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: Sow SO, Okoko BJ, Diallo A, et al. Immunogenicity and safety of a meningococcal A conjugate vaccine in Africans. N Engl J Med 2011;364:

2 CLINICAL STUDY PROTOCOL Protocol No. PsA-TT-002 A phase II, observer-blind, randomized, active controlled study to compare the safety, immunogenicity, and induction of immunological memory of a Meningococcal A Conjugate vaccine, a Meningococcal ACYW Polysaccharide vaccine and a Hib Conjugate vaccine, administered in healthy toddlers months of age Protocol Version Final Version 2 20 April 2006 Amendment 1 18 May 2006 Amendment 2 03 July 2006 Amendment 3-25 June 2008 Amendment 4-13 October 2008 Meningococcal A Tetanus Toxoid Conjugate Vaccine (PsA-TT), manufactured by Serum Institute of India Limited (SIIL) Phase Sponsors II Serum Institute of India Limited (SIIL) 212/2, Hadapsar, Pune , India Phone: ext Fax: Program for Appropriate Technology in Health (PATH) 1455 NW Leary Way Seattle, WA 98107, U.S.A. Phone: Fax: Confidential & Proprietary Information 1/83 The present document is to be used by investigators, medical authorities, institutional review boards and independent ethics committees. This document is confidential and cannot be communicated to third parties or used for publications or conferences without written approval of the sponsors (SIIL & PATH).

3 Meningitis Vaccine Project Protocol No. PsA-TT-002 Amendment No October2008 Authors Co-Authors Marie-Pierre Preziosi, MD, World Health Organization Medical Officer Meningitis Vaccine Project PATH 13, chemin du Levant Bâtiment Avant-Centre Ferney-Voltaire, France Elisa Marchetti, PhD, Clinical Operations Manager Meningitis Vaccine Project PATH 13, chemin du Levant Bâtiment Avant-Centre Ferney-Voltaire, France Prasad Kulkarni, MD, Deputy Medical Director Serum Institute of India Limited (SIIL) 212/2, Hadapsar, Pune , India Principal Investigators Study Monitor Vasudeo Ginde, MD, Pharmacovigilance Medical Coordinator Varsha Parulekar, Biostatistician igate Clinical Research International Pvt. Ltd. 101/102, Alpha, Hiranandani Gardens Powai, Mumbai , India Samba Sow, MD Centre pour les Vaccins en Développement (CVD) Mali, Centre National d'appui à la Lutte contre la Maladie (CNAM), Ministère de la Santé, Ex- Institut Marchoux, BP 251, Bamako. Mali Phone/Fax: ssow@medicine.umaryland.edu Adebayo Akinsola, MD Medical Research Council (MRC) Laboratories, Fajara, PO Box 273. Phone: Fax: aakinsola@mrc.gm Véronique Mazarin Diop, PhD Agence Africaine de Recherche en Santé Humaine BP , Dakar, Yoff, Senegal Phone: +221 (76) Fax: +221 (33) s:mazarin.diop@aarsh.com Confidential & Proprietary Information 2

4 Meningitis Vaccine Project Protocol No. PsA-TT-002 Amendment No October2008 WHO PROTOCOL PsA-TT-002 SIGNATURE PAGE WHO Medical Officer: Dr. Marie-Pierre Preziosi, MD, PhD Date: Signature: PATH Clinical Operations Manager: Dr. Elisa Marchetti, PhD Date: Signature: SIIL Deputy Medical Director: Dr. Prasad Kulkarni, MD Date: Signature: igate Pharmacovigilance Medical Coordinator: Dr. Gandhali Paranjape, MD Date: Signature: Biostatistician: Ms. Varsha Parulekar Date: Signature: Confidential & Proprietary Information 3

5 Meningitis Vaccine Project Protocol No. PsA-TT-002 Amendment No October2008 INVESTIGATOR S SIGNATURE PAGE By my signature below, I, Dr., hereby confirm that I will conduct the study described in Protocol No.PsA-TT 002 in compliance with ICH/GCP and the version of such protocol agreed to by the applicable regulatory authority(ies) and approved by all reviewing IRBs/IECs. Investigator (Name and Title): Date: Signature: Institution (address and phone number, or stamp): Confidential & Proprietary Information 4

6 Meningitis Vaccine Project Protocol No. PsA-TT-002 Amendment No October2008 STUDY SYNOPSIS Title of the Study: A phase II, observer-blind, randomized, active controlled study to compare the safety, immunogenicity, and induction of immunological memory of a Meningococcal A Conjugate vaccine, a Meningococcal ACYW Polysaccharide vaccine and a Hib Conjugate vaccine, administered in healthy toddlers months of age Name of the Sponsors: Serum Institute of India Limited (SIIL)/Program for Appropriate Technology in Health (PATH) Principal Investigators: Dr. Samba Sow MD, Bamako, Mali Dr. Adebayo Akinsola MD, Basse, The Gambia Co-investigators: Dr. Milagritos Tapia MD, Bamako, Mali Prof. Richard Adegbola PhD, Fajara, The Gambia Study Sites: Two study sites, one in Mali and one in The Gambia: Site 1: Centre pour les Vaccins en Développement (CVD) Mali, Centre National d'appui à la Lutte contre la Maladie (CNAM), Ministère de la Santé, Ex-Institut Marchoux, BP 251, Bamako, Mali Site 2: Medical research Council (MRC) Laboratories, Basse, The Gambia. Clinical Development Phase: Phase II Name of the Study Vaccine: Meningococcal A Conjugate Vaccine (Serum Institute of India Limited) referred to as PsA-TT vaccine Protocol No.: PsA-TT-002 Study Rationale: MVP, a partnership between WHO and PATH, was created in 2001 through core funding from the Bill and Melinda Gates Foundation with the goal of eliminating meningococcal epidemics in sub-saharan Africa through the development and use of conjugate meningococcal vaccines. Serogroup A meningococcus is the predominant cause of epidemic meningitis in sub-saharan Africa. Currently, three vaccines are available to protect against epidemic meningococcal A disease. All three are polysaccharide (Ps) vaccines: a bivalent (serogroups A and C), a trivalent (serogroups A, C, and W 135 ), and a tetravalent (serogroups A, C, W 135 and Y) vaccine. A fourth vaccine, a conjugate tetravalent Confidential & Proprietary Information 5

7 Meningitis Vaccine Project Protocol No. PsA-TT-002 Amendment No October2008 vaccine (serogroups A, C, W 135 and Y), has recently been licensed in the United States of America for the prevention of meningococcal disease among adolescents and adults aged 11 to 55 years. The candidate vaccine PsA-TT, manufactured by SIIL, is a conjugate vaccine composed of a serogroup A Neisseria meningitidis capsular polysaccharide, conjugated to a protein carrier, tetanus toxoid, with aluminum phosphate as an adjuvant. Because PsA-TT is a conjugate vaccine, a longer-lasting immune response and a boostable memory response are expected when compared to polysaccharide vaccines. The recent introduction of a conjugate meningococcal C vaccine in the United Kingdom showed significant benefit over polysaccharide vaccines, including decreased disease rates among unvaccinated children and adults (herd immunity effect). The study vaccine, PsA-TT, is intended for the prevention of meningococcal disease due to serogroup A N. meningitidis in African countries where the disease is endemic and periodically causes large epidemics of acute meningitis. The target population consists of children, adolescents, and adults aged 1 to 29 years. The vaccine has been tested in a phase I clinical study (PsA-TT-001, safety and immunogenicity of one dose of 10 µg of PsA-TT vaccine) and was found to be safe and immunogenic among healthy adult volunteers in India. A comprehensive review of SIIL PsA-TT vaccine is contained in the Investigator s Brochure supplied by the sponsor. The present study is a pivotal study and will be performed to evaluate the safety and immunogenicity of one dose of 10 µg of PsA-TT vaccine, and the immunological memory induced by one booster dose of 10 µg of PsA-TT vaccine given eights months later, in children 12 to 23 months of age. The booster study is expected to provide evidence that the PsA-TT conjugate vaccine is able to prime immunological memory. Antibody persistence will be evaluated at eight months (i.e. before the booster dose), one year and two years after the 1 st dose. Primary Objective: To compare the immunogenicity of a single dose of the PsA-TT vaccine with that of the Men A component of the PsACYW vaccine at 28 days after vaccination. Secondary Objectives: Safety: To evaluate the safety of a single dose of the PsA-TT vaccine, of the PsACYW vaccine and of the PRP-TT conjugate vaccine (Hib) at 4, 28 and 224 days after vaccination To evaluate the safety of a booster dose administered eight months after a primary dose of the PsA-TT vaccine, of the PsACYW vaccine (one fifth -1/5 th - of a dose as booster), and of the PRP-TT conjugate vaccine (Hib) after 7, 28, 140 and 504 days To evaluate the safety of one dose of PsA-TT vaccine in subjects who previously received one dose of PsACWY vaccine Immunogenicity: To evaluate the induction of immunological memory at 7 and 28 days after administration of the PsACWY vaccine (1/5 th of a dose as booster) eight months after a single dose of PsA-TT vaccine or PsACWY vaccine Confidential & Proprietary Information 6

8 Meningitis Vaccine Project Protocol No. PsA-TT-002 Amendment No October2008 To evaluate the immunogenicity at 7 and 28 days after a booster dose administered eight months after a primary dose, of the PsA-TT vaccine as compared to the immunogenicity after a booster dose of the PsACYW vaccine (1/5 th of a dose as booster) To evaluate the immunogenicity at 7 and 28 days after the administration of one dose of PsA- TT vaccine in subjects who previously received one dose of PsACWY vaccine To evaluate the immunogenicity at 7 and 28 days after one dose of PsA-TT vaccine in subjects months of age To evaluate the antibody persistence at 224, 364 (1 year) and 728 days (2 years) after a single dose of the PsA-TT vaccine as compared to the antibody persistence after a single dose of the PsACYW vaccine. To evaluate the antibody persistence at 140 and 504 days after a booster dose of the PsA-TT vaccine as compared to a booster dose of the PsACYW vaccine (1/5 th of a dose for booster) Study Design: This Phase II, observer-blind, randomized, comparative clinical study is designed to evaluate the immunogenicity and the safety of a single intramuscular injection of the study vaccine. Immunological memory and persistence of antibodies (Ab) induced by a single intramuscular injection of the study vaccine will also be evaluated. The immunogenicity will be assessed against that of a licensed meningococcal polysaccharide ACYW vaccine. The three-group design will allow comparison of the PsA-TT vaccine (study vaccine group) safety profile with that of two licensed vaccines: the meningococcal ACYW tetravalent polysaccharide vaccine (reference vaccine group), and the Hib-conjugate vaccine (control vaccine group). The latter control vaccine also contains a tetanus toxoid component (TT) which binds the Hib polysaccharide (PRP), as does the study vaccine where the TT binds the MenA polysaccharide. A single booster injection of the study vaccine will allow the assessement of the induced immunological memory. This is a pivotal clinical study for the evaluation of the study vaccine, i.e. the study is designed to demonstrate the functionality of the antibody response, the ability to trigger memory and the two-year persistence of the vaccine-induced antibodies. The study will be performed in toddlers 12 to 23 months of age, the youngest age-group of the target population (1 to 29 years of age), and one that is more capable of discriminating between meningococcal conjugate and polysaccharide vaccines and will constitute a marker for the older age-groups. Toddlers will be recruited from African sites (Mali and The Gambia). Permanent residents of the study areas will be randomized just before vaccination. Each participant will undergo a total of 7 visits for the vaccine and booster periods (of a total duration of 36 weeks), including one vaccination visit and 2 follow-up visits, and one booster vaccination visit and 3 follow-up visits. Each participant will undergo two additional visits at a one year and two year followup. Thus, a total of 9 visits will be proposed to each participant over a total duration of 104 weeks (2 years). Reactogenicity and short-term safety will be assessed during home visits or consultations at the immunization clinic at 4 and 28 days after the initial and booster dose. Diary cards will be used and all adverse events will be followed. Serious adverse events will be monitored during the entire study duration. Likewise, blood draws to assess immunogenicity will be proposed at baseline (just before the primary injection), at 28 days after the primary injection and 7 and 28 days after the booster injection. Indeed, the additional blood draw at 7 days after the booster dose will allow a better assessment of the Confidential & Proprietary Information 7

9 Meningitis Vaccine Project Protocol No. PsA-TT-002 Amendment No October2008 kinetics of the secondary immune response and will be proposed to all subjects. In addition, one blood draw will be proposed at 32 weeks (just before the booster injection) to assess the persistence of the immune response induced by the study vaccine and the pre-booster baseline. Thus, a total of five blood draws will be proposed over a period of 36 weeks, the total duration for one subject of the vaccine and booster periods. Two additional blood draws will be proposed one and two years after initial dose. Thus, a total of maximum 7 blood draws will be proposed to each participant over a two-year period. Each participant will receive one single primary intramuscular injection and one single booster injection eight months after the primary injection and will be followed up for at least 104 weeks (2 years) after the primary injection. At Visit 9all subjects who: 1. did not receive any dose of a MenA-containing vaccine (subjects in the control vaccine group 3C)or 2. did receive a Men A-containing vaccine but with an anti-menpsa rsba titer < 1:128, as measured 28 days after the booster immunization* will be offered a dose of PsA-TT vaccine and will be followed for safety evaluation for an additional 56 days (2 months) after this vaccination**. *or as measured just prior to booster immunization (10 months after primary immunization) if for any reason the 28-day post-booster result is not available. If no results are available (missing information at both time points), the subjects will be offered a dose." **If the relevant subjects refuse to be followed for an additional 56 days, they will still be offered a dose of the licensed polysaccharide PsACWY vaccine Duration of Study Participation/Study period: Note: The initial study period will be referred to as the vaccine period and will include the four weeks following the administration of 1 st dose of vaccine. The booster period will include the four weeks following the administration of the booster. An additional vaccine period will include the four weeks following the administration of the PsA-TT vaccine at Visit 9. The duration of study participation for each subject is 36 weeks for the initial vaccine and booster period: a 4-week vaccine period and a 32-week follow-up period (including a 4-week booster period). An additional 68 weeks will be required for the follow-up period (antibody persistence 1 year and 2 years after initial dose). For subjects who will receive a dose of PsA-TT vaccine at Visit 9, 8 additional weeks will be required for the safety follow-up: a 4-week vaccine period and a 4-week follow-up period. The estimated duration of the recruitment period is 12 weeks. Therefore, the total duration of the study is expected to be 128 weeks from the enrollment of the first subject until the end of follow-up of the last subject enrolled. Number of Subjects: A total of at least 465 evaluable subjects (155 for each vaccination group) is necessary. Considering possible drop-outs, the sample size will be increased by 22.5 %, i.e. a total of 600 eligible subjects (200 for each vaccination group) will be randomized in a 1:1:1 ratio, into one of the following 3 groups: Group 1: Study vaccine (PsA-TT) Group 2: Reference vaccine (GSK Mencevax ACWY ) Group 3: Control vaccine (GSK Hiberix ) Confidential & Proprietary Information 8

10 Meningitis Vaccine Project Protocol No. PsA-TT-002 Amendment No October2008 In addition, subjects in each group will be randomized in a 1:1:1 ratio into one of the following 3 subgroups: Group a: Study vaccine (PsA-TT) Group b: 1/5 th Reference vaccine (GSK Mencevax ACWY ) Group c: Control vaccine (GSK Hiberix ) Subjects will be randomly assigned to one of the following 9 groups: Group 1a PsA-TT conjugate+ PsA-TT conjugate booster Group 1b PsA-TT conjugate+ 1/5th ACWY polysaccharide booster Group 1c PsA-TT conjugate+ Hib booster Group 2a ACWY polysaccharide + PsA-TT conjugate booster Group 2b ACWY polysaccharide + 1/5th ACWY polysaccharide booster Group 2c ACWY polysaccharide + Hib booster Group 3a Hib+ PsA-TT conjugate booster Group 3b Hib+ 1/5 th ACWY polysaccharide booster Group 3c Hib+ Hib booster All subjects who receive at least one vaccine dose will be included in safety analyses. Subjects who provide evaluable blood samples at day 0 and 28 will be included in immunogenicity analyses. Subjects who also provide evaluable blood samples before and 28 days after the booster will be included in immunological memory analyses. Study Population: Inclusion Criteria A subject will be eligible for inclusion if ALL of the following apply at the time of enrollment: 1. Age 12 to 23 months of age (both included) 2. Written informed consent obtained from the mother, father, or guardian of the child 3. Free of obvious health problems as established by medical history including physical examination and clinical judgment of the investigator 4. Mother, father, or guardian capable and willing to bring their child or to receive home visits for their child for all follow-up visits 5. Residence in the study area 6. Fully vaccinated according to local EPI schedule Exclusion Criteria Subjects with any of the following criteria at study entry will not be eligible for participation: 1. Previous vaccination against serogroup A Neisseria meningitidis 2. Known exposure to serogroup A Neisseria meningitidis during the three previous months 3. History of allergic disease or known hypersensitivity to any component of the three study vaccines Confidential & Proprietary Information 9

11 Meningitis Vaccine Project Protocol No. PsA-TT-002 Amendment No October History of Serious Adverse Reactions following administration of vaccines included in the local program of immunization 5. Administration of any other vaccine within 60 days prior to administration of study vaccines or planned vaccination during the first four weeks after the study vaccination 6. Use of any investigational or non-registered drug within 90 days prior to the administration of study vaccines 7. Administration of immunoglobulins and/or any blood products since birth or planned administration during the vaccine period 8. Chronic administration (defined as more than 14 days) of immunosuppressants or other immune-modifying agents since birth (Including systemic or inhaled corticosteroids, this means prednisone, or equivalent, 0.5 mg/kg/day; topical steroids are allowed.) 9. A family history of congenital or hereditary immunodeficiency 10. History of meningitis or seizures or any neurological disorder 11. Major congenital defects or serious chronic illness, including malnutrition (as per investigator s judgment) 12. Acute disease at the time of enrollment (acute disease is defined as the presence of a moderate or severe illness with or without fever) is a temporary exclusion. 13. Acute or chronic, clinically significant pulmonary, cardiovascular, hepatic, or renal functional abnormality, as determined by medical history, physical examination or laboratory tests, which in the opinion of the investigator, might interfere with the study objectives 14. Any condition or criteria that in the opinion of the investigator might compromise the well being of the subject or the compliance with study procedures or interfere with the outcome of the study 15. Non residence in the study area or intent to move out within 1 year Vaccines formulation / Dose/ Route of administration/ Dosage regimen: Study vaccine: Serum Institute of India Ltd (SIIL) PsA-TT vaccine, 0.5 ml/dose Active substances: Purified polysaccharide from Group A Neisseria meningitidis 10 µg Tetanus Toxoid µg Excipients: Tris(hydroxymethyl)aminoethane 0.6 mg. Diluent: aluminum phosphate 1.35 mg, thiomersal 0.01%, sodium chloride 0.9%, sterile water q.s. for injection Note: One 0.5 ml dose out of a decadose vial of PsA-TT vaccine will be injected IM in the right thigh as primary vaccination and IM in the right deltoid when given as a booster. Reference vaccine: GlaxoSmithKline Biologicals (GSK) Mencevax ACWY, 0.5 ml/dose Active substances: Confidential & Proprietary Information 10

12 Meningitis Vaccine Project Protocol No. PsA-TT-002 Amendment No October2008 Purified polysaccharide from Group A Neisseria meningitidis 50 µg Purified polysaccharide from Group C Neisseria meningitidis 50 µg Purified polysaccharide from Group Y Neisseria meningitidis 50 µg Purified polysaccharide from Group W-135 Neisseria meningitidis 50 µg Diluent: lactose, sodium chloride, water for injection. Note: One full dose of PsACWY vaccine will be injected IM in the right thigh as primary vaccination while one reduced dose (1/5 th of the full dose) will be injected SC in the right deltoid when vaccine is given as a booster. Control vaccine: GlaxoSmithKline Biologicals (GSK) Hiberix, 0.5 ml/dose Active substances: 10 µg of purified PRP capsular polysaccharide of Haemophilus influenzae type b conjugated to µg of tetanus protein. Diluent: lactose, sodium chloride, water for injection. Note: One 0.5 ml dose of Hib vaccine will be injected IM in the right thigh as primary vaccination and IM in the right deltoid when given as a booster. Concomitant vaccines: None are allowed within 60 days prior to administration of study, control or reference vaccines, and within the 28 days post-vaccination and/or post-booster. Endpoints: Primary Endpoint The percentage of subjects who show a seroconversion for anti-meningococcal Polysaccharide A (MenPsA) antibodies, i.e. a 4-fold increase in post-immunization serum titer with respect to preimmunization serum titer, at 28 days after a single vaccine dose, as measured by rsba assay. Secondary Endpoints Safety 1. The percentage of subjects with local and systemic adverse events, including solicited adverse reactions and events, and SAEs, as measured at 4 and 28 days after the primary vaccination (reactogenicity and short-term safety); 2. The percentage of subjects with local and systemic adverse events, including solicited adverse reactions and events, and SAEs, as measured at 4 and 28 days after the booster vaccination (reactogenicity and short-term safety); 3. The percentage of subjects with SAEs during the entire study duration, as measured at 32, 52 weeks and 104 weeks (long-term safety). An additional analysis will be performed on safety data collected over an additional study period in the subset of study subjects who will be offered and who will actually receive a dose of PsA-TT vaccine at the end of the trial. This analysis will comprise the percentage of subjects with local and systemic adverse events (including solicited adverse reactions and events) as measured at 4 and 28 days (reactogenicity and short-term safety) and with SAEs as measured at 56 days after the administration of the PsA-TT vaccine dose. Confidential & Proprietary Information 11

13 Meningitis Vaccine Project Protocol No. PsA-TT-002 Amendment No October2008 Immunogenicity 1. The percentage of subjects with anti-menpsa titer 1:8 (defined as seroprotection to MenA) at 28 days after a single vaccine dose, as measured by rsba assay. The percentage of subjects with anti-menpsa titer 1:128 (defined as long-term seroprotection to MenA) will be also considered. 2. GMTs for anti-menpsa antibodies at 28 days after a single vaccine dose, as measured by rsba assay. 3. Evaluation of reverse cumulative distribution curves for MenPsA antibody titers at 28 days after a single vaccine dose, as measured by rsba assay. 4. The percentage of subjects who show a seroconversion for anti-menpsa total IgG, i.e. a 2-fold increase in post-immunization serum concentration with respect to pre-immunization serum concentration, at 28 days after a single vaccine dose, as measured by ELISA. The percentage of subjects with a 4-fold increase in post-immunization serum concentration with respect to preimmunization serum concentration will be also considered. 5. The percentage of subjects with anti-menps A total IgG concentration 2 µg/ml (discriminatory cut-off) 28 days after a single vaccine dose, as measured by ELISA. 6. GMCs for anti-menpsa total IgG at 28 days after a single vaccine dose, as measured by ELISA. All the above immunogenicity endpoints (1 to 6) will also be evaluated at 32 (pre-booster), 52 and 104 weeks (1 year and 2 years) after the single primary vaccine dose, and at 7 and 28 days after the single booster dose. 7. Immunogenicity endpoints for anti-hib Polysaccharide (PRP) and anti-tetanus Toxoid (TT) response will also be evaluated on a sub-sample of subjects: (1) the percentage of subjects with anti-prp titer 0.15 µg/ml (defined as seroprotection to Hib) and 1.0 µg/ml (long-term seroprotection) at different time points (baseline, weeks 4, 32, 36, 52 and 104), as measured by ELISA; (2) the percentage of subjects with antibody levels against tetanus toxoid 0.1 IU/ml at different time points (baseline, weeks 4, 32, 36, 52 and 104), as measured by ELISA. 8. Other assays might be done to further characterize the immune response to the study vaccine. These exploratory analyses will be done on a sub-sample of subjects' sera, volume permitting, to evaluate anti-menpsa: (1) Antibody Avidity Indices, as measured by ELISA; (2) SBA with Human Complement; (3) Opsonophagocytic activity. A thick film to measure malaria parasitaemia will be processed each time blood sampling is done. Statistical Methods: Criteria for assessing the primary objective The difference in proportion of four-fold response in anti-menpsa titer, as measured by rsba, 28 days after a single vaccine dose with respect to baseline titer (sero-responders) will be estimated for the PsA-TT and ACYW polysaccharide vaccine groups with the 95% confidence limits. A total number of 600 subjects (i.e. 200 per groups) will be enrolled in order to obtain 155 evaluable Confidential & Proprietary Information 12

14 Meningitis Vaccine Project Protocol No. PsA-TT-002 Amendment No October2008 subjects per group which accounts for a projected 22.5% dropout rate by the end of the study. This sample size was calculated assuming the proportion of responders for the ACYW polysaccharide vaccine is 90%. 155 subjects per group is required to detect a 10% difference in proportions of responders between the two vaccines and assumes 80% power and an alpha level = Hypothesis The primary objective is to demonstrate that the PsA-TT vaccine elicits antibody responses that are not inferior (i.e., noninferior ) to that achieved by the PsACYW vaccine in subjects 28 days after a single dose. A non-inferiority equivalence boundary of 0.10 is assumed. The antibody response of the PsA-TT vaccine will be judged to be noninferior to the PsACYW vaccine if the upper 95% confidence bound of the difference in proportions of sero-responders (4-fold titer increase) is less than 0.10 (10%). This corresponds to a 5.0% level of significance. The null hypothesis for this study is that the PsA-TT conjugate vaccine will have a significantly lower proportion of 4-fold response in post-immunization serum titer with respect to pre-immunization serum titer than the ACWY polysaccharide vaccine. The alternative hypothesis is that PsA-TT vaccine is noninferior to the ACWY polysaccharide vaccine. If the null hypothesis is rejected (if the upper 95% confidence bound of the difference in proportions of sero-responders between the two vaccines is <0.10) then we may conclude, with stated power and level of significance, that the PsA-TT vaccine is noninferior to the PsACYW vaccine in terms of the amount of anti-menpsa rsba antibody elicited by the two vaccines. Early Safety Analysis An independent Data Safety Monitoring Board (DSMB) composed of three experts will be set up prior to initiating the study. An early safety analysis will be performed after administration of the first vaccine dose to the first 60 subjects. Data from this analysis (all adverse events, local and systemic reactions and SAEs), will be reviewed and analyzed by the DSMB. This analysis will be descriptive in nature and will not include any statistical testing. No unblinding will be done unless deemed necessary by DSMB members. Study stopping rules will be described in the DSMB charter. After this early safety analysis, the DSMB will regularly revise all Serious Adverse Events (related and not related, irrespective of severity) and all related and severe Adverse Events. A detailed description of DSMB procedures is provided in the DSMB charter. Confidential & Proprietary Information 13

15 Randomization Booster Group 1a: PsA-TT conjugate vaccine (study vaccine group) PsA-TT Vaccinatio Group 1: PsA-TT conjugate vaccine (study vaccine group) Group 1b: 1/5th ACWY polysaccharide vaccine (reference vaccine group) Group 1c: Hib vaccine (control vaccine group) Vaccination and follow-up Group 2: ACWY polysaccharide vaccine (reference vaccine Group 2a: PsA-TT conjugate vaccine (study vaccine group) group) Group 2b: 1/5th ACWY polysaccharide vaccine (reference vaccine group) Group 3: Hib vaccine (control vaccine group) Group 2c: Hib vaccine (control vaccine group) Group 3a: PsA-TT conjugate vaccine (study vaccine group) Group 3b: 1/5th ACWY polysaccharide vaccine (reference vaccine group) Only for subject from control vaccine group and subjects with anti- MenPsA antibody rsba<1:128 at 28 days after the booster Group 3c: Hib vaccine (control vaccine group) Confidential & Proprietary Information 14/83 The present document is to be used by investigators, medical authorities, institutional review boards and independent ethics committees. This document is confidential and cannot be communicated to third parties or used for publications or conferences without written approval of the sponsors (SIIL & PATH).

16 OVERALL STUDY DESIGN Meningitis Vaccine Project Protocol No. PsA-TT-002 Amendment No October2008 Study Week Wk 0 Wk 1 Wk 4 Wk 32 Wk 33 Wk 33 Wk 36 Wk 52 Wk 104 Wk 105 Wk 108 Wk 112 Visit 1 Visit 2 Visit 3 Visit 4 Visit 5 Visit 6 Visit 7 Visit 8 Visit 9 Visit 10 ** Visit 11 ** Visit 12 ** Study Day D 0 D 0+x D 0+28 D D D D D D D 0+732** D 0+760** D 0+788** Interval (days) [+4; +7] Post-1 st dose [+28; +35] Post-1 st dose [+224; +280] Post-1 st dose [+4; +7] Post-booster [+7; +10] Post-booster [+28; +38] Post-booster [+140; +196] Post-booster [+504; +560] Post-booster [+4; +7] Post- PsA-TT dose [+28;+35] Post-PsA- TT dose [+56;+90] Post-PsA-TT dose Blood sampling 1 Blood sampling 2 Blood sampling 3 Blood sampling 4 Blood sampling 5 Blood sampling 6 Blood sampling 7 Immunogenicit y (baseline) Safety Home visit Safety, Immunogenicit y post 1 st dose Safety, Immunogenicit y post 1 st dose, pre booster Safety Home visit Immunogenicit y (memory kinetics) Safety, Immunogenicit y post booster Ab persistence 1 year Ab persistence 2 years Malaria Parasitaemia Malaria Parasitaemia Malaria Parasitaemia Malaria Parasitaemia Malaria Parasitaemia Malaria Parasitaemia Malaria Parasitaemia Reportin g All Adverse Events All medications related to AE Serious Adverse Events All medications related to SAE **only for subjects who will receive a dose of PsA-TT at Visit 9. All Adverse Events Serious Adverse Events All Adverse Events All medications related to AE All medications related to SAE All medications related to AE Serious Adverse Events All medications related to SAE Confidential & Proprietary Information 15

17 Meningitis Vaccine Project Protocol No. PsA-TT-002 Amendment No October2008 SCHEDULE OF VISITS AND PROCEDURES Periods of the Study Evaluation Timing in weeks in days interval in days Age of the subject [12-23 months] Collect informed consent Randomization Assignment of subject s number Vaccine period Follow-up period Follow up period Vaccine period Reactogenicity Short- term safety Reactogenicity Short-term safety Long-term safety Short-and long term safety Immune response Immune persistence and booster Immunological memory Safety Wk 0 Wk 0 +0 Wk 0 +4 Wk Wk Wk Wk Wk Wk Wk ** Wk ** D 0 D 0+4 D 0+28 D D D D D D D 0+732* D 0+760* D 0+788* [+4; +7] Post-1 st dose [+28; +35] Post-1 st dose [+224; +280] Post-1 st dose [+4; +7] Post-booster [+7; +10] Post-booster [+28; +38] Post-booster [+140; +196] Post-booster [+504; +560] Post-booster [+4; +7] Post-PsA-TT dose [+28; +35] Post-PsA-TT dose Wk ** [+56;+90 ] Post-PsA-TT dose Check inclusion/exclusion criteria * Check discontinuation criteria * Medical history Physical examination ** ** ** Body temperature ( C) ** ** Vaccination ** Blood sampling Recording of local and systemic reactions (D 0 /+4 and D 224 /+4) ** Recording of AEs ** ** Filling diary cards ** ** ** Record concomitant medication * Report SAEs * End of follow-up ** *as needed at each subsequent visit **only for subjects who will receive a dose of PsA-TT at Visit 9. Confidential & Proprietary Information 16

18 TABLE OF CONTENTS CLINICAL STUDY PROTOCOL...1 PROTOCOL PSA-TT-002 SIGNATURE PAGE...3 INVESTIGATOR S SIGNATURE PAGE...4 STUDY SYNOPSIS...5 OVERALL STUDY DESIGN...15 SCHEDULE OF VISITS AND PROCEDURES...16 TABLE OF CONTENTS...17 LIST OF ABBREVIATIONS INTRODUCTION BACTERIAL MENINGITIS VACCINES AGAINST MENINGITIS MENINGOCOCCAL A TETANUS TOXOID CONJUGATE VACCINE (PSA-TT) STUDY OBJECTIVES AND ENDPOINTS PRIMARY OBJECTIVE SECONDARY OBJECTIVES PRIMARY ENDPOINT SECONDARY ENDPOINTS STUDY DESIGN STUDY RATIONALE AND OVERALL STUDY DESIGN DISCUSSION OF OVERALL STUDY DESIGN STUDY POPULATION Study sites Target Population Planned Duration of the Study Recruitment SELECTION OF STUDY POPULATION Inclusion Criteria...32 Confidential & Proprietary Information 17/83 The present document is to be used by investigators, medical authorities, institutional review boards and independent ethics committees. This document is confidential and cannot be communicated to third parties or used for publications or conferences without written approval of the sponsors (SIIL & PATH).

19 Meningitis Vaccine Project Protocol No. PsA-TT-002 Amendment No October Exclusion Criteria DISCONTINUATION CRITERIA DURING THE STUDY CONTRAINDICATIONS TO REPEATED VACCINATION VACCINES RATIONALE FOR DOSE SELECTION PREPARATION, ADMINISTRATION AND PRECAUTIONS Study Vaccine PsA-TT-Meningococcal A Tetanus Toxoid Conjugate Vaccine (SIIL) Preparation of a Single Dose of PsA-TT vaccine (0.5 ml) Reference Vaccine - Mencevax ACWY (GSK Biologicals) Preparation of the Reference Vaccine Control Vaccine - Hiberix (GSK Biologicals) Preparation of Control Vaccine PRECAUTIONS TO BE OBSERVED IN ADMINISTERING STUDY VACCINES PACKAGING, LABELING, AND STORAGE OF VACCINES CLINICAL STUDY SUPPLY, DISPENSING, AND ACCOUNTABILITY METHOD OF ASSIGNING SUBJECTS TO VACCINATION GROUPS Blinding Methods for Emergency Unblinding Adherence to Randomization List CONDUCT OF THE STUDY GENERAL STUDY ASPECTS ENPOINTS ASSESSMENT Prior and concomitant therapy...42 *only for subjects who will receive a dose of PsA-TT vaccine at Visit Subject s Diary Card...43 *only for subjects who will receive a dose of PsA-TT vaccine at Visit Safety Assessment by the Investigator...44 *only for subjecst who will receive a dose of PsA-TT vaccine at Visit Assessment of Severity/Intensity for Post-Immunization Reactions Assessment of Severity for Adverse Events...44 *only for subjects who will receive a dose of PsA-TT vaccine at Visit Documentation of Meningitis Case Assessments of Immunogenicity Serum Bactericidal Activity (SBA) Anti-PsA IgG (ELISA) Anti-PRP Antibodies (ELISA) Anti-TT Antibodies (ELISA) VISITS SCHEDULE Visit 1 (Day 0) Visit 2 (+4 days after vaccination, window +3)...47 Confidential & Proprietary Information 18

20 Meningitis Vaccine Project Protocol No. PsA-TT-002 Amendment No October Visit 3 (+28 days after vaccination, window +7 days) Visit 4 (Booster vaccination 8 to 10 months after initial vaccination) Visit 5 (+4 days after booster vaccination, window +3) Visit 6 (+7 days after booster vaccination, window +3) Visit 7 (+28 days after booster vaccination, window +10 days) Visit 8 (5 months after booster vaccination, window +56 days) Visit 9/Study Termination Visit or PsA-TT vaccination Visit (18 months after booster vaccination, window +56 days) Visit 10 (4 days after PsA-TT vaccination, window +3 days) Visit 11 (28 days after PsA-TT vaccination, window +7 days) Visit 12/Study Termination Visit (56 days after PsA-TT vaccination, window +34 days) SAFETY EVALUATION DATA SAFETY MONITORING BOARD POST-IMMUNIZATION REACTIONS Local (injection site) post-immunization reactions Systemic post-immunization reactions Assessment of post-immunization reactions severity ADVERSE EVENTS Documenting Adverse Events Assessment of Adverse Events severity Recording adverse events Assessment of causality Follow-up of adverse events and assessment of outcome SERIOUS ADVERSE EVENTS Definition of a serious adverse event Reporting Serious Adverse Events Follow-up of serious adverse events Causality assessment of serious adverse events Treatment of AE and SAEs Management and transmission of SAEs Post-study events UNANTICIPATED PROBLEMS STUDY DISCONTINUATION REASONS FOR PREMATURE TERMINATION EARLY TERMINATION EVALUATION VISIT STATISTICAL CONSIDERATIONS AND ANALYSIS PLAN STUDY CONDUCT CONSIDERATIONS Sample Size...60 Confidential & Proprietary Information 19

21 Meningitis Vaccine Project Protocol No. PsA-TT-002 Amendment No October Randomization Blinding Procedures TIMING OF ANALYSIS DEFINITION OF ANALYSIS SETS ASSESSMENT ENDPOINTS ANALYSIS METHODS Statistical Model Hypothesis Testing and Tests Types of Analysis ANALYSIS PLAN Analysis of Demographics and Other Baseline Characteristics Analysis of Overall Safety QUALITY CONTROL AND QUALITY ASSURANCE PRE-STUDY DOCUMENTATION MONITORING DATA MANAGEMENT AND PROCESSING Data Collection Data Management Procedures Data Verification Procedures Coding Procedures for Analysis of Consistency and Medical Plausibility Data to be recorded directly into the CRF STUDY AND SITE CLOSURE AUDITS AND INSPECTIONS DATA PROTECTION REGULATORY AND ETHICAL REQUIREMENTS INSTITUTIONAL REVIEW BOARDS INDEPENDENT ETHICS COMMITTEES SUBJECT INFORMATION AND INFORMED CONSENT SUBJECT CONFIDENTIALITY NOTIFICATION OF PRIMARY CARE PHYSICIAN RECORD RETENTION PARTICIPANTS AND COMMUNITY INFORMATION ON OUTCOME OF THE RESEARCH ADMINISTRATIVE MATTERS PROTOCOL AMENDMENTS INSURANCE CONFIDENTIALITY REFERENCES...73 Confidential & Proprietary Information 20

22 Meningitis Vaccine Project Protocol No. PsA-TT-002 Amendment No October2008 APPENDIX A: WORLD MEDICAL ASSOCIATION DECLARATION OF HELSINKI APPENDIX B: LABELLING, HANDLING AND SHIPPING OF LABORATORY SPECIMENS...80 APPENDIX B: LABELLING, HANDLING AND SHIPPING OF LABORATORY SPECIMENS...81 APPENDIX C: BACTERIAL MENINGITIS AND MENINGOCOCCAL DISEASE...82 APPENDIX D: SERIOUS ADVERSE EVENT REPORTING PROCESS (INITIAL REPORT)...83 Confidential & Proprietary Information 21

23 Meningitis Vaccine Project Protocol No. PsA-TT-002 Amendment No October2008 LIST OF ABBREVIATIONS Ab AE ANOVA CDC CI CRA CRF CNAM CV CVD DCGI DSMB ELISA EPI ERC FDA GCP GMC GMT Hib HPA HSPC ICH IEC igate IgG IM Antibodies Adverse event Analysis of Variance Centers for Disease Control and Prevention Confidence Interval Clinical Research Associate Case report form Centre National d'appui à la Lutte contre la Maladie Control Vaccine Centre pour le Développement des Vaccins Drug Controller General of India Data Safety Monitoring Board Enzyme-linked immunosorbent assay Expanded Programme on Immunization Research Ethics Review Committee, WHO Food & Drug Administration Good Clinical Practice Geometric Mean Concentration Geometric Mean Titer Haemophilus influenzae type b Health Protection Agency PATH Human Subjects Protection Committee International Conference on Harmonization Independent Ethics Committee igate Clinical Research International Pvt. Ltd. Immunoglobulin G Intra-Muscular Confidential & Proprietary Information 22

24 Meningitis Vaccine Project Protocol No. PsA-TT-002 Amendment No October2008 IRB ITT IU LSLV MedDRA MenA MenPsA MLE MRC MVL/CDC MVP PATH PP PRP PS PsA Institutional Review Board Intention to treat International unit Last Subject Last Visit Medical Dictionary for Regulatory Activities Meningococcal A Meningococcal polysaccharide A Maximum Likelihood Estimation Medical Research Council Meningitis Vaccine Laboratory, Centers for Disease Control and Prevention, Atlanta, U.S.A. Meningitis Vaccine Project Program for Appropriate Technology in Health Per protocol Polyribosyl-ribitol-phosphate Polysaccharide Meningococcal A polysaccharide PsA-TT q. s. Quantity Sufficient Meningococcal A Tetanus Toxoid Conjugate Vaccine RV SAP SV SOP SAE SBA rsba SIIL TT UNK Reference Vaccine Statistical Analysis Plan Study Vaccine Standard Operating Procedure Serious adverse event Serum bactericidal activity Serum bactericidal activity using baby rabbit complement Serum Institute of India Limited Tetanus toxoid Unknown Confidential & Proprietary Information 23

25 Meningitis Vaccine Project Protocol No. PsA-TT-002 Amendment No October2008 VED/HPA WHO WMA Vaccine Evaluation Department, Health Protection Agency, Manchester, U.K. World Health Organization World Medical Association Definitions of Terms End of Trial: The End of Trial corresponds to the last visit or last follow-up of the last subject undergoing the trial (LSLV, Last Subject Last Visit). Sponsor: An individual, company, institution, or organization which takes responsibility for the initiation, management, and/or financing of a clinical trial. SIIL and PATH are both sponsor of this study. Adverse Event: An adverse event (AE) is any untoward medical occurrence in a patient or clinical investigation subject administered a pharmaceutical product at any dose that does not necessarily have to have a causal relationship with this treatment. An AE can, therefore be any unfavorable and unintended sign (including an abnormal laboratory finding, for example), symptom, or disease temporally associated with the use of an investigational product, whether or not considered related to the investigational product. This definition includes intercurrent illnesses or injuries and exacerbation of pre-existing conditions. Local and Systemic Reactions: Selected local and systemic AEs are routinely monitored in vaccine clinical trials as indicators of vaccine reactogenicity. It is recognized that each of these events, and particularly those of a systemic nature, may under some circumstances, in any individual subject, have a cause that is unrelated to the study vaccine. However, as a matter of convenience and in accordance with common clinical practice, all such events occurring within 4 and/or 7 days after immunization are herein termed local and systemic reactions and therefore considered related to the study vaccines. Month, Week, Day: Study months are based upon 28 days cycles. Weeks are based upon 7 days cycles. The study day refers to the number of days after vaccination, with the day of 1 st vaccination being designated day 0. Serious Adverse Event: Any experience or reaction that suggests a significant hazard, contraindication, side effect, or precaution. These events include any experience that is fatal or lifethreatening, requires or prolongs inpatient hospitalization, is permanently disabling, leads to a congenital abnormality, requires intervention to prevent permanent impairment or damage, or is an important and significant medical event that, based upon appropriate medical judgment, may jeopardize the subject. Vaccine failure should also be considered as a serious adverse event (SAE). WHO Medical Officer: The WHO Medical Officer is the sponsor physician responsible for trialrelated medical questions or problems and providing the appropriate medical expertise required for the design and conduct of the trial. Study Monitor: Qualified and appropriately trained individual designated by the sponsors to monitor all aspects of the study. Confidential & Proprietary Information 24

26 Meningitis Vaccine Project Protocol No. PsA-TT-002 Amendment No October INTRODUCTION 1.1 Bacterial Meningitis Bacterial meningitis is a life-threatening medical emergency. Morbidity includes hearing loss, chronic seizures, and learning disability. Up to 25 per cent of meningitis patients who recover suffer permanent central nervous system injury. Meningitis in children is a leading cause of acquired mental retardation. (1,2) The principal pathogens of bacterial meningitis are Neisseria meningitidis, Streptococcus pneumoniae and Haemophilus influenzae. Neisseria meningitidis is particularly feared because it has the capability of causing large outbreaks of disease. Endemic meningococcal disease occurs worldwide and is mostly caused by meningococci of serogroups A, B, C, W 135 and Y. (1) The serogroup A meningococcus is the predominant cause of large epidemics. (3) In the so-called African meningitis belt, major serogroup A epidemics occur at intervals of 5-12 years and result in excessive morbidity and mortality among children and young adults. (3, 4, 5) In recent years, serogroup W 135 meningococci have caused outbreaks in this region (6) and in Saudi Arabia, whereas several Western countries have experienced outbreaks caused by serogroup C strains. (7) In India, the most common serogroup is A. It was responsible for an epidemic among young adults in the 80s (8), and more recently in (9) Meningococcal disease is associated with high case-fatality rates (5%-15%) even where adequate medical services are available. Chemoprophylactic measures can only prevent the disease in close contacts of the index case, but are not recommended for the control and prevention of the disease at the population level. (10) From 1980 to 1990, 114 epidemics of meningitis, the majority due to serogroup A meningococcus, occurred in the African meningitis belt, where at least one country experienced a major epidemic every year. The median annual incidence ranges from 0 to 20 cases per 100,000, but the attack rates can exceed more than 1,200 per 100,000 of population. In 1996, an epidemic involving (4, 5) more than 180,000 cases occurred in the African meningitis belt. 1.2 Vaccines against Meningitis The best available option for the control and prevention of meningococcal meningitis is through vaccination. It is known that immunity following meningococcal infection is serogroup specific. Meningococcal polysaccharide vaccines are recommended for controlling epidemics of meningococcal disease caused by the serogroups present in the vaccine, through large-scale emergency immunization of the population at risk. Current internationally marketed polysaccharide meningococcal vaccines are combination vaccines as A/C, A/C/W135, or A/C/Y/W135. When meningococcal A+C vaccines are (11, 12) given in response to an epidemic, the vaccine can be given to children under two years of age. However, as polysaccharide vaccines are B-dependent antigen, they are not able to prime immunological memory and are poorly immunogenic in infants and young children. The coupling of polysaccharide antigens to proteins transforms the antigen, so called conjugate polysaccharide, into a T-cell dependent antigen, capable of priming immunological memory and be immunogenic in infants and young children. (13, 14), This technology has been widely used to develop several conjugate (15, polysaccharide vaccines such as those against Haemophilus influenzae type b (Hib) 16) Streptoccoccus pneumoniae (17), Neisseria meningitidis serotype C (18), and more recently A,C, W 135,Y. (19) Confidential & Proprietary Information 25