multi-centre phase 3 study is planned by This will randomize 16,000 children, with 30 months follow up time. Hence, at the planned time of an application for licensure there will be no study including more than 30 months of follow up. Measures of immunity to malaria. The public health burden of malaria is felt in terms of presentations to routine health facilities, either dispensaries in the community or hospitals, identifying a spectrum of disease from mild febrile episodes to severe malaria and death. However, presentation during passive surveillance depends of a variety of individual, geographical and social factors 16,17 and will miss episodes of malaria. Accurate estimates of the actual burden of disease in the population have been extrapolated from the results of active surveillance 1. However, active surveillance may be more prone to miss-identifying self-limiting disease and chronic parasitaemia as cases of malaria. We have shown that a parasite density threshold can be used 18 to improve the specificity of the case definition, and we have recently refined the active case detection methodology to reduce the sampling of children with subjective illness 19. When fever is reported but not documented objectively, we use careful and repeated observation without taking a malaria slide or giving treatment. This methodology has proven highly successful in defining correlates of immunity to malaria 20-25, in identifying children prone to multiple episodes 26 and in estimating the relative environmental and genetic components of risk 27. The potential for rebound malaria There are parallels between an insecticide treated net (ITN) and a pre-erythrocytic vaccine. Both protect from disease by preventing a blood stage infection from becoming established, and so may interfere with the acquisition of immunity to malaria. This was a substantial concern for ITNs given that the relationship between malaria transmission and morbidity is not linear, and that communities with lower rates of infection in young children may have overall higher cumulative rates of infection due to the slower acquisition of immunity 28. Furthermore, children who sleep under ITNs have lower antibodies associated with blood stage immunity 29. However, in long term follow up studies it appears there is not a rebound effect (i.e. malaria incidence does not appear to rise after an initial fall with ITN use) It is unclear why this should be, but the explanation may be that there is an overall KEMRI protocol. Extended follow up of a Phase IIb vaccine trial with RTS,S in Kilifi District, Kenya. Version 1 26 th July

6 decrease in transmission seen within the community following widespread ITN use, probably as a result of reducing mosquito survival 33,34. One is unlikely to see a rebound in the context of falling transmission 35, and this may then be quite different from the situation in communities that have been under stable transmission conditions for years 28. However, it is unclear whether even widespread use of RTS,S will protect a community in the same way as ITNs do. RTS,S might reduce gametocytaemia, but will have no direct effect on the mosquito population. Furthermore, unlike ITN use, the protection provided by vaccination is likely to reduce over time. Rebound effects have been seen after discontinuing chemoprophylaxis against malaria 36, and so despite the reassuring data on long term ITN use, long term data on a new, partially effective vaccine is critical. Immunity to malaria On the other hand, it is possible that exposing children to lower inoculums of parasites allow them to form a more rapid and sustained immune response. Malaria may be immunosuppressive 37,38 and small repeated inoculums of naïve volunteers generate immunity rapidly 39, although this latter observation may be complicated by chemotherapy effects 40. Early intermittent presumptive treatment in infants trials demonstrated long lived protection 41, and the use of intermittent presumptive treatment has been associated with higher merozoite antibody responses 42. However, other trials have not demonstrated long lived protection after presumptive treatment 43. The protection seen during 18 months follow up after RTS,S in Mozambique has nevertheless raised the possibility that post-vaccination acquisition of blood stage immunity might contribute to protection. Although ITN use is associated with reduced responses to blood stage antigens 29, this may not be the case with RTS,S. Although some infections will be completely blocked, some will be partially blocked, with a reduction in the number of infected hepatocytes releasing merozoites into the blood 44. There is good evidence that antigen dose plays an important role in the deviation of T-cell and B-cell responses. Excessive antigen can result in rapid mediated unresponsiveness of antigen presenting cells and early exhaustion of T-cells. Furthermore, some evidence suggests that prolonged presentation of antigen-derived peptides by B-cells induces T-cell tolerance. Finally, high but not excessive antigen-doses tend to induce a primarily Th-1 response. Children who have acquired partial immunity to falciparum liver stages may have an additional advantage in their acquisition of falciparum bloodstage immunity in that they induce a qualitatively and quantitatively different immune response against falciparum blood stage antigens. Study of acquired immunity to malaria has focussed on two main targets in Kilifi. Variant surface antigen is a highly polymorphic antigen expressed on the surface of the infected red cell. These molecules are implicated in the pathogenesis of disease 45, and children specifically seroconvert following exposure 46. Nevertheless, cross-reactivity does exist, and children with antibodies to red cells infected with a laboratory strain have a 4 fold reduction in the incidence of febrile disease during subsequent follow up 25. There is now mounting evidence that var genes encoding PfEMP-1 on the red cell surface fall into different groups which are preferentially expressed in non-immune hosts or in hosts with considerable prior exposure to the parasite. In addition, some PfEMP-1 variants may induce long-lasting immune responses whereas others tend to be more short-lived. However, the underlying mechanisms are not clear. For instance, it may be that either frequent boosting or increased KEMRI protocol. Extended follow up of a Phase IIb vaccine trial with RTS,S in Kilifi District, Kenya. Version 1 26 th July

7 immunogenicity of some variants results in a more stable immune response against that variant. Studies in Kilifi 23,47,48, in Burkina Faso 49 and Senegal 50 suggest that antibodies to a number of merozoite surface antigens are associated with a reduced risk of febrile malaria during follow up. Although individual studies suggest greater or lesser roles for individual antigens, it may be that antibodies to different antigens are partially protective, and that the overall breadth of the response determines protection 23,51. However, protective responses are slowly acquired and often short-lived 52. The RTSS cohort provides an opportunity determine whether bloodstage immunity to PfEMP-1 and to merozoite antigens develops in the same way or at a reduced rate in a group of children living in the same area but with different exposure rates due to RTSS-induced liver stage immunity. It also provides the opportunity to study the longer-term effects on reinfection rates, and infection lengths, both of which will have a significant impact on the herd immunity effect of the vaccine. 5) Justification The worldwide mortality and morbidity from Plasmodium falciparum malaria is high. Although the implementation of insecticide treated nets and newer anti-malarials have reduced this, further interventions to sustain these gains are required. The clinical development plan for the candidate vaccine RTS,S is designed to lead to a rapid licensure, and does not provide for long term follow up. The utility of a partially effective vaccination depends heavily on the overall effect of malaria incidence during a child s acquisition of natural immunity. However, other cohorts vaccinated in Phase II studies have already discontinued follow up for episodes of malaria, and the planned Phase III studies will run for at most 30 months post vaccination. The established immunology programme in Kilifi, and the infrastructure for monitoring and quality assuring clinical trials offer an opportunity to obtain this long term data, which may prove critical in informing public health policy once the vaccine is licensed. This would provide the most long term data on the effect of vaccination that will be available before Phase 4 studies can be conducted, with detailed data on febrile malaria episodes (monitored by active surveillance) and immunological markers of naturally acquired immunity. 6) Null Hypotheses There is no difference in frequency of malaria episodes during long term followup between children vaccinated with RTS,S and children vaccinated with a rabies control vaccine. 7) Objectives General Objectives To examine the long term outcome of vaccination with RTS,S on episodes of febrile malaria. KEMRI protocol. Extended follow up of a Phase IIb vaccine trial with RTS,S in Kilifi District, Kenya. Version 1 26 th July

8 Specific objectives 1. To compare the number of episodes of febrile malaria during long term follow up following vaccination with the candidate malaria vaccine RTS,S with the episodes following control vaccination. 2. To compare the age distribution of episodes of malaria following vaccination with RTS,S with the age distribution following control vaccination. 3. To compare the in vitro markers of naturally acquired immunity in vaccinated and unvaccinated children, and assess the associations of these markers with subsequent episodes of febrile malaria. 4. To assess safety of vaccination by continued reporting of SAEs. 5. To determine the average length of asymptomatic infection, and re-infection rates in the vaccinated vs. unvaccinated groups. 6. To compare the whole-transcriptome expression profiles of parasites found in unvaccinated vs. vaccinated individuals in order to seek evidence of vaccine-induced selection of parasite populations. 8) Design Study site In Kenya, the investigation will take place at the KEMRI Centre for Geographic Medicine Research Coast, Kilifi. There are several dispensaries suitable for such a study in Kilifi district. We currently use Junju dispensary and Pingalikani dispensary (also known as Gongoni dispensary), 30km and 20km south of KEMRI Centre for Geographic Medicine Research. Kilifi District experiences long rains in May-July and short rains in November/December. Measured Entomological Inoculation Rates in the area vary from per year. The dispensaries are the primary source of antimalarial treatment in the area: there are very few shops or private dispensaries in the area. The local administrative structure involves an area chief, a sublocation chief, a dispensary health committee and a clinical officer based in the dispensary. Study population The local population are predominantly from Mijikenda (Chonyi) ethnic group and majority are subsistence farmers. Children aged 5-17 months were eligible for the trial. 450 children were vaccinated in the original study (SCC 1132), and all would be eligible for extended follow up under this protocol. Sampling The original study was powered to detect 30% efficacy over 4 months of surveillance, during which it was estimated that 36% of children would have been affected by a malaria episode. This required 400 subjects per group, and 900 were recruited between Kilifi and Korogwe to allow for loss to follow up. Of the 450 recruited in Kilifi, 420 remain under active follow up. KEMRI protocol. Extended follow up of a Phase IIb vaccine trial with RTS,S in Kilifi District, Kenya. Version 1 26 th July

9 Although the models and analyses will be more sophisticated, the power available can be illustrated by a comparison of proportions. If more than 50% of children have more than 2 episodes of malaria during a further 4 years follow up, the power to detect efficacy against multiple episodes would be 93%. This is for a single proportion. For models studying the distribution of the number of episodes, the power will be greater. Sensitisation and recruitment Study subjects have already been recruited to SSC 1132, and no additional recruiting outside this cohort will take place. However, the study extension will be administered as a new separate study, i.e. a full explanation of the proposed study will be offered to parents, and their consent will be sought for continued follow up. It will be made clear that there is no compulsion to enter this new study. Outline of Procedures For subjects whose parents give informed consent, active surveillance for episodes of malaria will then continue. Children will be seen weekly, their temperature measured, and their parents asked about symptoms. Those who are afebrile but symptomatic will have their temperature measured again 6-10 hours later. Febrile children will have a blood film made, a rapid test for malaria parasites, and treated with anti-malarials if positive. Clinical disease based on febrile illness with blood film confirmed parasitaemia above a defined threshold will be the primary endpoint. The primary analysis will be the rate of acquisition of the first episode of clinical disease. Full details of these procedures are given below. Single unblinding (i.e. of GSK biologicals) will have already occurred for SSC 1132, and one of the investigators (PB) will participate in the analysis of a database (which will not contain names or household names of the subjects). The investigators involved in direct clinical supervision of follow up will remain blind (i.e. single blind) until the full follow up has been completed. Role of the Local Dispensary We will assess adverse events and episodes of malaria at the local dispensary, when mothers bring their children for attendance. We have extended the facilities in the local dispensary to allow for the space required, and employ two clinical officers to conduct this work. The clinical officers also assist with the clinical care of non-study children when they attend the centre. Screening visit. We will confirm the identity of the child, and take a clinical history to identify and any serious adverse events (SAEs) that may have occurred. Inclusion criteria Enrollment and vaccination in SCC 1132 KEMRI protocol. Extended follow up of a Phase IIb vaccine trial with RTS,S in Kilifi District, Kenya. Version 1 26 th July

10 Written or oral, signed or thumb-printed and witnessed informed consent obtained from the parent(s)/guardian(s) of the child. Exclusion criteria Moving out of the study area, so that follow up is impractical. Follow up. Surveillance for Serious Adverse Events (all subjects) Morbidity surveillance for the detection of SAEs will be in place through the health care system in place for this trial. Details of all outpatient attendances and admissions to health facilities will be reviewed by study clinicians to identify those meeting the criteria for SAE reporting. In the case of a death which has occurred at home, supplementary information will be gained using the verbal autopsy technique. The verbal autopsy will be conducted according to previously published methods and detailed in the SOPs on file with the investigators. Field workers will also be readily available to subject s families during the course of the trial. Field workers will have access to a study clinician for consultation by mobile phone 24 hours a day, 7 days a week and transport is available 24 hours a day, 7 days a week if necessary. Junju and Pingilikani Dispensaries are open during normal working hours. All subjects will be seen by clinically qualified personnel. Transfer to Kilifi District Hospital will be facilitated if necessary. Any expenses including transport incurred by the parents/guardians of study participants for the purpose of obtaining a diagnosis of an adverse event as well as for clinical care related to acute conditions will be borne by the study. Long-term care for chronic conditions unrelated to study procedures will be delivered following local guidelines with no financial support from the study. Management and treatment of malaria Children with malaria that can be treated with oral medication will receive a 6-dose regimen of Coartem (artemether/lumefantrine, Novartis AG, Zurich, Switzerland), as advocated by WHO and the governments of Kenya and Tanzania. Children who require parenteral treatment will be admitted and receive treatment with quinine, which is standard practice and effective therapy in both Kenya and Tanzania. Blood sampling for experimental studies KEMRI protocol. Extended follow up of a Phase IIb vaccine trial with RTS,S in Kilifi District, Kenya. Version 1 26 th July

11 Venous blood sampling will be performed on all children once per year, by fingerprick two months later each year, and by venous sampling at each clinical episode during the first year of the study. At the annual venous blood sampling, the routine enquiry used for serious adverse events during the course of the year will be made again. A home visit within 4 weeks of the bleed will be used to document ITN use. The following blood tests will be conducted hematology (complete blood count). serology (anti-blood-stage humoral immunity). Separating peripheral blood mononuclear cells for studies on cellular immunity Rapid test for malaria parasites (only if subject is febrile). Blood slide for malaria parasites. Blood test for malaria parasite DNA studies. Sickle cell testing (first cross-sectional bleed only) In practice, blood slides for malaria parasites in well children will not be read in real time. It will take several weeks for the results to be available. During that time, children will be undergoing regular surveillance for fever, and when a fever is documented, a rapid blood test for malaria parasites will be carried out, and treatment given as described below. Any serum, plasma or blood cells not immediately used in immunological assays will be stored at -20 C or less and would be used to assess the immune response to vaccination or to malarial antigens, to assess any potential toxicity of the vaccine, and to measure the density of asexual parasites and gametocytes in the blood. The latter will be carried out using quantitative real-time PCR using established techniques. In addition we will conduct a further fingerprick cross-sectional bleed 2 months later. This blood will be used for; Blood slide for malaria parasites. Blood test for malaria parasite DNA studies. By comparing the positivity rates at these two timepoints, we will be able to estimate the clearance rate of asymptomatic infection, and hence estimate the average length of infection. Upon every clinical episode (see below), we will take a venous blood sample. A team of fieldworkers skilled in phlebotomy are available during working hours to take these samples. In order to ensure uniformity of follow up, we propose to take such acute samples in the present application (Extended follow up of a Phase IIb vaccine trial with RTS,S in Kilifi District, Kenya). These samples will allow investigation of the immediate cellular responses to acute falciparum malaria, and a subsequent analysis by vaccination. This sample will be cultured in vitro for gene expression profiling by microarray analysis. For these studies, 50 KEMRI protocol. Extended follow up of a Phase IIb vaccine trial with RTS,S in Kilifi District, Kenya. Version 1 26 th July

12 samples from control vs. vaccinated children will be required. Peripheral Blood Mononuclear Cells will be separated to study cellular responses to malaria infection. These studies will be either from fresh samples or after storage in liquid nitrogen. Active case detection for malaria At each visit the axillary temperature of each subject will be taken and if 37.5C a blood slide and a rapid test (OptiMal, Flow Incorporated, Oregon, USA) to determine malaria parasitemia will be taken. If the child is not febrile, no sample will be taken. If the child is documented to be febrile and the rapid test is positive, the field worker will give the child antimalarial chemotherapy and inform a study physician. Mobile phones will be provided to study staff. Appropriate to the clinical features of the illness, transfer of children will be arranged to a health facility. If the child is documented to be febrile and the rapid test is negative, antimalarial chemotherapy will not be administered by the field worker. A study physician will be informed. Appropriate to the clinical features of the illness, the child s parent(s)/guardian(s) will be advised to attend their nearest health facility to be reviewed by clinically qualified personnel (medical or nursing) or transfer will be arranged to the secondary care facility. If the parents/guardians report the child has had a fever but the axillary temperature is < 37.5C, the field worker will revisit the child between 6 to 12 hours later to recheck the temperature and if the temperature is measured as 37.5C a blood slide and rapid malaria test will be taken. If the test is positive, the child s case will be managed as above. If no fever is documented at the revisit, a study physician will be informed. Appropriate to the clinical features of the illness, the child s parent(s)/guardian(s) may be advised to attend their nearest health facility to be reviewed by clinically qualified personnel (medical or nursing), or transfer will be arranged to the secondary care facility or on a study physician s advice, no further action is required. The parent(s)/guardian(s) will be informed that if they have concerns, they may always attend their nearest health facility to have their child reviewed by clinically qualified personnel. Treatment will be indicated by rapid test result. Blood slides for parasitaemia will not be read in real time to guide case management routinely. Blood slides will always be taken in duplicate. At any time at which a child is referred to a health facility, procedures for PCD will be followed. When a subject is presented to a health facility within the study areas, the child will be reviewed by clinically qualified personnel (medical or nursing) and treated as required. The temperature will be recorded and if 37.5C a rapid test and blood slide will be performed. Antimalarial medication will be recorded and study personnel will be informed. Blood slides for parasitemia will not be read in real time to guide case management routinely. Blood slides will always be taken in duplicate. Families will also be informed that KEMRI protocol. Extended follow up of a Phase IIb vaccine trial with RTS,S in Kilifi District, Kenya. Version 1 26 th July

13 field workers will be readily available to facilitate access to care during the course of the trial. Field workers will have access to a study clinician for consultation by mobile phone 24 hours a day, 7 days a week and transport is available 24 hours a day, 7 days a week if necessary. Sponsor The sponsor of SSC 1132 was GlaxoSmithKline Biologicals, Rue de l Institut 89, 1330 Rixensart, Belgium. The extension proposed here is investigator initiated. KEMRI, though CGMR-C, Kilifi, will take on the role of database management for active and passive case detection and monitoring of laboratory and field studies. However, SAEs will be reported to GSK and GSK will retain responsibility for SAE data management. The final analysis will be conducted by the investigators, but the analysis plan will be discussed with and approved by GSK before it is implemented. Data Safety Monitoring Board (DSMB) An independent committee consisting of experts in malaria, pediatrics, statistics and other appropriate disciplines has been appointed to oversee ethical and safety aspects of the study conduct. A quorum of 3 members is required at scheduled meetings. The role of the DSMB includes the review of the implementation and progress of the study. It provides initial, regular, and closing advice on safety-related issues to GSK Biologicals. Its advice is based on the interpretation of study data with reference to the study protocol. The DSMB may, if deemed necessary, convene a meeting with, or request further information from the Principal Investigators, the Medical Monitor/Local Safety Monitors and GSK Biologicals and MVI at PATH s designated project representatives at any stage of the study. The DSMB is empowered to suspend the enrollment to the trial and/or vaccination on the trial pending review of potential safety issues arising in this trial or other relevant trials of the same product. The process will be described in study-specific SOPs. Data Reviewed by the DSMB The DSMB will be informed of: All SAEs. All withdrawals of study subjects by the Principal Investigator or the parent(s)/guardian(s) of a subject due to adverse events. New information that may affect adversely the safety of the subjects or the conduct of the study. All subsequent protocol amendments, informed consent changes or revisions of other documents originally submitted for review. All subsequent protocol modifications (for information). KEMRI protocol. Extended follow up of a Phase IIb vaccine trial with RTS,S in Kilifi District, Kenya. Version 1 26 th July

14 Local Safety Monitor (LSM) The overall role of the Local Safety Monitors (LSM), who are experienced clinicians based in-country, will be to support the clinical investigators and to act as a link between the investigators and the DSMB. The LSM s role will include: Acting as the study volunteer s advocate. Promptly communicating relevant safety information to the DSMB. Providing advice to the investigators on whether a set of clinical circumstances in a study warrants formal notification to the DSMB. Unblinding a subject if deemed necessary to allow for adequate treatment. Liaising closely with the chair of the DSMB throughout the course of the trial. Data Reviewed by the LSM The relevant LSM must be informed by the investigator on an as received basis of: All SAEs. All withdrawals of study subjects by the Principal Investigator or the parent(s)/ guardian(s) of a subject due to adverse events. 9) Data Management Storage The source documents for episodes of malaria detected in the field will be electronic. This uses handheld devices, and data is synchronised centrally on a daily basis. Paper source documents will be used to report SAEs, and for the yearly scheduled clinic visits. Copies of anonymous SAE reports will be sent to GSK biologicals for data entry. All study documentation containing personal information relating to study subjects will be kept in a secure locked area at the investigator s sites. Such documentation will only be made available to authorized personnel. All electronic data kept at the investigator s site are kept secure. Computer access is only available to authorized personnel. Data Analysis The main analysis will be conducted at the end of 4 years. However, an early indication of results would be valuable to policy makers, and so we will conduct an interim analysis after the first 2 years of follow up. Both these analyses will follow the pattern described below, and be described in a prospectively agreed analysis plan. The first analysis will be to tabulate the number of children with 0, 1, 2, 3 etc. episodes of malaria during follow up against vaccine allocation. This will provide immediately relevant information regarding the utility of vaccination. Significance testing will be using Poisson regression. Although this simple analysis may be the most immediately informative in public KEMRI protocol. Extended follow up of a Phase IIb vaccine trial with RTS,S in Kilifi District, Kenya. Version 1 26 th July

15 health terms, furthering our understanding of the outcome will require more complex studies. It is difficult to be confident about waning efficacy using survival analyses on Kaplan Meier plots or Cox regression models. Survival plots will tend to converge once the pool of susceptible subjects without an event are reduced, and this can be mistaken for waning efficacy, as indeed has occurred with studies on RTS,S 54. A presumptive adjustment can be made for this by fitting shared frailties 12, but with malaria the pool of susceptible subjects may well change from year to year, thus violating the assumptions made in fitting shared frailties. We will explore this, by comparing estimates of the changing survival function over time with estimates made on restricted survival analyses from yearly starting points. We will also examine Poisson regression models, where the unit of analysis is a subject year. Hierarchical models clustering on subject and geographical area will then allow us to explore interactions between vaccination status and time. The advantage of these models over survival analyses is that multiple events contribute to analysis, and there is no depletion of susceptible subjects from the analysis. Our analysis of febrile episodes of malaria in an observational study shows us that, in common with many infectious diseases, episodes of febrile malaria are overdispersed by individual; that is, some individuals have many more episodes than expected. This violates one of the assumptions of Poisson models. One solution would be to use a negative binomial model to conduct the same analysis, but one can also use the Poisson model to identify those individuals with significantly more episodes of malaria than expected. This group comprises 20% of the population, but experience 50% of the episodes of malaria 26. Further analysis would examine the distribution of this population by vaccination status. Estimates of reinfection and parasite clearance rates from the 2-month interval studies will be obtained using well-established techniques, and will be analysed in response to vaccination status, village and bednet use, and antibody status.. For gene expression studies, data from the whole transcriptome generated by microarrays (using the 70mer oligonucleotide array from Singapore) will be analysed for differences between the control and vaccinated groups using modified t-tests with adjustment for multiple comparisons. Individual genes that differ in expression between the two groups will be followed up using RT real-time PCR. For validation of the results, these candidate genes will then be tested for differences from fingerprick or venous samples from children found to be parasitized later in the study. 10) Time Frame Pilot Study Not applicable Definitive study Screening October 2008 KEMRI protocol. Extended follow up of a Phase IIb vaccine trial with RTS,S in Kilifi District, Kenya. Version 1 26 th July

16 Surveillance Oct 2008-Oct ) Ethical Considerations The protocol will be submitted to both the KEMRI National Ethics Committee, Oxford Tropical Research Ethics Committee and Western IRB in the US. Changes to the protocol requested by either committee will be referred to the other for approval. The Ethics Committees will be informed of serious and/or unexpected adverse experiences occurring during the study, and any new information that questions the safety of the subjects or the conduct of the study. Human subjects Confidentiality All notes will be held securely in a filing cabinet in a locked office. Data sent to the sponsor will not contain any personal or identifying data. Duty to minimise risks to children The risk from any new vaccination is likely to be minimised by careful extended follow up. Blood will be taken after adequate training and supervision to minimise the distress caused. Duty to assure high standards of informed consent We will provide detailed information about the study for distribution to parents. Each parent will have an opportunity to ask questions or delay a decision should the wish. Inducement We would not offer any financial incentive to attend. There will be a perception of benefit from physical examination, laboratory screening and aspects of routine care such as treatment of iron deficiency. Health screening, and the early detection of malaria have been perceived as benefits in the current study. We do not feel that these are benefits excessive, and all could be considered routine community based health care. Animal subjects Not applicable 12) Expected Application of Results The current failure of control measures, the difficulty of providing and maintaining bed nets, the rise in drug resistance to commonly available antimalarials and the increase in malaria attributable mortality over recent years argue strongly the need for a malaria vaccine. The is current encouraging evidence that the RTS,S vaccines are efficacious. Measuring efficacy over extended follow up is important to evaluating the public health impact of the vaccine. KEMRI protocol. Extended follow up of a Phase IIb vaccine trial with RTS,S in Kilifi District, Kenya. Version 1 26 th July

17 13) References 1. Snow RW, Guerra CA, Noor AM, Myint HY, Hay SI. The global distribution of clinical episodes of Plasmodium falciparum malaria. Nature 2005;434(7030): Hay SI, Guerra CA, Tatem AJ, Noor AM, Snow RW. The global distribution and population at risk of malaria: past, present, and future. Lancet Infect Dis 2004;4(6): Snow RW, Craig M, Deichmann U, Marsh K. Estimating mortality, morbidity and disability due to malaria among Africa's non-pregnant population. Bull World Health Organ 1999;77(8): Lengeler C. Insecticide-treated bednets and curtains for preventing malaria (Cochrane Review). Cochrane Database Syst Rev 2004(1). 5. White NJ, Olliaro P. Artemisinin and derivatives in the treatment of uncomplicated malaria. Med Trop (Mars) 1998;58(3 Suppl): White N, Nosten F, Bjorkman A, Marsh K, Snow RW. WHO, the Global Fund, and medical malpractice in malaria treatment. Lancet 2004;363(9415): Okiro EA, Hay SI, Gikandi PW, et al. The decline in paediatric malaria admissions on the coast of Kenya. Malar J 2007;6: Greenwood BM, Fidock DA, Kyle DE, et al. Malaria: progress, perils, and prospects for eradication. J Clin Invest 2008;118(4): Malkin E, Dubovsky F, Moree M. Progress towards the development of malaria vaccines. Trends Parasitol 2006;22(7): Stoute JA, Slaoui M, Heppner DG, et al. A preliminary evaluation of a recombinant circumsporozoite protein vaccine against Plasmodium falciparum malaria. RTS,S Malaria Vaccine Evaluation Group. N Engl J Med 1997;336(2): Stoute JA, Kester KE, Krzych U, et al. Long-term efficacy and immune responses following immunization with the RTS,S malaria vaccine. J Infect Dis 1998;178(4): Bojang KA, Milligan PJ, Pinder M, et al. Efficacy of RTS,S/AS02 malaria vaccine against Plasmodium falciparum infection in semi-immune adult men in The Gambia: a randomised trial. Lancet 2001;358(9297): Alonso PL, Sacarlal J, Aponte JJ, et al. Efficacy of the RTS,S/AS02A vaccine against Plasmodium falciparum infection and disease in young African children: randomised controlled trial. Lancet 2004;364(9443): Alonso PL, Sacarlal J, Aponte JJ, et al. Duration of protection with RTS,S/AS02A malaria vaccine in prevention of Plasmodium falciparum disease in Mozambican children: single-blind extended followup of a randomised controlled trial. Lancet 2005;366(9502): Aponte JJ, Aide P, Renom M, et al. Safety of the RTS,S/AS02D candidate malaria vaccine in infants living in a highly endemic area of Mozambique: a double blind randomised controlled phase I/IIb trial. Lancet 2007;370(9598): Guyatt HL, Snow RW. The management of fevers in Kenyan children and adults in an area of seasonal malaria transmission. Trans R Soc Trop Med Hyg 2004;98(2): Molyneux CS, Murira G, Masha J, Snow RW. Intra-household relations and treatment decision-making for childhood illness: a Kenyan case study. J Biosoc Sci 2002;34(1): Mwangi TW, Ross A, Snow RW, Marsh K. Case definitions of clinical malaria under different transmission conditions in Kilifi District, Kenya. J Infect Dis 2005;191(11): Bejon P, Mwacharo J, Kai O, et al. A Phase 2b Randomised Trial of the Candidate Malaria Vaccines FP9 ME-TRAP and MVA ME-TRAP among Children in Kenya. PLoS Clin Trials 2006;1(6):e Mwangi TW, Ross A, Marsh K, Snow RW. The effects of untreated bednets on malaria infection and morbidity on the Kenyan coast. Trans R Soc Trop Med Hyg 2003;97(4): Kinyanjui SM, Mwangi T, Bull PC, Newbold CI, Marsh K. Protection against clinical malaria by heterologous immunoglobulin G antibodies against malaria-infected erythrocyte variant surface antigens requires interaction with asymptomatic infections. J Infect Dis 2004;190(9): Osier FH, Polley SD, Mwangi T, Lowe B, Conway DJ, Marsh K. Naturally acquired antibodies to polymorphic and conserved epitopes of Plasmodium falciparum merozoite surface protein 3. Parasite Immunol 2007;29(8): Osier FH, Fegan G, Polley SD, et al. Breadth and magnitude of antibody responses to multiple Plasmodium falciparum merozoite antigens are associated with protection from clinical malaria. Infect Immun 2008;76(5): KEMRI protocol. Extended follow up of a Phase IIb vaccine trial with RTS,S in Kilifi District, Kenya. Version 1 26 th July

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