A Phase II, Randomized Study on an Investigational DTPw-HBV/Hib-MenAC Conjugate Vaccine Administered to Infants in Northern Ghana

Background Combining meningococcal vaccination with routine immunization in infancy may reduce the burden of meningococcal meningitis, especially in the meningitis belt of Africa. We have evaluated the immunogenicity, persistence of immune response, immune memory and safety of an investigational DTPw-HBV/Hib-MenAC conjugate vaccine given to infants in Northern Ghana. Methods and Findings In this phase II, double blind, randomized, controlled study, 280 infants were primed with DTPw-HBV/Hib-MenAC or DTPw-HBV/Hib vaccines at 6, 10 and 14 weeks of age. At 12 months of age, children in each group received a challenge dose of serogroup A+C polysaccharides. Antibody responses were assessed pre, and one month-post dose 3 of the priming schedule and pre and 1 month after administration of the challenge dose. One month post-dose 3, 87.8% and 88.2% of subjects in the study group had bactericidal meningococcal serogroup A (SBA-MenA) and meningococcal serogroup C (SBA-MenC) antibody titres ≥1∶8 respectively. Seroprotection/seropositivity rates to the 5 antigens administered in the routine EPI schedule were non-inferior in children in the study group compared to those in the control group. The percentages of subjects in the study group with persisting SBA-MenA titres ≥ 1∶8 or SBA-MenC titres ≥1∶8 at the age of 12 months prior to challenge were significantly higher than in control group (47.7% vs 25.7% and 56.4% vs 5.1% respectively). The administration of 10 μg of serogroup A polysaccharide increased the SBA-MenA GMT by 14.0-fold in the DTPW-HBV/HibMenAC-group compared to a 3.8 fold increase in the control-group. Corresponding fold-increases in SBA-MenC titres following challenge with 10 μg of group C polysaccharide were 18.8 and 1.9 respectively. Reactogenicity following primary vaccination or the administration of the challenge dose was similar in both groups, except for swelling (Grade 3) after primary vaccination which was more frequent in children in the vaccine than in the control group (23.7%; 95%CI [19.6–28.1] of doses vs 14.1%; 95% CI [10.9–17.8] of doses). Fifty-nine SAEs (including 8 deaths), none of them related to vaccination, were reported during the entire study. Conclusions Three dose primary vaccination with DTPw-HBV/Hib-MenAC was non-inferior to DTPw-HBV/Hib for the 5 common antigens used in the routine EPI schedule and induced bactericidal antibodies against Neisseria meningitidis of serogroups A and C in the majority of infants. Serogroup A and C bactericidal antibody levels had fallen below titres associated with protection in nearly half of the infants by the age of 12 months confirming that a booster dose is required at about that age. An enhanced memory response was shown after polysaccharide challenge. This vaccine could provide protection against 7 important childhood diseases (including meningococcal A and C) and be of particular value in countries of the African meningitis belt. Trial Registration Controlled-Trials.com ISRCTN35754083


Secondary
• At 12 months of age, to evaluate the antibody persistence induced by the primary vaccination with Tritanrix ™ -HepB/Hib-MenAC (new vaccine) and Tritanrix ™ -HepB/ Hiberix TM (control vaccine) with respect to the immunogenicity of all antigens administered. • At 12 months of age, to evaluate the immune memory induced by the primary vaccination with Tritanrix ™ -HepB/Hib-MenAC vaccine by administering 10 µg of each meningococcal A and C polysaccharide (one fifth of a dose of Mencevax™ AC), using the unprimed subjects of the Tritanrix ™ -HepB/ Hiberix ™ group as control.
• To assess the reactogenicity and safety of the primary vaccination after each vaccine dose and overall in the two study groups. • At 12 months of age, to assess the reactogenicity and safety of the 10 µg meningococcal A and C polysaccharides when given to subjects primed with either Tritanrix™-HepB/Hib-MenAC or with Tritanrix™-HepB/ Hiberix™. Study design • Experimental design: 2 parallel groups of 140 subjects each to receive either of the following vaccines: -Tritanrix ™ -HepB/Hib-MenAC -Tritanrix ™ -HepB/Hiberix ™ • Active control group (Tritanrix ™ -HepB/Hiberix ™ ) • Indication: primary vaccination of infants according to the Expanded Program of Immunization (EPI) schedule (6, 10, 14 weeks of age).
• BCG and OPV vaccines can be given at birth or up to at least 2 weeks before study start. OPV can then be given concomitantly with the study vaccines at 6, 10 and 14 weeks of age or at any time during the study, whichever is more appropriate. At 9 months of age, all subjects will receive measles and yellow fever vaccines. • At 12 months of age, all subjects will be evaluated for antibody persistence and will be administered 10 µg of each plain meningococcal serogroup A and C polysaccharides (one fifth of a dose of Mencevax™ AC) to evaluate the immune memory induced by vaccination.
• After study end, one dose of Mencevax™ ACWY will be offered to the Tritanrix ™ -HepB/Hiberix ™ control group to protect them against meningococcal diseases of these serogroups: Mencevax™ ACWY will be given when the child is two years of age, or, if a meningitis epidemic occurs, earlier in the second year of life.

Glossary of Terms
Adverse Event: Any untoward medical occurrence in a patient or clinical investigation subject administered a pharmaceutical product and which does not necessarily have a causal relationship with the pharmaceutical product (as defined by ICH Guideline for Good Clinical Practice).

Blinding:
A procedure in which one or more parties to the trial are kept unaware of the treatment assignment in order to reduce the risk of biased study outcomes. In a single-blind trial, the investigator and/or his staff are aware of the treatment assignment but the subject is not. When the investigator and sponsor staff who are involved in the treatment or clinical evaluation of the subjects and review/analysis of data are also unaware of the treatment assignments, the study is double blind. This level of blinding is maintained throughout the conduct of the trial, and only when the data are cleaned to an acceptable level of quality will appropriate personnel be unblinded or when required in case of a serious adverse event.

Eligible:
Qualified for enrolment into the study based upon strict adherence to inclusion/exclusion criteria.

Evaluable:
Meeting all eligibility criteria, complying with the procedures defined in the protocol, and, therefore, included in the according-to-protocol (ATP) analysis (see Sections 4.4 and 10.4 for details on criteria for evaluability).

eTrack
GlaxoSmithKline's clinical trials tracking tool

Investigational Product:
A pharmaceutical form of an active ingredient or placebo being tested or used as a reference in a clinical trial, including a product with a marketing authorization when used in a way different from the approved form, or when used for an unapproved indication, or when used to gain further information about an approved use.

Protocol Amendment:
Any change in a clinical protocol which affects the safety of subjects, the scope, design, assessments or scientific 759346

Protocol Modification:
Strictly, any change to a clinical protocol that is not considered to be a protocol amendment. In essence, changes to clarify (but not alter) existing design features of a clinical investigation or to encourage greater compliance with the intent of the clinical protocol may be issued as modifications rather than amendments. A protocol modification addresses logistical or administrative aspects of the study (e.g., change of monitor(s), telephone number(s).

Site Monitor:
An individual assigned by GSK Biologicals who is responsible for assuring proper conduct of a clinical study at one or more investigational sites.

Local Safety Monitor:
The overall role of the Local Safety Monitor, an experienced physician based in-country, will be to support the study investigators and to act as a link between the investigators and the Independent Data Monitoring Committee (IDMC) (see Section 5.1.3 for further details).

Solicited Adverse Event:
Adverse events (AEs) to be recorded as endpoints in the clinical study. The presence/occurrence/intensity of these events is actively solicited from the subject or an observer during a specified post-vaccination follow-up period.

Study Monitor:
An individual assigned by and centrally located at GSK Biologicals who is responsible for assuring proper conduct of a clinical study, i.e., Central Study Coordinator.

Subject(s):
Term used throughout the protocol to denote the enrolled individual(s).

Unsolicited Adverse Event:
Any adverse event (AE) reported in addition to those solicited during the clinical study. Also any "solicited" symptom with onset outside the specified period of followup for solicited symptoms will be reported as an unsolicited adverse event.

Introduction
Meningococcal disease, including meningitis and meningococcaemia (meningococcal septicaemia), usually follows invasive infection by Neisseria meningitidis (meningococcus) and is a major cause of death and morbidity throughout the world. Despite the availability of appropriate treatment, the mortality rate is still 7-19% 1 . The risk of meningococcal disease is inversely related to age with 49% of cases occurring in children of four years and younger in Europe 2 . However, older children, adolescents and adults are more often affected during epidemics 3 , 4 .
The disease is endemic in developed countries, such as the US and Europe, and developing countries 5 . Epidemics occur regularly worldwide with highest attack rates in sub-Saharan countries. Serogroups A, B, C, W-135 and Y are the most common causes of invasive meningococcal disease worldwide; serogroups B and C account for most of the cases in Europe and Latin America, while B, C and Y are the 3 prevalent serogroups in the USA. In Asia, serogroups A and C are most common 2, 5, 6 .
African countries, and more specifically the countries of the meningitis belt, face regular epidemics of meningococcal disease, including meningitis with incidence as high as 1000/100,000 and a case-fatality rate of 10 % but much higher death rates are reported in remote areas where the population have a poor access to health care services. Except for outbreaks of serogroup W-135 that spread in Burkina Faso in 2002 and 2003, serogroup A and to a less extent serogroup C are the major serogroups responsible for N. meningitidis epidemics in African countries. Outbreaks hit all age-groups, children aged 5-14 years being the most affected 7 .
The disease presentation may be fulminant with no time for antibiotics to be effective, and permanent neurological damage can occur in children after infection, despite antibiotic therapy. Meningococcal strains with relative or absolute resistance to penicillin have been reported in many countries. In Africa, oily chloramphenicol is the drug of choice and although no cases of resistance have been reported in Africa, some few cases have been reported in Vietnam.

Background
Currently available meningococcal vaccines consisting of pure capsular polysaccharides (serogroup A, C, W-135 and Y meningococcal vaccines) are safe and efficacious in adults and children over two years of age. However they do not induce a satisfactory response in infants and young children under two years of age, the age group most at risk of 23 meningococcal infections. Indeed in this age group, maturation of B-cells is incomplete which leads to a defective B-cell activation. In addition, polysaccharide vaccines do not reduce mucosal carriage and cannot consequently confer herd immunity; they do not induce immunological memory and the associated affinity/avidity maturation of antibodies.
Polysaccharide antigens can however be made to induce a T-cell response and immunological memory, by their covalent coupling with proteins which, in association with Major Histocompatibility Complex (MHC) class II molecules, will be presented to T-helper cells on the surface of Antigen Presenting Cells. This principle was the key to the successful development of Haemophilus influenzae type b (Hib)-conjugate vaccines immunogenic in young children and able to induce immunological memory. 8 , 9 , 10 , 11 To date, vaccine manufacturers have been developing meningococcal conjugate vaccines using the same carrier proteins as the one they use for their Hib conjugate vaccine. Based on this principle, meningococcal serogroup C conjugate vaccines using a non-toxic mutant of diphtheria toxin (CRM 197 ) or tetanus toxoid (TT) as carrier proteins have been recently licensed in Europe, Canada, Australia and Latin America. The first data available after the introduction of the meningococcal serogroup C conjugate vaccines in a mass vaccination campaign in the United Kingdom have proven the effectiveness of these vaccines in all age-groups including infants 12 , 13 , 14 .
N. meningitidis of serogroup A being important in Africa, meningococcal serogroup A + C conjugate vaccines using carrier proteins derived from the diphtheria toxoid have been developed and evaluated in clinical trials in African countries such as Niger and The Gambia. They were found to be safe in these populations. 15 , 16 .
The immunogenicity of meningococcal conjugate vaccines in infants is best shown by measuring bactericidal antibodies one month after primary vaccination. Demonstrating that meningococcal serogroup A and C conjugate vaccines prime for immune memory is an important mechanism to support the long-term protection effect of these vaccines. Such priming for memory was shown for the meningococcal serogroup C conjugate vaccine 17 and for a preliminary meningococcal serogroup A+C conjugate candidate vaccine evaluated in the United Kingdom 18 . However, in another trial in the Gambia, strong memory to the C component was induced but evidence of memory induction for the A component was less convincing 15 . Priming for memory was demonstrated by administering a small dose (10 µg) of unconjugated polysaccharide C six to twelve months after priming. The dose of 10 µg polysaccharide was selected after three adverse events (allergic reactions) had been reported in 10 subjects initially vaccinated with the full polysaccharide vaccine (50 µg/polysaccharide). No further allergic reactions were reported with the reduced dose of 10 µg 18 . 759346 In a previous set of studies, GSK Biologicals had evaluated a Hib-MenAC conjugate vaccine (the meningococcal serogroup A and C polysaccharides were conjugated to another carrier protein than tetanus toxoid) and this combination, given as such or extemporaneously mixed with the Tritanrix™-Hep B vaccine was found to be safe (total N: 259 infants).
Recently, GSK Biologicals has developed a MenC and a Hib-MenC conjugate vaccine using also -TT as carrier protein. Both vaccines were shown to be safe and immunogenic in infants.
In the first three-dose primary vaccination study performed in the Philippines, three formulations of the combined Hib-Men AC vaccine (using TT as carrier protein) each extemporaneously mixed with Tritanrix™-HepB were evaluated and the Tritanrix™-HepB/Hib-MenAC combination using the Hib-MenAC formulation with a dosage of 2.5 µg of each of the MenA, MenC and Hib conjugate antigens was selected for further clinical development: the selected formulation was well tolerated; it induced similar antibody levels against the diphtheria, tetanus, pertussis, hepatitis B, Hib and meningococcal serogroup C antigens as the benchmark vaccines (Tritanrix™-HepB/Hiberix™ and Meningitec™) and 97.7 % subjects had meningococcal serogroup A serum bactericidal antibody titres of at least 1:8.

Rationale for the study
African countries, and more specifically the countries of the meningitis belt, face regular epidemics of meningococcal disease, including meningitis with an incidence as high as 1000/100,000. Except for an outbreak of serogroup W-135 that spread in Burkina Faso in 2002 and 2003, serogroup A and to a lesser extent serogroup C are largely responsible for N. meningitidis epidemics in African countries. Children aged 5-14 years are the most 759346 affected. The existing meningococcal plain polysaccharide vaccines are less immunogenic in small children; also the duration of protection induced by these vaccines is short-lived as polysaccharides vaccine do not induce immune memory. Therefore, there is a need to develop new generation meningococcal vaccines that protect against meningococcal disease in early childhood and induce immune memory.
Recently, meningococcal serogroup C conjugate vaccines have been developed and have proven effective in small children and infants.
In order to protect children from early childhood against N. meningitidis of the two most prevalent serogroups in Africa, and to facilitate the incorporation of such a vaccine in the immunization calendar, GSK Biologicals is developing a combined Hib-MenAC conjugate vaccine to be extemporaneously mixed with the Tritanrix™-HepB vaccine for administration as a 3-dose primary vaccination course in infants aged 6 weeks and over.
The new Tritanrix ™ -HepB/Hib-MenAC vaccine has been evaluated in a feasibility trial in the Philippines. The combination using the Hib-MenAC formulation with a dosage of 2.5 µg of each of the MenA, MenC and Hib conjugate antigens has been selected for further clinical development: the selected formulation was well tolerated; it induced similar antibody levels against the diphtheria, tetanus, pertussis, hepatitis B, Hib and meningococcal serogroup C antigens as the benchmark vaccines (Tritanrix™-HepB/Hiberix™ and Meningitec™) and 97.7 % subjects had meningococcal serogroup A serum bactericidal antibody titres of at least 1:8. As feasibility of the new vaccine is has now been shown, the present study plans to evaluate the use of GSK Biologicals' Tritanrix ™ -HepB/Hib-MenAC vaccine in a high endemicity region in Africa.
The study will evaluate the immunogenicity induced by three doses of the vaccine given at 6, 10 and 14 weeks of age by measuring the immune response one month postvaccination to ensure the vaccine is effective in the youngest children when the disease burden is highest. In order to assess whether the vaccine has induced long-term protection, the persistence of the immune response will be measured when the child is one year of age, and a small dose of plain polysaccharide (1/5 th of a dose of Mencevax™ AC, i.e. 10µg/polysaccharide) will be given to evaluate whether immune memory was induced by priming.

Study site
The to October (average annual rainfall 850-950 mm) and a long dry season from November to April. The district covers an area of 1,675 km 2 with a population of about 140,000. The entire population of the district is included in a demographic surveillance system, the Navrongo Demographic Surveillance System (NDSS), in which births, deaths, in and out migrations and other demographic parameters are recorded in a database and updated every ninety days. 19 There are four health centers in the district, one each in the northern, southern, eastern and western parts of the district. The district hospital, the War Memorial Hospital (WMH), is situated in the central part of the district. It is a 120-bed hospital. Immunization services are conducted in all the health facilities as well as outreach clinics. The Expanded Programme of Immunization (EPI) calendar is as follows: BCG and OPV are given at birth. Tritanrix-HepB/Hib (DTPw-HepB/Hib) and OPV vaccines are given as 3 doses at 6, 10 and 14 weeks of age. Measles and yellow fever vaccines are given at 9 months.
The Navrongo Health Research Center (NHRC) has the mandate to research into health problems facing the people of the Sahelian ecological belt with a view to informing policy decisions. Over the years, it has conducted research on Vitamin A supplementation, use of impregnated bednets, Community Health and Family planning services, malaria, diarrhoea and meningitis. The findings from many of these studies have been made into policies in the country.
Ongoing work on meningitis in collaboration with the Swiss Tropical Institute, Basel, Switzerland has involved studies on carriage 20 , 21 , risk factors 22 , survival and sequelae 23 and aetiological agents of meningitis in the district 24 .

Coordination with the EPI
The vaccine trial will be conducted at several EPI clinics in the Kassena-Nankana district where routine immunization is taking place.
All vaccinees in the EPI system have the so-called "Growth Monitoring Cards" on which the vaccines they receive are recorded. The subjects will be recruited at the time they are to receive the routine DTP, Hib and HepB vaccines (at 6 weeks of age). The vaccines given will be recorded on a specific immunization card (yellow card) and also on the Growth Monitoring Cards, i.e. the name of the study vaccine will be recorded in the box where DTP, Hib and HepB vaccines are usually recorded. Therefore, a duplication of vaccination is prevented as the Growth Monitoring Cards are always brought to the EPI clinics by the parents/guardians. 759346
Criteria to achieve the co-primary objectives: • Immunogenicity of SBA-MenA and SBA-MenC: lower limit of the 95% CI for the percentage of subjects with SBA-MenA titers (respectively SBA-MenC titers) ≥ 1:8 is greater than 80%.
• Non-inferiority for all common antigens: upper limit of the 95% CI for the group difference in terms of the percentage of subjects with anti-PRP concentration ≥ 1 µg/ml (respectively, anti-diphtheria concentration ≥ 0.1 IU/ml, anti-tetanus 759346 28 concentration ≥ 0.1 IU/ml, anti-HBs concentration ≥ 10 mIU/ml) is below 10% and upper limit of the 95% CI for the group GMC ratio in anti-BP is below 1.5.
Refer to Section 10.1 for definition of the co-primary endpoints.

Secondary objectives
• At 12 months of age, to evaluate the antibody persistence induced by the primary vaccination with Tritanrix ™ -HepB/Hib-MenAC (new vaccine) and Tritanrix ™ -HepB/ Hiberix ™ (control vaccine) with respect to the immunogenicity of all antigens administered.
• At 12 months of age, to evaluate the immune memory induced by the primary vaccination with Tritanrix ™ -HepB/Hib-MenAC vaccine by administering 10 µg of each meningococcal A and C polysaccharide (one fifth of a dose of Mencevax™ AC), using the unprimed subjects of the Tritanrix ™ -HepB/ Hiberix ™ group as control.
• To assess the reactogenicity and safety of the primary vaccination after each vaccine dose and overall in the two study groups. • At 12 months of age, to assess the reactogenicity and safety of the 10 µg meningococcal A and C polysaccharides when given to subjects primed with either Tritanrix™-HepB/Hib-MenAC or with Tritanrix™-HepB/ Hiberix™. Refer to Section 10.2 for definitions of secondary endpoints. 759346 Note: BCG and OPV vaccines can be given at birth (or up to at least 2 weeks before the subject's first visit) according to the nationally recommended immunization schedule. OPV can also be given at 6, 10, 14 weeks of age concomitantly with the study vaccines or at any time during the study, whichever is more appropriate.
• Experimental design: 2 parallel groups of 140 subjects each to receive either of the • To comply with the Ghanean Immunisation Calendar, at 9 months of age, all subjects will receive measles and yellow fever vaccines.
• At 12 months of age, all subjects will be evaluated for antibody persistence and will be administered 10 µg of each plain meningococcal serogroup A and C polysaccharides (one fifth of a dose of Mencevax™ AC) to evaluate the immune memory induced by vaccination.
• Active control group (Tritanrix ™ -HepB/Hiberix ™ ) • As the control group will not be properly immunised with 1/5 th of a dose of Mencevax™ AC, after study end, one dose of Mencevax™ ACWY will be offered to the Tritanrix ™ -HepB/Hiberix ™ control group to protect them against meningococcal diseases of these serogroups: Mencevax™ ACWY will be given when the child is two years of age, or, if a meningitis epidemic occurs, earlier in the second year of life.
• Treatment allocation: randomized (1:1). Refer to Section 6.3 for a detailed description of the randomization method.
• Blinding: Double-blind. Refer to Section 6.4 for details of blinding procedure.
• Study duration per subject: approximately 22.5 months to include the retrospective follow-up of SAE(s) up to the time that subjects are at least 24 months of age.
• Data collection: Hard copy Case Report Form (CRF) • For all subjects: 4 blood samples (3.5 ml each): − immediately before the first dose of the primary vaccination course − one month after the third dose of the primary vaccination course − just prior to administration of the plain polysaccharides of serogroups A and C − one month after the administration of the plain polysaccharides of serogroups A and C.: • 4-day follow-up of solicited local and general symptoms after each dose.
• One month follow-up for unsolicited symptoms after each dose.
• Reporting of serious adverse events during the entire study period. 759346

Number of subjects
Target enrolment will be 280 healthy male and female infants (140 subjects per group) to provide 220 evaluable subjects (110 per group) for the immunogenicity analysis. The study will be conducted at the Navrongo Health Research Center in Ghana (War Memorial Hospital and several health centres of Navrongo health district).
Enrolment will be terminated when 280 subjects have been enrolled.
Refer to Section 10.3 for a detailed description of the criteria used in the estimation of sample size.
Details of recruitment at each health center (immunization point), including any criteria for termination of enrolment, will be discussed in the recruitment plan, which is summarized in Appendix C of this document.
If at the time of the initiation of the booster phase any parent/guardian declines participation of his/her child, refusal will be documented as instructed in the "subject tracking document" provided by GSK Biologicals. A copy of the completed tracking document will be forwarded to GSK Biologicals' Study Monitor. The information will be entered in the GSK Biologicals' clinical database for use in identification of any safety issue that may have prevented a subject's participation.

Inclusion criteria
All subjects must satisfy the following criteria at study entry: • Subjects for whom the investigator believes that their parents/guardians can and will comply with the requirements of the protocol (e.g., completion of the diary cards, return for follow-up visits) should be enrolled in the study. 759346

Exclusion criteria for enrolment
The following criteria should be checked at the time of study entry. If any apply, the subject must not be included in the study: • Use of any investigational or non-registered drug or vaccine other than the study vaccine(s) within 30 days preceding the first dose of study vaccine, or planned use during the study period.
• Chronic administration (defined as more than 14 days) of immunosuppressants or other immune-modifying drugs since birth. (For corticosteroids, this will mean prednisone, or equivalent, ≥ 0.5 mg/kg/day. Inhaled and topical steroids are allowed.) • Planned administration/ administration of a vaccine not foreseen by the study protocol within 30 days before each dose of vaccine, with the exception of OPV.
• Hepatitis B and BCG vaccine given within two weeks prior to vaccination. Note: Subjects with fever at the time of first vaccination (i.e., axillary temperature ≥ 37.5°C or rectal temperature ≥ 38.0°C) need not necessarily be excluded but can be vaccinated at a later date after the subject recovers and if the subject still meets all eligibility criteria.
• Administration of immunoglobulins and/or any blood products since birth or planned administration during the study period.
• Other conditions which in the opinion of the investigator may potentially interfere with interpretation of study outcomes.

Elimination criteria during the study
The following criteria should be checked at each visit subsequent to the first visit. If any become applicable during the study, it will not require withdrawal of the subject from the study but does determine a subject's evaluability in the according-to-protocol (ATP) analysis. See Section 10.4 for definition of study cohorts to be evaluated.
• Use of any investigational or non-registered drug or vaccine other than the study vaccine(s) during the study period.
• Chronic administration (defined as more than 14 days) of immunosuppressants or other immune-modifying drugs during the study period. (For corticosteroids, this will mean prednisone, or equivalent, ≥ 0.5 mg/kg/day. Inhaled and topical steroids are allowed).
• Administration of a vaccine not foreseen by the study protocol during the period starting from 30 days before the first dose of vaccine(s) and ending 30 days after the last dose of vaccine(s) with the exception of OPV.
• Administration of a meningococcal vaccine not foreseen by the study protocol during the entire study period.
• Administration of immunoglobulins and/or any blood products during the study period.

Contraindications to subsequent vaccination
The following adverse events (AEs) constitute absolute contraindications to further administration of the study vaccines; if any of these AEs occur during the study, the subject must not receive additional doses of vaccine but may continue other study procedures at the discretion of the investigator (see Section 9). The subject must be followed until resolution of the event, as with any AE (see Section 8.7): • Anaphylactic reaction following the administration of vaccine(s). 759346 The following AEs constitute contraindications to administration of the study vaccines at that point in time; if any one of these AEs occurs at the time scheduled for vaccination, the subject may be vaccinated at a later date, within the time window specified in the protocol (see Section 5.3) or withdrawn at the discretion of the investigator (see Section 9). The subject must be followed until resolution of the event, as with any adverse event (see Section 8.7): • Acute disease at the time of vaccination. (Acute disease is defined as the presence of a moderate or severe illness with or without fever. All vaccines can be administered to persons with a minor illness such as diarrhoea, mild upper respiratory infection with or without low-grade febrile illness, i.e., axillary or oral temperature < 37.5°C/rectal temperature < 38°C).
• Axillary or oral temperature ≥ 37.5°C/rectal temperature ≥ 38°C at the time of vaccination.

DTPw-HBV (Tritanrix™-HepB)
The following AEs associated with DTP vaccination constitute absolute contraindications to further administration of DTP. If any of these AEs occur during the study, the subject must be withdrawn and must be followed until resolution of the event, as with any adverse event (see Section 8.7).
• Encephalopathy (not due to another identifiable cause). This is defined as an acute, severe central nervous system disorder occurring within 7 days following vaccination, and generally consisting of major alterations in consciousness, unresponsiveness, generalized or focal seizures that persist more than a few hours, with failure to recover within 24 hours. Even though causation by DTP vaccine cannot be established, no subsequent doses of pertussis vaccine should be given. In these circumstances the vaccination course should be continued with DT and HB vaccines.

Special warnings and special precautions for use
If any of the following events occur in temporal relation to receipt of Tritanrix™-HepB, the decision to give subsequent doses of vaccine containing the pertussis component should be carefully considered. 759346 There may be circumstances, such as a high incidence of pertussis, when the potential benefits outweigh possible risks.
A history of febrile convulsions, a family history of convulsions, a family history of Sudden Infant Death Syndrome (SIDS) and a family history of an adverse event following Tritanrix™-HepB vaccination do not constitute contraindications.
As with all injectable vaccines, appropriate medical treatment should always be readily available in case of anaphylactic reactions following the administration of the vaccine. For this reason, the vaccinee should remain under medical supervision for 30 minutes after vaccination.
Tritanrix™-HepB should be administered with caution to subjects with thrombocytopenia or a bleeding disorder since bleeding may occur following an intramuscular administration to these subjects.
Tritanrix™-HepB should under no circumstances be administered intravenously.

Hiberix™
The Hiberix™ vaccine should not be administered to subjects with known hypersensitivity to any component of the vaccine, or to subjects having shown signs of hypersensitivity after previous administration of Hib vaccines.

Ethics and regulatory considerations
The study will be conducted according to Good Clinical Practice (GCP), the 1996 version of the Declaration of Helsinki (Protocol Appendix A), and local rules and regulations of the country. The study protocol will be submitted for ethical review to the Navrongo Health Center Ethics Committee, WHO Ethical Review Board, the Ethics Committee of the London School of Hygiene and Tropical Medicine. 759346 The IRB/IEC must be constituted according to the local laws/customs of each participating country. The ICH Harmonized Tripartite Guideline for Good Clinical Practice recommends that the IRB/IEC should include: (a) At least five members. (b) At least one member whose primary area of interest is in a non-scientific area. (c) At least one member who is independent of the institution/ study site. Only those IRB/IEC members who are independent of the investigator and the sponsor of the study should vote/ provide opinion on a study-related matter.
A list of IRB/IEC members and their qualifications should be obtained by the investigator.
This protocol and any other documents that the IRB/IEC may need to fulfil its responsibilities, including subject recruitment procedures and information about payments and compensation available to subjects, will be submitted to the IRB/IEC by the investigator. Written unconditional approval of the IRB/IEC must be in the possession of the investigator and GSK Biologicals before commencement of the study. Relevant GSK Biologicals' data will be supplied by the investigator to the hospital/ university/ independent IRB/IEC for review and approval of the protocol. Verification of IRB/IEC unconditional approval of the protocol and the written informed consent statement will be transmitted by the investigator to the Site Monitor using the standard notification form, prior to shipment of vaccine supplies and CRFs to the site. This approval must refer to the study by exact protocol title and number, and should identify the documents reviewed and state the date of review.
No deviations from, or changes to, the protocol should be initiated without prior written IRB/IEC approval/ favourable opinion of an appropriate amendment, except when necessary to eliminate immediate hazards to the subjects or when the change(s) involves only logistical or administrative aspects of the study (e.g., change of monitor[s], telephone number[s].) Modifications are submitted to the IRB/IEC for information only. However, written verification that the modification was submitted should be obtained. Approvals/ verifications must be transmitted in writing to the Site Monitor, by the investigator.
The IRB/IEC must be informed by the investigator of: • an annual update and/or request for re-approval, where required, • when the study has been completed, where required.

Informed consent
In obtaining and documenting informed consent, the investigator should comply with the applicable regulatory requirement(s), and should adhere to GCP and to the ethical principles that have their origin in the 1996 version of the Declaration of Helsinki. Prior to the beginning of the trial, the investigator should have the IRB/IEC's written approval/favourable opinion of the written informed consent form and any other written information to be provided to the subjects' parents/guardians. Information should be given in both oral and written form whenever possible and as deemed appropriate by the IRB/IEC. An investigator or designate will describe the protocol to potential subjects' parents/guardians face to face. The Subject Information and Consent Form may be read to the subjects' parents/guardians, but, in any event, the investigator or designate shall give the subjects' parents/guardians ample opportunity to inquire about details of the study and ask any questions before dating and signing the Consent Form.
Subject information and consent forms must be in a language fully comprehensible to the prospective subjects' parents/guardians. Informed consent shall be documented by the use of a written consent form approved by the IRB/IEC and signed/thumbprinted and dated by the parent/guardian, and by the person who conducted the informed consent discussion. The signature/thumbprint confirms the consent is based on information that has been understood. All illiterate individuals will have the study, the Subject Information and Consent Form explained to them point by point by the interviewer in the presence of an impartial witness. The witness will personally sign and date the consent form. Oral witnessed consent will replace written consent only in countries where the local custom is contrary or if the parents'/guardians' illiteracy precludes this and provided that the local legal obligations are fulfilled.
Each subject's signed/ thumb printed informed consent form must be kept on file by the investigator for possible inspection by Regulatory Authorities and/or GSK Biologicals' professional and Regulatory Compliance persons. The parents/guardians should receive a copy of the signed/ thumb printed and dated written informed consent form and any other written information provided to the subjects' parents/guardians, and should receive copies 759346 38 of any signed and dated consent form updates. Any amendments to the written information will be provided to subjects' parents/guardians.
Both the informed consent discussion and the written informed consent form and any other written information to be provided to the subjects' parents/guardians should include explanations of the following: (a) That the trial involves research. (b) The purpose of the trial. (c) The trial treatment(s) and the probability for random assignment to each treatment. (d) The trial procedures to be followed, including all invasive procedures. (e) The subject's parents'/guardians' responsibilities. (f) Those aspects of the trial that are experimental. (g) The reasonably foreseeable risks or inconveniences to the subjects and, when applicable, to an embryo, fetus or nursing infant. (h) The reasonable expected benefits. When there is no intended clinical benefit to subjects, the subjects and/or subjects' parents/guardians should be made aware of this. (i) The alternative procedure(s) or course(s) of treatment/ methods of prevention that may be available to subjects, and their important potential benefits and risks. (j) The compensation and/or treatment available to subjects in the event of trial-related injury. (k) The anticipated prorated payment, if any, to subjects' parents/guardians for participating in the trial. (l) The anticipated expenses, if any, to subjects' parents/guardians for participating in the trial. (m) That the subjects' participation in the trial is voluntary and subjects' parents/guardians may refuse to participate or withdraw from the trial, at any time, without penalty or loss of benefits to which subjects are otherwise entitled. (n) That the monitor(s), the auditor(s), the IRB/IEC, and the regulatory authority(ies) will be granted direct access to the subject's original medical records for verification of clinical trial procedures and/or data, without violating the confidentiality of subjects, to the extent permitted by the applicable laws and regulations and that, by signing a written informed consent, the subject's parents/guardians is authorizing such access. (o) That records identifying subjects will be kept confidential and, to the extent permitted by the applicable laws and/or regulations, will not be made publicly available. If the results of the trial are published, subjects' identity will remain confidential. (p) That the subjects' parents/guardians will be informed in a timely manner if information becomes available that may be relevant to the subjects' parents/guardians willingness for their continued participation in the trial. (q) The person(s) to contact for further information regarding the trial and the rights of trial subjects, and who to contact in the event of trial-related injury. GSK Biologicals will prepare a representative subject information sheet/informed consent document which will embody all the elements described above. While it is strongly recommended that this representative document be followed as closely as possible, the informed consent requirements given in this document are not intended to pre-empt any local regulations which require additional information to be disclosed for informed consent to be legally effective. Clinical judgement, local regulations and requirements should guide the final structure and content of the document.
The investigator has the final responsibility for the final presentation of the subject information sheet/informed consent document, respecting the mandatory requirements of local regulations. The consent form generated by the investigator with the assistance of the local sponsor's representative, must be approved (along with the protocol, and any other necessary documentation) by the IRB/IEC and be acceptable to GSK Biologicals.

Composition of the IDMC
An independent committee consisting of up to five experts in meningococcal diseases, paediatrics, vaccines, statistics and other appropriate disciplines will be appointed to oversee ethical and safety aspects of the study conduct.
The Charter (terms of references) of the IDMC will be written after the first meeting of the IDMC members, prior to study start.

Role of the IDMC
The role of the IDMC 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 IDMC will confer before the initiation of the study (pre-initiation review), during the study proper and at the close out of the study to review any relevant safety data; after study end, the IDMC will have a first assessment report. Other unscheduled meetings may be required. Meetings must be documented and minutes made available for the study files The IDMC must be informed by the investigator of: • all subsequent protocol amendments, informed consent changes or revisions of other documents originally submitted for review • systemic grade 3 adverse events (AEs) related to vaccination, serious adverse events and adverse events suggesting a lack of efficacy of the vaccine occurring during the study (transmitted through GSK Biologicals for SAEs and through the Local safety Monitor for the other adverse events) • all subsequent protocol modifications (for information) • new information that may affect adversely the safety of the subjects or the conduct of the study.
The IDMC will be empowered to put the study on hold pending review of potential safety issues. All SAEs, including death, will be reported by the Principal Investigator to the local safety monitor, to the Manager of Clinical Safety Vaccines at GSK Biologicals, to WHO and to the WHO safety monitor. GSK Biologicals will be responsible to provide all information related to the SAEs to the IDMC according to the IDMC Charter.

Reporting to IDMC -Role of Local Safety Monitor
A Local Safety Monitor (LSM) will act independently from the investigational team. The overall role of the Local Safety Monitor (LSM), who will be an experienced clinician based in-country, will be to support the clinical investigators and to act as a link between the investigators and the IDMC. All systemic grade 3 AEs probably or suspected to be related to vaccination, all SAEs and all AEs suggesting a lack of efficacy of the vaccine will be reported to him/her. His/her involvement will be particularly important when decisions have to be made quickly. In exceptional circumstances, for example a death possibly related to vaccination, he/she will have the authority to suspend the whole or any specific aspect of the trial pending discussion with the IDMC.
The LSM's role will include: • acting as the study volunteer's advocate • provide the IDMC and the medical monitor of GSK Biologicals with a blinded listing of non-serious systemic grade 3 symptoms (solicited and unsolicited) that are related with vaccination, and of non-serious adverse events suggesting a lack of efficacy of the vaccine, this according to the IDMC Charter (note: GSK Biologicals will send all 759346 The LSM will liaise closely with the chair of the IDMC throughout the course of the trial.

General study aspects
The maximum distance between a subject's compound and the nearest health facility will be 10 km. Each visit to the clinic for vaccination will last approximately 4 hours; each home visit by a field worker will last about 15 minutes.
The dates of administration of a dose of hepatitis B vaccine and/or Bacille Calmette-Guérin (BCG) and/or Oral Polio Vaccine (OPV) vaccine as well as any other vaccines other than the study vaccines that may be given to the subjects from birth until the end of the study will be documented in the concomitant vaccination section of the individual CRF.
Any meningococcal vaccine given to the mother of the child from one year before the first study vaccine dose administration up to the first study vaccine administration will be documented in the concomitant vaccination section of the individual CRF (see section 6.8 for details).
One specific ancillary study will be done on the subjects participating in this study by Abraham Hodgson, Navrongo Health Research Center (NHRC), Navrongo, Ghana; Gerd Pluschke, Molecular Immunology, Swiss Tropical Institute (STI), Basel, Switzerland. GSK Biologicals is not sponsor of this ancillary study but the protocol of this ancillary study is attached as an Appendix to this protocol (refer Appendix H for further details) for operational reasons (only one subject information sheet will be written and one informed consent will be submitted to the approval of the parents/guardians of the child, however, any parent/guardian accompanying the child at visits subsequent to the first visit who has not already signed an Informed Consent for this study will also have to sign an 759346 Informed Consent prior to providing any throat swab). This ancillary study will be reported separately by the investigators of the ancillary study. This ancillary study requires collection of throat swabs from the parent/guardian and infant prior to the first dose (6-8 weeks) and at 10 weeks, 18 weeks, 9 months and 12 months of age. The collection of throat swab will be detailed in the study procedures (section 5. 3) and the subject information sheet. 759346  • denotes a procedure which must be recorded in the individual CRF. ° denotes a procedure which need not be recorded in the individual CRF. # any parent/guardian accompanying the child at visits subsequent to the first visit who has not already signed an Informed Consent for this study will have to sign an Informed Consent prior to providing any throat swab .## Contraindications for measles and yellow fever vaccines. ### Relevant medication/treatment will include meningococcal vaccine, any investigational or non-registered product (drug or vaccine), any immunosuppressant(s) or other immune-modifying drug(s), immunoglobulins and/or any blood product(s). * a minimum of 2 ml may be taken at prevaccination timepoint. See Section 5.5.2; ** from the subject and subject's parents/guardians; ***Measles, yellow fever vaccine ****1/5 th of dose of Mencevax™ AC Note: BCG and OPV vaccines are given at birth (or at least 2 weeks before the subject's first visit) according to the nationally recommended immunization schedule. OPV vaccine can be administered to the subjects at 6, 10 and 14 weeks of age concomitantly with the study vaccines or at any time during the study, whichever is more appropriate. It is the investigator's responsibility to ensure that the intervals between visits/contacts are strictly followed. These intervals determine each subject's evaluability in the according-to-protocol analyses (see Sections 4.4 and 10.4 for details of criteria for evaluability and cohorts to be analyzed).

Detailed description of study stages/visits
A description of each stage of the study, as summarized in the schedule of assessments (see Table 5-1) is given hereafter. This includes also the recording of any meningococcal vaccine given to the mother during the year preceding the visit 1. • Randomization (See Section 6.3). • Provision of temporary study identification card. • Collection of blood for serology: it is understood that collection of blood in infants aged 6 weeks may be technically difficult. Therefore a minimum of 2.0 ml of whole blood, which is the minimum volume of blood that will allow the essential serology to be undertaken, has to be taken according to instructions in Appendix D. However, whenever possible a 3.5 ml sample should be collected to provide the 1.5 ml sample of serum that is needed for full serological assessment of the response to the vaccine and for validation assays. See Section 5.5.2. Technically skilled and trained individuals will draw blood to avoid unnecessary pain and technical failure as much as possible. When materials are provided by GSK Biologicals, it is MANDATORY that all clinical samples (including serum samples) will be collected and stored using exclusively those materials in the appropriate manner. HepB/Hib-MenAC or Tritanrix™-HepB/Hiberix™ according to the guidelines set out in Section 6.1.6. The vaccinees will be observed closely for at least 30 minutes, with appropriate medical treatment readily available in case of a rare anaphylactic reaction following the administration of vaccines. • The field worker will be given a diary card and will visit the subjects at their home to record, on the day of vaccine injection (day 0) and during the 3 subsequent days (days 1-3) or up to resolution of the symptoms, whichever is longer, the subject's body temperature and any local (at the injection site) or general adverse events (see section 8.5.1). • The field worker will visit the subjects' parents/guardians one month after each vaccination to remind them to go for the next visit. • The subjects' parents/guardians will be instructed to contact the investigator/study contact person at the health centre immediately should the subject manifest any signs or symptoms they perceive as serious.

Visit 2: 10 weeks of age: Study Month 1 (28-42 days after Visit 1): Vaccination
• Provision of definitive study ID card (plastified ID card including picture of parent/guardian and infant). • The investigator will verify the diary cards provided by the field workers. He will transcribe the information into the appropriate sections of the case report form (CRF), in English. • Check of elimination criteria (see Section 4.4).
• Check of any contraindications (see Section 4.5). • Recording of any concomitant medication administered since the last visit (see Section 6.8). • Recording of any serious and any unsolicited adverse events which might have occurred since the last visit. • Physical examination by the investigator including measurement and recording of pre-vaccination temperature. • Collection of throat swabs from the subject and subject's parent/guardian (any parent/guardian accompanying the child at visits subsequent to the first visit who has not already signed an Informed Consent for this study will have to sign an Informed Consent prior to providing any throat swab). • Vaccination: intramuscular administration of one dose of Tritanrix™-HepB/Hib-MenAC or Tritanrix™-HepB/Hiberix™ according to the guidelines set out in Section 6.1.6. The vaccinees will be observed closely for at least 30 minutes, with appropriate medical treatment readily available in case of a rare 759346 47 anaphylactic reaction following the administration of vaccines. • The field worker will be given a diary card and will visit the subjects at their home to record, on the day of vaccine injection (day 0) and during the 3 subsequent days (days 1-3) or up to resolution of the symptoms, whichever is longer, the subject's body temperature and any local (at the injection site) or general adverse events (see section 8.5.1). • The field worker will visit the subjects' parents/guardians one month after each vaccination to remind them to go for the next visit. • The subjects' parents/guardians will be instructed to contact the investigator immediately should the subject manifest any signs or symptoms they perceive as serious. N.B. The parent/guardian of the study subject will be asked to present the study ID card every time they attend the hospital / health centers.
Note: Any parent/guardian accompanying the child at visits subsequent to the first visit who has not already signed an Informed Consent for this study will have to sign an Informed Consent prior to providing a throat swab.

Visit 3: 14 weeks of age: Study Month 2 (28-42 days after Visit 2): Vaccination
• The investigator will verify the diary cards provided by the field workers. He will transcribe the information into the appropriate sections of the CRF, in English. • Check of elimination criteria (see Section 4.4).
• Check of any contraindications (see Section 4.5). • Recording of any concomitant medication administered since the last visit (see Section 6.8). • Recording of any serious and any unsolicited adverse events which might have occurred since the last visit. • Physical examination by the investigator including measurement and recording of pre-vaccination temperature. • Vaccination: intramuscular administration of one dose of Tritanrix™-HepB/Hib-MenAC or Tritanrix™-HepB/Hiberix™ according to the guidelines set out in Section 6.1.6. The vaccinees will be observed closely for at least 30 minutes, with appropriate medical treatment readily available in case of a rare anaphylactic reaction following the administration of vaccines. • The field worker will be given a diary card and will visit the subjects at their home to record, on the day of vaccine injection (day 0) and during the 3 subsequent days (days 1-3) or up to resolution of the symptoms, whichever is 48 longer, the subject's body temperature and any local (at the injection site) or general adverse events (see section 8.5.1). • The field worker will visit the subjects' parents/guardians one month after each vaccination to remind them to go for the next visit. • The subjects' parents/guardians will be instructed to contact the investigator/study contact person at the health centre immediately should the subject manifest any signs or symptoms they perceive as serious.

Visit 4: 18 weeks of age: Study Month 3 (30-42 days after Visit 3): Followup visit
• The investigator will verify the diary cards provided by the field workers. He will transcribe the information into the appropriate sections of the CRF, in English. • Recording of any unsolicited adverse events which might have occurred within one month (minimum 30 days) following the previous vaccination (see Section 8.7). • Recording of any serious adverse events which might have occurred since the last visit. • Check of elimination criteria (see Section 4.4). • Recording of any concomitant medication administered since the last visit (see Section 6.8). • Physical examination by the investigator. • Collection of throat swabs from the subject and subject's parent/guardian (any parent/guardian accompanying the child at visits subsequent to the first visit who has not already signed an Informed Consent for this study will have to sign an Informed Consent prior to providing any throat swab). • Collection of blood for serology: a minimum of 3.5 ml of whole blood to provide a minimum of 1.5 ml of serum according to instructions in Appendix D.
Technically skilled and trained individuals will draw blood to avoid unnecessary pain and technical failure as much as possible. When materials are provided by GSK Biologicals, it is MANDATORY that all clinical samples (including serum samples) will be collected and stored using exclusively those materials in the appropriate manner. The use of other materials could result in the exclusion of the subject from the ATP analysis (See Section 10.4 for definition of study cohorts to be evaluated). The investigator must ensure that his/her personnel and the laboratory(ies) under his/her supervision comply with this requirement. However, when GSK Biologicals does not provide material for collecting and storing clinical samples, then appropriate materials from the investigator's site are to be used. 759346 Refer to Appendix D and Appendix E. • The subjects' parents/guardians will be instructed to contact the investigator/study contact person at the health centre immediately should the subject manifest any signs or symptoms they perceive as serious.
Note: Any parent/guardian accompanying the child at visits subsequent to the first visit who has not already signed an Informed Consent for this study will have to sign an Informed Consent prior to providing a throat swab • Study conclusion of the primary phase

Visit 5: 9 months of age (8-10 months) -Vaccination with Measles and Yellow Fever vaccine
• If at the time of the initiation of the booster phase any parent/guardian declines participation of his/her child, refusal will be documented as instructed in the "subject tracking document" provided by GSK Biologicals. A copy of the completed tracking document will be forwarded to GSK Biologicals' Study Monitor. The information will be entered in the GSK Biologicals' clinical database for use in identification of any safety issue that may have prevented a subject's participation. • Recording of any serious adverse events which might have occurred since the last visit. • Check of elimination criteria (see Section 4.4). • Recording of concomitant medication administered since the last visit (see Section 6.8). • Physical examination by the investigator including measurement and recording of pre-vaccination temperature. • Check of contraindications for measles and yellow fever vaccine (this will not be documented in the individual CRF) • Retrospective recording of any relevant concomitant medication/ vaccination since the last visit according to instructions provided in Section 6.8. Relevant vaccines will include meningococcal vaccines and investigational or non registered vaccines and will be recorded with trade name, route of administration and date(s) of administration. Relevant medications/treatments will include any investigational or non-registered product(s), any immunosuppressant(s) or other immun-modifying drug(s), immunoglobulins and/or any blood products. All such medication/treatment is to be recorded with generic name of the medication (trade names are allowed for combination drugs, i.e. multi-component drugs), medical indication, total daily dose, route of administration, start and end dates of treatment. • Collection of throat swabs from the subject and subject's parent/guardian (any 759346 50 parent/guardian accompanying the child at visits subsequent to the first visit who has not already signed an Informed Consent for this study will have to sign an Informed Consent prior to providing any throat swab). • Vaccination: intramuscular/subcutaneous administration of one dose of measles vaccine and intramuscular administration of one dose of yellow fever vaccine. The vaccinees will be observed closely for at least 30 minutes, with appropriate medical treatment readily available in case of a rare anaphylactic reaction following the administration of vaccines. • The subjects' parents/guardians will be instructed to contact the investigator/study contact person at the health centre immediately should the subject manifest any signs or symptoms they perceive as serious.

Visit 6: 12 months of age (11-13 months) -Booster vaccination
• Recording of any serious adverse events which might have occurred since the last visit. • Check of elimination criteria (see Section 4.4). • Recording of any concomitant medication administered since the last visit (see Section 6.8). • Physical examination by the investigator including measurement and recording of pre-vaccination temperature. • Collection of throat swabs from the subject and subject's parent/guardian (any parent/guardian accompanying the child at visits subsequent to the first visit who has not already signed an Informed Consent for this study will have to sign an Informed Consent prior to providing any throat swab). • Collection of blood for serology: a minimum of 3.5 ml of whole blood to provide a minimum of 1.5 ml of serum according to instructions in Appendix D.
Technically skilled and trained individuals will draw blood to avoid unnecessary pain and technical failure as much as possible. When materials are provided by GSK Biologicals, it is MANDATORY that all clinical samples (including serum samples) will be collected and stored using exclusively those materials in the appropriate manner. The use of other materials could result in the exclusion of the subject from the ATP analysis (See Section 10.4 for definition of study cohorts to be evaluated). The investigator must ensure that his/her personnel and the laboratory(ies) under 759346 • Vaccination: intramuscular administration of 1/5 th of a dose of Mencevax™ AC according to the guidelines set out in Section 6.1.6. The vaccinees will be observed closely for at least 30 minutes, with appropriate medical treatment readily available in case of a rare anaphylactic reaction following the administration of vaccines. • The field worker will be given a diary card and will visit the subjects at their home to record, on the day of vaccine injection (day 0) and during the 3 subsequent days (days 1-3) or up to resolution of the symptoms, whichever is longer, the subject's body temperature and any local (at the injection site) or general adverse events (see section 8.5.1). • The field worker will visit the subjects' parents/guardians one month after each vaccination to remind them to go for the next visit. • The subjects' parents/guardians will be instructed to contact the investigator/study contact person at the health centre immediately should the subject manifest any signs or symptoms they perceive as serious.
Note: Any parent/guardian accompanying the child at visits subsequent to the first visit who has not already signed an Informed Consent for this study will have to sign an Informed Consent prior to providing a throat swab.

Visit 7: Study Month 11.5 (30-42 days after Visit 6): Blood Sampling and Follow-up visit
• The investigator will verify the diary cards provided by the field workers. He will transcribe the information into the appropriate sections of the CRF, in English. • Recording of any serious adverse events which might have occurred since the last visit. • Check of elimination criteria (see Section 4.4). • Recording of any concomitant medication administered since the last visit (see Section 6.8). • Physical examination by the investigator. • Collection of blood for serology: a minimum of 3.5 ml of whole blood to provide a minimum of 1.5 ml of serum according to instructions in Appendix D.
Technically skilled and trained individuals will draw blood to avoid 759346

The study personnel will contact the parents/guardians of the subjects and will question them in a RETROSPECTIVE manner if the subject has suffered any unreported SAE(s) in the period between the last study visit (study conclusion of the booster phase) and this follow-up contact; the study personnel will also consult the child health card whenever available. If SAE(s) had occurred, further investigations will be performed on the subject's hospital/health centre records (if available). Details of these SAE(s) will be reported on the SAE forms. If a death had occurred, a verbal
autopsy report will be obtained whenever possible.

As subjects will be at least two years of age at this safety follow-up visit, the study staff will also remind parents/guardians that the children part of the control group and those with sub-optimal response to the MenA and/or MenC component of the vaccines given to the DTPw-HepB/Hib-MenAC group, will be offered a dose of Mencevax™ ACWY vaccine to protect them against meningococcal diseases (serogroups A, C, W and Y).
This will be the study conclusion for the extended safety follow-up phase.

Laboratory assays
All serological assays will be performed at GlaxoSmithKline Biologicals' central laboratory or in a validated laboratory designated by GlaxoSmithKline Biologicals using standardized, validated procedures with adequate controls.
For all subjects, a 3.5 ml sample of whole venous blood will be collected at visits 1, 4, 6 and 7 using tubes with serum separator. Since the investigator has suggested that the collection of this amount of blood at Visit 1 may be technically difficult in such small infants, the quantity collected at this visit can be reduced to 2.0 ml, as a strict minimum, however this will not allow for any validation/retesting etc that may be required when the testing is performed. After blood centrifugation and serum separation, samples will be stored at −20°C until collection by the sponsor. The aliquots of serum (approximately 1.5 ml) will be sent to GlaxoSmithKline Biologicals for the following tests according to the serology plan (refer to Section 5.5.3): 1. Functional anti-meningococcal serogroups A and C activity (SBA-MenA and SBA-MenC) will be determined by a serum bactericidal test according to the CDC protocol 27 using rabbit complement. The cut-off of the test is a dilution of 1:8. Titers will be expressed as the reciprocal of the dilution resulting in 50% inhibition. The use of 1:8 and 1:128 as SBA endpoints when using rabbit complement for SBA-MenC has been discussed at a recent WHO conference, they are now being used in the literature to report on the immunogenicity of meningococcal C conjugate vaccines 28 More recently, efficacy data from postlicensure surveillance has been shown to validate the SBA-MenC 1:8 cut-off as a serological correlate of protection for meningococcal serogroup C conjugate vaccines 29 .
2. Meningococcal polysaccharide C specific IgG will be measured by ELISA; the assay is based on the CDC protocol 30 . The assay cut-off is 0.30 µg/ml. The use of the ELISA endpoint of 2 µg/ml is based on the correlate of protection proposed for the plain meningococcal C polysaccharide vaccine. The same cut-offs will be employed for meningococcal A polysaccharide. 3. Total antibodies to the Hib polysaccharide PRP will be measured by ELISA using an in-house assay. The cut-off is 0.15 µg/ml. 4. Specific antibodies against diphtheria and tetanus toxoid will be measured by ELISA techniques. The cut-off of the tests is 0.1 IU/ml 31 , 32 . Post-vaccination serum samples with ELISA anti-D antibody concentrations <0.1 IU/ml will be re-tested using a VERO-cell neutralization assay 33,34 . The cut-off of the VERO-cell assay is 0.016 IU/mL. 759346 6. Antibodies to HBs antigen will be measured using a commercially available kit (AUSAB  EIA test, Abbott Laboratories or equivalent). Antibody concentrations = 10 mIU/ml are considered as protective.
Note: Details of assays for throat swabs are available in Appendix H.

Serology plan
Pre-vaccination (Day 0) serum samples will be tested in all subjects for antibodies against SBA-MenA, SBA-MenC, anti-PSA, anti-PSC, diphtheria toxoid, pertussis antigen and recombinant DNA hepatitis B surface antigen (no pre-vaccination testing of anti-TT and anti-PRP is foreseen as immunogenicity results of the previous clinical study were highest for these two antibodies).
Post-vaccination III and pre-booster serum samples will be tested in all subjects for antibodies against all vaccine antigens (SBA-MenA, SBA-MenC, anti-PRP, anti-PSA, anti-PSC, diphtheria and tetanus toxoids, pertussis antigen and recombinant DNA hepatitis B surface antigen). 759346 The post-booster serum sample taken from all children will be tested for SBA-MenA and SBA MenC, anti-PSA and anti-PSC antibodies.
In case of insufficient blood sample volume to perform assays for all antibodies, they will be analyzed according to the following priority ranking: Any additional serology on antigens contained in the study vaccines may be performed if deemed necessary by GlaxoSmithKline Biologicals, or if any findings in the present study or in other studies necessitate investigation of the immunogenicity of the vaccine. In this case, the ranking above may also be changed.

Endpoints for suboptimal response
Suboptimal response will be defined as either of the following: SBA-MenA < 1:8 SBA-MenC < 1:8 anti-PRP concentration < 0.15 µg/ml anti-HBs concentration < 10 mIU/ml anti-BP concentration < 15 EL.U/ml anti-diphtheria concentration < 0.016 (Vero-cell assay) anti-tetanus concentration < 0.1 IU/ml Subjects who demonstrate a suboptimal response to one or more of the following antigen(s) -D, T, Pw, HBV or Hib one month after the primary series will receive a booster dose of licensed Tritanrix™-HepB/Hiberix™ vaccine in the second year of life.
Subjects who demonstrate a suboptimal response to the MenA or MenC antigens will receive a dose of Mencevax™ ACWY to be provided by GSK Biologicals. Mencevax™ ACWY will be given when the child is two years of age or, if a meningitis epidemic occurs, earlier in the second year of life. 759346 The Quality Control Standards and Requirements for each candidate vaccine are described in separate release protocols and the required approvals have been obtained.
Commercial vaccines are assumed to comply with the specifications given in the manufacturer's Summary of Product Characteristics.

GSK Biologicals' Haemophilus influenzae type b meningococcal AC-TT (Hib-MenAC) conjugate vaccine
One lot of the vaccine will be used. The vaccine will be supplied as a white freeze dried pellet in monodose vials to be reconstituted with Tritanrix™-HepB before use. Each vial of the Hib-MenAC (5/5/5) vaccine needs to be reconstituted with 2 vials of the Tritanrix™-HepB before use (0.5 ml per vial). Only half of the dose (i.e. 0.5 ml will be administered. For details of the dose dilution see Section 6.1.7. One dose (0.5 ml) of the reconstituted vaccine contains the following components: Haemophilus influenzae type b capsular polysaccharide (PRP) conjugated to tetanus toxoid :

µg
Neisseria meningitidis A capsular polysaccharide conjugated to tetanus toxoid :

GSK Biologicals' Haemophilus influenzae type b conjugate vaccine:
Hiberix    The vaccine will be supplied as a white freeze-dried pellet in monodose vials to be reconstituted with one 0.5 ml vial of Tritanrix-HepB before use. One dose (0.5 ml) of the reconstituted vaccine contains the following components: Haemophilus influenzae type b capsular polysaccharide conjugated to tetanus toxoid : 10 µg Refer to Appendix G for details of vaccine supplies.

GSK Biologicals' Mencevax™ AC vaccine, used as booster vaccine
Mencevax™ AC is a commercial vaccine which is manufactured by GSK Biologicals. The vaccine will be supplied as a freeze dried pellet to be reconstituted before use with 1.0 ml of the appropriate diluent, supplied in 2 separate vials or ampoules each containing 0.5 ml of diluent. One dose (1 ml) of the reconstituted vaccine will contain: Note: Only one fifth of a dose of Mencevax™ AC (0.2 ml) will be administered to each subject as detailed below: After reconstitution, 0.8 ml (four fifths) of a dose will be discarded and the remaining 0.2 ml, i.e., one fifth (1/5) of a dose, will be administered. 759346

Commercial Measles and Yellow Fever vaccines
The Measles and Yellow Fever vaccines used will be those available commercially in Ghana at the time of the study.

Dosage and administration
An overview of the vaccines to be administered during the study is provided in Table 6-1.

Injection technique
In order to ensure proper intramuscular injection of the study vaccines, a needle of at least 1 inch (2.54 cm) length will be used. The following injection technique is recommended 35 : The needle should be inserted in the upper lateral quadrant of the thigh, directed inferiorly at an angle of 45 degrees with the long axis of the leg, and posteriorly at a 45-degree angle to the tabletop, with the subject supine. During the injection, the tissues of the injection site are compressed with the free hand, increasing the penetrable muscle mass and stabilising the extremity.
The vaccinees will be observed closely for at least 30 minutes following the administration of vaccines, with appropriate medical treatment readily available in case of a rare anaphylactic reaction.

Extemporaneous mixing of Tritanrix™-HepB and Hib-MenAC vaccines or Hiberix™
Tritanrix™-HepB and Hiberix™ The liquid Tritanrix™-HepB vaccine should always be shaken before use. The full content of the Tritanrix™-HepB vaccine vial should be extracted and injected into the vial containing the lyophilized Hiberix™ vaccine. The vial should be agitated until the lyophilized vaccine pellet has completely dissolved. The mixed vaccines will appear white. The reconstituted mixed vaccines should be used promptly after reconstitution (within 30 minutes): • withdraw one dose of 0.5 ml of the mixed vaccines from the vial into the syringe; • a new needle should be used for injection; One dose (0.5 ml) of the mixed vaccines should be administered by intramuscular injection into the anterolateral quadrant of the left thigh.
Tritanrix™-HepB and Hib-MenAC : dose dilution to provide Hib-MenAC (2.5/2.5/5.5) The Hib-MenAC (2.5/2.5/2.5) formulation containing 2.5 µg of each polysaccharide will be used in this study. This will be obtained by dilution of the supplied Hib-MenAC • withdraw one dose of 0.5 ml of the mixed vaccines from the vial; • a new needle should be used for injection; One dose (0.5 ml) of the mixed vaccines should be administered by intramuscular injection into the anterolateral quadrant of the left thigh.
Note: The reconstituted vaccine remaining in the vial after 1 dose (0.5 ml) has been withdrawn is to be retained by the investigator for purposes of vaccine accountability and should not be reused. See Appendix G for more details.

Storage
All vaccines must be stored in a safe and locked place with no access for unauthorized personnel. They must be kept in the refrigerator (+2°C to +8°C/ 36°F to 46°F) and must not be frozen. Storage temperature should be monitored and documented at least once per day. It is advisable to have a back-up refrigerator/ freezer in case of power failure/ breakdown. Procedures must be in place to ensure that the vaccine is kept at the indicated temperature range at all times.
The study monitor must be contacted if the vaccines become frozen or if refrigeration fails.
Storage conditions for transport of vaccines from country medical department or dispatch center to study sites or between sites are described in Appendix G.

Treatment allocation and randomization
The target sample size is 280 enrolled subjects (220 evaluable for immunogenicity analysis, with 110 subjects in each group).
The randomization will be performed in GlaxoSmithKline Biologicals, Rixensart, using a standard Statistical Analysis System (SAS  ) program.
A randomization list will be generated using a randomization blocking scheme (1:1 ratio). This list will be used to ensure balance between treatments and will be used to number the vaccines: a randomization number will uniquely identify the vaccine doses to be administered to the same subject. 759346 The vaccine doses will be distributed to the study center while respecting the randomization block size. On Day 0 (Month 0), subjects will be administered the vaccine dose with the lowest number still available at the study center. The vaccine number will also be used as subject identification for all data collected under the study.

Method of blinding and breaking the study blind
The study will be double blind. The study vaccine and the control vaccine will have the same whitish appearance after reconstitution. However, since the vaccines used in this study are different in their packaging volumes (Tritanrix™-HepB and Hiberix™ = 1 vial of 0.5ml of each vaccine, Tritanrix™-HepB and Hib-MenAC = 2 vials of 0.5 ml of Tritanrix™-HepB and 1 vial of 0.5 ml of Hib-MenAC), special precautions will be taken to ensure blinding. For each vaccination during the course of the study, a Syringe Filler and Vaccinator will work as a vaccination team: the Syringe Filler will prepare the vaccine for administration to a specific subject and the Vaccinator will administer the vaccine to each subject, according to the investigator's SOP. To ensure blinding, a vaccination team will perform no other function in the study. The subject's parent/guardian as well as those responsible for the evaluation of safety or immunogenicity study parameters will all be unaware which vaccine preparation was administered to a particular subject.
A set of individual codes will be held at the GSK Biologicals' Central Safety Office and by the Local Safety Monitor. The code will be broken by the GSK Clinical Safety physician or Local Safety Monitor only in the case of medical events that the investigator/physician in charge of the subject feels cannot be treated without knowing the identity of the study vaccine.
GSK Biologicals' policy (incorporating ICH E2A guidance, EU Clinical Trial Directive and Federal Regulations) is to unblind any serious adverse event (SAE) report associated with the use of the investigational product, which is unexpected and attributable/suspected, prior to regulatory reporting. The Clinical Safety physician is responsible for unblinding the treatment assignment in accordance with specified time frames for expedited reporting of SAEs (Refer to Section 8.9).

Replacement of unusable vaccine doses
Additional vaccine doses will be provided to replace those that are unusable (see Appendix G for details of supplies). 759346 In addition to the vaccine doses provided for the planned number of subjects, at least 5% additional doses of Tritanrix™-HepB and Hiberix™ will be supplied. In case a vaccine dose is broken or unusable, the investigator should replace it with a replacement vaccine dose. Although GlaxoSmithKline Biologicals need not be notified immediately in these cases, documentation of the use of the replacement vaccine must be recorded by the investigator on the vaccine administration page of the CRF and on the vaccine accountability form.

Packaging
See Appendix G.

Vaccine accountability
See Appendix G. Any concomitant medication administered prophylactically in anticipation of reaction to the vaccination must be recorded in the CRF with generic name of the medication (trade names are allowed for combination drugs only), total daily dose, route of administration, start and end dates of treatment and coded as 'Prophylactic'.
Concomitant medication administered for the treatment of an AE or SAE must be recorded in the CRF with generic name of the medication (trade names are allowed for combination drugs only), medical indication (including which AE/SAE), total daily dose, route of administration, start and end dates of treatment. Refer to Section 8.2 for definition of SAE.

Recording of concomitant medication/treatment during the booster phase of the study
At Visit 5, the investigator will question the parents/guardians in a RETROSPECTIVE manner if the subject received any relevant medication/treatment or vaccine in the time period since the last study visit of the primary phase of the study (Visit 4). Relevant vaccines will include meningococcal vaccines and investigational or non registered vaccines and will be recorded with trade name, route of administration and date(s) of administration. Relevant medications/ treatments will include any investigational or nonregistered product(s), any immunosuppressant(s) or other immune-modifying drug(s), immunoglobulins and/or any blood products. All such medication/treatment is to be recorded with generic name of the medication (trade named are allowed for combination drugs, i.e. multi-component drugs), medical indication, total daily dose, route of administration, start and end dates of treatment.
In addition, all concomitant medication, with the exception of vitamins and/or dietary supplements, administered at ANY time during the period starting with administration of each dose and ending one month (minimum 30 days) after each dose are to be recorded with generic name of the medication (trade names are allowed for combination drugs, i.e., multi-component drugs), medical indication, total daily dose, route of administration, start and end dates of treatment. 759346 65 Any treatments and/or medications specifically contraindicated, e.g., any immunoglobulins, other blood products and any immune modifying drugs administered at any time during the study period are to be recorded with generic name of the medication (trade names are allowed for combination drugs only), medical indication, total daily dose, route of administration, start and end dates of treatment. Refer to Section 4.4.
Any vaccine not foreseen in the study protocol administered throughout the study period are to be recorded with trade name, route of administration and date(s) of administration, with the exception of OPV that can be given throughout the study. Refer to Section 4.4.
Any concomitant medication administered prophylactically in anticipation of reaction to the vaccination must be recorded in the CRF with generic name of the medication (trade names are allowed for combination drugs only), total daily dose, route of administration, start and end dates of treatment and coded as 'Prophylactic'.
Concomitant medication administered for the treatment of an AE or SAE must be recorded in the CRF with generic name of the medication (trade names are allowed for combination drugs only), medical indication (including which AE/SAE), total daily dose, route of administration, start and end dates of treatment. Refer to Section 8.2 for definition of SAE.

Health Economics
Not applicable.

Adverse Events, Serious Adverse Events and vaccine failure
The investigator is responsible for the detection and documentation of events meeting the criteria and definition of an adverse event (AE) or serious adverse event (SAE) as provided in this protocol. During the study, when there is a safety evaluation, the investigator or site staff will be responsible for detecting AEs and SAEs, as detailed in this section of the protocol.
Each subject's parents/guardians will be instructed to contact the investigator immediately should the subject manifest any signs or symptoms they perceive as serious. 759346

Definition of an adverse event
An AE is any untoward medical occurrence in a clinical investigation subject, temporally associated with the use of a medicinal product, whether or not considered related to the medicinal product.
An AE can therefore be any unfavourable and unintended sign (including an abnormal laboratory finding), symptom, or disease (new or exacerbated) temporally associated with the use of a medicinal product. For marketed medicinal products, this also includes failure to produce expected benefits (i.e., lack of efficacy), abuse or misuse.
Examples of an AE include: • Significant or unexpected worsening or exacerbation of the condition/indication under study. See Section 8.3 'Lack of Efficacy' for additional information.
• Exacerbation of a chronic or intermittent pre-existing condition including either an increase in frequency and/or intensity of the condition.
• New conditions detected or diagnosed after investigational product administration even though it may have been present prior to the start of the study.
• Signs, symptoms, or the clinical sequelae of a suspected interaction.
• Signs, symptoms, or the clinical sequelae of a suspected overdose of either investigational product or a concurrent medication (overdose per se should not be reported as an AE/SAE).
• Signs, symptoms temporally associated with vaccine administration.
Examples of an AE DO NOT include: • Medical or surgical procedure (e.g., endoscopy, appendectomy); the condition that leads to the procedure is an AE.

•
Situations where an untoward medical occurrence did not occur (social and/or convenience admission to a hospital).
• Anticipated day-to-day fluctuations of pre-existing disease(s) or condition(s) present or detected at the start of the study that do not worsen.

•
The disease/disorder being studied, or expected progression, signs, or symptoms of the disease/disorder being studied, unless more severe than expected for the subject's condition. 759346 AEs may include pre-or post-treatment events that occur as a result of protocol-mandated procedures (i.e., invasive procedures, modification of subject's previous therapeutic regimen).
N.B. AEs to be recorded as endpoints (solicited events) are described in Section 8.5.1. All other AEs will be recorded as UNSOLICITED AES.
Example of events to be recorded in the medical history section of the CRF: • Pre-existing conditions or signs and/or symptoms present in a subject prior to the start of the study (i.e. prior to the first study procedure) should be recorded in the medical history section of the subject's CRF as instructed by the GSK Biologicals' Study Monitor.

Lack of efficacy
"Lack of efficacy" per se will not be reported as an AE. The signs and symptoms or clinical sequelae resulting from lack of efficacy will be reported if they fulfil the AE or SAE definition (including clarifications). Any case of diphtheria, tetanus, pertussis, hepatitis B, invasive diseases due to Haemophilus influenzae type b, measles, yellow fever or Neisseria meningitidis of serogroup A and C will be reported to the Independent Data Monitoring Committee (IDMC).

Clinical laboratory parameters and other abnormal assessments qualifying as adverse events and serious adverse events
Abnormal laboratory findings (e.g., clinical chemistry, haematology, urinalysis) or other abnormal assessments (e.g., ECGs, X-rays, vital signs, etc.) that are judged by the investigator to be clinically significant will be recorded as AEs or SAEs if they meet the definition of an AE, as defined in Section 8.1 or SAE, as defined in Section 8.2. Clinically significant abnormal laboratory findings or other abnormal assessments that are detected during the study or are present at baseline and significantly worsen following the start of the study will be reported as AEs or SAEs. However, clinically significant abnormal laboratory findings or other abnormal assessments that are associated with the disease being studied, unless judged by the investigator as more severe than expected for the subject's condition, or that are present or detected at the start of the study and do not worsen, will not be reported as AEs or SAEs. 759346 The investigator will exercise his or her medical and scientific judgement in deciding whether an abnormal laboratory finding or other abnormal assessment is clinically significant.

Time period, frequency, and method of detecting adverse events and serious adverse events
All AEs occurring within one month (minimum 30 days) following administration of each dose of vaccine/ comparator must be recorded on the Adverse Event form in the subject's CRF, irrespective of severity or whether or not they are considered vaccinationrelated.
The standard time period for collecting and recording SAEs will begin at randomization or the first receipt of vaccine/ comparator and will end one month (minimum 30 days) following administration of the last dose of study vaccine/ comparator for each subject. See Section 8.8 for instructions for reporting and recording SAEs.
Additionally, in order to fulfil international reporting obligations, SAEs that are related to study participation (e.g. procedures, invasive tests, a change from existing therapy) or are related to a concurrent medication will be collected and recorded from the time the subject consents to participate in the study until she/he is discharged.

•
The investigator will inquire about the occurrence of AEs/SAEs at every visit/contact during the study.
All AEs either observed by the investigator or one of his clinical collaborators or reported by the subject's parent/guardian spontaneously or in response to a direct question will be evaluated by the investigator. AEs not previously documented in the study will be recorded in the Adverse Event form within the subject's CRF. The nature of each event, date and time (where appropriate) of onset, outcome, intensity and relationship to vaccination should be established. Details of any corrective treatment should be recorded on the appropriate page of the CRF. Refer to Section 6.8.
As a consistent method of soliciting AEs, the subject's parent/guardian should be asked a non-leading question such as: "Has your child acted differently or felt different in any way since receiving the vaccine or since the last visit?" N.B. The investigator should record only those AEs having occurred within the time frame defined above. 759346 N.B. If an AE changes in frequency or intensity during the specified reporting period, a new record of the event will be entered.
When an AE/SAE occurs, it is the responsibility of the investigator to review all documentation (e.g., hospital progress notes, laboratory, and diagnostics reports) relative to the event. The investigator will then record all relevant information regarding an AE/SAE on the CRF or SAE Report Form as applicable. It is not acceptable for the investigator to send photocopies of the subject's medical records to GSK Biologicals in lieu of the appropriate completed AE/SAE pages. However, there may be instances when copies of medical records for certain cases are requested by GSK Biologicals. In this instance, all subject identifiers will be blinded on the copies of the medical records prior to submission to GSK Biologicals.
The investigator will attempt to establish a diagnosis of the event based on signs, symptoms, and/or other clinical information. In such cases, the diagnosis should be documented as the AE/SAE and not the individual signs/symptoms.

There will be a retrospective follow-up of SAE(s) (i.e. occurrence of any SAE(s) since the last study visit [Visit 7] up to the time that subjects are at least 24 months of age).
(Amendment 7: 12 April 2006)

Local (injection site) adverse events
A four-day follow-up (day 0-3) of solicited local adverse events at each injection site will be performed after vaccination. Data concerning the following adverse events will be solicited using diary cards provided by the sponsor.

General (systemic) adverse events
A four-day follow-up (day 0-3) of solicited general adverse events will be performed after vaccination. Data concerning the following adverse events will be solicited using diary cards provided by the sponsor.

Evaluating adverse events and serious adverse events
Field workers will be trained on the definition of adverse events including SAEs as per protocol definition, under the supervision of the principal investigator.

Assessment of intensity
Intensity of the following AEs will be assessed as described: Crying more than usual/ no effect on normal activity 2 Crying more than usual/ interferes with normal activity 3 Crying that cannot be comforted/ prevents normal activity Drowsiness 0 Behavior as usual 1 Drowsiness easily tolerated 2 Drowsiness that interferes with normal activity 3 Drowsiness that prevents normal activity Loss of appetite 0 Normal 1 Eating less than usual/ no effect on normal activity 2 Eating less than usual/ interferes with normal activity 3 Not eating at all *Fever is defined as axillary temperature ≥ 37.5°C.
The maximum intensity of local injection site swelling will be scored at GSK Biologicals as follows: 0 : The maximum intensity of fever* will be scored at GSK Biologicals as follows: Axillary 0 : <37.5°C 1 : ≥37.5°C -≤38.5°C
For each solicited symptom, the subjects' parents/guardians will be asked if they sought medical advice (i.e., contact with a member of medical personnel) for this symptom.
The investigator will make an assessment of intensity for all other AEs, i.e. unsolicited symptoms, including SAEs reported during the study. The assessment will be based on the investigator's clinical judgement. The intensity of each AE and SAE recorded in the CRF or SAE Report Form, as applicable, should be assigned to one of the following categories: An AE that is assessed as Grade 3 (severe) should not be confused with a SAE. Grade 3 is a category utilized for rating the intensity of an event; and both AEs and SAEs can be assessed as Grade 3. An event is defined as 'serious' when it meets one of the pre-defined outcomes as described in Section 8.2.

Assessment of causality
The investigator is obligated to assess the relationship between investigational product and the occurrence of each AE/SAE. The investigator will use clinical judgement to determine the relationship. Alternative causes, such as natural history of the underlying diseases, concomitant therapy, other risk factors and the temporal relationship of the event to the investigational product will be considered and investigated. The investigator will also consult the Investigator Brochure and/or Product Information, for marketed products, in the determination of his/her assessment.
There may be situations when a SAE has occurred and the investigator has minimal information to include in the initial report to GSK Biologicals. However, it is very important that the investigator always makes an assessment of causality for every event prior to transmission of the SAE Report Form to GSK Biologicals. The investigator may change his/her opinion of causality in light of follow-up information, amending the SAE Report Form accordingly. The causality assessment is one of the criteria used when determining regulatory reporting requirements.
In case of concomitant administration of multiple vaccines, it may not be possible to determine the causal relationship of general AEs to the individual vaccines administered. The investigator should, therefore, assess whether the AE could be causally related to vaccination rather than to the individual vaccines.
All solicited local (injection site) reactions will be considered causally related to vaccination. Causality of all other AEs should be assessed by the investigator using the following question: YES : There is a reasonable possibility that the vaccine contributed to the AE.
Non-serious and serious AEs will be evaluated as two distinct events. If an event meets the criteria to be determined "serious" (see Section 8.2 for definition of serious adverse event), it will be examined by the investigator to the extent to be able to determine ALL contributing factors applicable to each serious adverse event.
Other possible contributors include: • Medical history • Other medication • Protocol required procedure • Other procedure not required by the protocol • Lack of efficacy of the vaccine • Erroneous administration • Other cause (specify)

Follow-up of adverse events and serious adverse events and assessment of outcome
After the initial AE/SAE report, the investigator is required to proactively follow each subject and provide further information to GSK Biologicals on the subject's condition.
All AEs and SAEs documented at a previous visit/contact and designated as not recovered/not resolved or recovering/resolving will be reviewed at subsequent visits/contacts.
Investigators will follow-up subjects: (1) with SAEs or subjects withdrawn from the study as a result of an AE, until the event has resolved, subsided, stabilized, disappeared, the event is otherwise explained, or the subject is lost to follow-up; 759346 (2) or, in the case of other non-serious AEs, until they complete the study or they are lost to follow-up.
Clinically significant laboratory abnormalities will be followed up until they have returned to normal, or a satisfactory explanation has been provided. Reports relative to the subsequent course of an AE noted for any subject must be submitted to the Study Monitor.
GSK Biologicals may request that the investigator perform or arrange for the conduct of supplemental measurements and/or evaluations to elucidate as fully as possible the nature and/or causality of the AE or SAE. The investigator is obliged to assist. If a subject dies during participation in the study or during a recognized follow-up period, GSK Biologicals will be provided with a copy of any available post-mortem findings, including histopathology. In the case of a death, supplementary information will be gained using the verbal autopsy technique. This will be conducted according to previously published methods and detailed in the SOPs on file with the investigators. The Verbal Autopsy Questionnaire will be completed and transmitted by the investigator to GSK Biologicals, WHO and the Local Safety Monitor, in addition to the SAE report, irrespective of relationship to vaccination and whether a written autopsy was performed or not. The Standard Verbal Autopsy Questionnaire will not replace a written autopsy.
New or updated information will be recorded on the originally completed SAE Report Form, with all changes signed and dated by the investigator. The updated SAE report form should be resent to GSK Biologicals within 24 hours of receipt of the follow-up information as outlined in Section 8.8.1.
All SAEs will be reported within 24 hours by the investigator to GSK Biologicals safety department, to the local safety monitor and to the WHO study contact for SAE reporting. GSK Biologicals will transmit the information to the IDMC according to the operating procedures of the IDMC.
In addition, the investigator will also provide a listing of all systemic grade 3 symptoms suspected to be related to vaccination and any case of diphtheria, tetanus, pertussis, hepatitis B, invasive diseases due to Haemophilus influenzae type b, measles, yellow fever or Neisseria meningitidis of serogroup A and C to the LSM. The Local Safety Monitor will send the listing and information related to the non-serious grade 3 systemic symptoms related with vaccination and non-serious adverse events suggesting a lack of efficacy of the vaccine to the IDMC and the GSK medical monitor (the GSK medical monitor will forward the information to the other members of the Steering Committee) according to the Charter for the IDMC. 759346

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

Regulatory reporting requirements for serious adverse events
The investigator will promptly report all SAEs to GSK Biologicals in accordance with the procedures detailed in Section 8.8. GSK Biologicals has a legal responsibility to promptly notify, as appropriate, both the local regulatory authority and other regulatory agencies about the safety of a product under clinical investigation. Prompt notification of SAEs by the investigator to the Study Contact for Reporting SAEs is essential so that legal obligations and ethical responsibilities towards the safety of other subjects are met.
The investigator, or responsible person according to local requirements, will comply with the applicable local regulatory requirements related to the reporting of SAEs to regulatory authorities and the IRB/IEC. Expedited Investigator Safety Reports (EISR) are prepared according to GSK Biologicals policy and are forwarded to investigators as necessary. An EISR is prepared for a SAE that is both attributable to the investigational product and unexpected. The purpose of the EISR is to fulfil specific regulatory and Good Clinical Practice (GCP) requirements, regarding the product under investigation.
An investigator who receives an EISR describing a SAE or other specific safety information from GSK Biologicals will file it with the Investigator Brochure and will notify the IRB/IEC, if appropriate according to local requirements.

Post study adverse events and serious adverse events
A post-study AE/SAE is defined as any event that occurs outside of the AE/SAE detection period defined in Section 8.5. Investigators are not obligated to actively seek AEs or SAEs in former study participants. 759346 However, if the investigator learns of any SAE, including a death, at any time after a subject has been discharged from the study, and he/she considers the event reasonably related to the investigational product, the investigator will promptly notify the Study Contact for Reporting SAEs.

Treatment of adverse events
Treatment of any adverse event is at the sole discretion of the investigator. Any AE will be treated for free according to good medical practice and standard of care in Ghana. Standard treatment of the common diseases in the study sites will be available in each facility (hospital, health centres) where the study will take place. Any medication administered for the treatment of an AE should be recorded in the subject's CRF. Refer to Section 6.8.

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

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

Subject withdrawal from the study
From an analysis perspective, a 'withdrawal' from the study is any subject who did not come back for the concluding visit foreseen in the protocol.
A subject qualifies as a 'withdrawal' from the study when no study procedure has occurred, no follow-up has been performed and no further information has been collected for this subject from the date of withdrawal/last contact. 759346 Investigators will make an attempt to contact those subjects who do not return for scheduled visits or follow-up.
Information relative to the withdrawal will be documented on the Study Conclusion page of the CRF. It will be specified which of the following possible reasons were responsible for withdrawal: • serious adverse event

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

There will be a retrospective follow-up of SAE(s) that occurred since the last study visit [Visit 7] up to the time that subjects are at least 24 months of age.
(

Screen and baseline failures
Not applicable.

Data Evaluation: Criteria for Evaluation of Objectives
Note: Data collected for the ancillary study will be provided to the investigators of the ancillary study for evaluation (refer to Appendix H and Section 5.2).

Local:
Pain and swelling at the injection site General: Drowsiness, Fever, Irritability/ Fussiness, Loss of appetite • Occurrence of unsolicited symptoms (during the 30-day follow-up period) Over the full course of the study: • Occurrence of serious adverse events

Estimated sample size
The target sample size is 280 enrolled subjects to reach 220 evaluable subjects in the ATP cohort for immunogenicity (110 evaluable subjects in each group). Refer to Section 10.4 for definition of study cohorts. Criteria to achieve the co-primary objectives: 1. Immunogenicity of SBA-MenA and SBA-MenC: lower limit of the 95% CI for the percentage of subjects with SBA-MenA titers (respectively SBA-MenC titers) ≥ 1:8 is greater than 80%. 2. Non-inferiority for all common antigens: upper limit of the 95% CI for the group difference in terms of the percentage of subjects with anti-PRP concentration ≥ 1 µg/ml (respectively, anti-diphtheria concentration ≥ 0.1 IU/ml, anti-tetanus concentration ≥ 0.1 IU/ml, anti-HBs concentration ≥ 10 mIU/ml) is below 10% and upper limit of the 95% CI for the group GMC ratio in anti-BP is below 1.5.
In order to cope with the multiplicity of the objectives, the criteria of the objectives will be assessed sequentially and will lead to a conclusion only if all previous primary criteria have been met (refer to Section 10.6.2). Table 10-1 and Table 10-2 present the order in which the criteria will be assessed (see Rank), the power to meet each criterion and the power to meet all previous criteria (see cumulative power). For instance, the power to meet the first primary objective and non-inferiority for anti-PRP will be 78.6% assuming that the 2 vaccine groups are identical with respect to the primary endpoints. 759346   *standard deviation of log10 (concentration) References: MenAC-Hib-001 for % of anti-PRP, Hib-065 for % of anti-tetanus, anti-diphtheria (by ELISA only) and anti-HBs, DTPw-HBV-033 for s.d. of log10 (concentration) for anti-BP.
It is acknowledged that the power to meet all primary objectives is low (at worst 49.0%). However, considering this is a Phase II feasibility study, the low power is deemed acceptable.

Study cohorts to be evaluated
A total of 6 cohorts are defined for the purpose of analysis.
• Three cohorts that will be used for the analysis of the primary vaccination course: ! the Total vaccinated cohort ! the ATP cohort for safety ! the ATP cohort for immunogenicity (used for the analysis of the co-primary objectives of immunogenicity) • Three cohorts that will be used for the analysis of the booster phase: ! the Total vaccinated cohort 759346

The Total vaccinated cohort (Primary vaccination)
The Total vaccinated cohort will include all subjects vaccinated in the primary phase of the study. For the Total analysis of safety, this will include all subjects vaccinated in the primary phase of the study with at least one vaccine administration documented in the primary phase. For the Total analysis of immunogenicity / efficacy, this will include subjects vaccinated in the primary phase of the study for whom data concerning immunogenicity/efficacy endpoint measures are available. The Total vaccinated cohort analysis will be performed per treatment actually administered.

According to protocol (ATP) cohort for safety (Primary vaccination)
The According to protocol (ATP) cohort for safety will include all subjects: • who have received at least one dose of study vaccine/control according to their random assignment, • with sufficient data to perform an analysis of safety, • for whom the administration route of study vaccine/control is known and correct, • who have not received a vaccine not specified or forbidden in the protocol, • who did not receive a replacement vaccine, during the primary vaccination course.

According to protocol (ATP) cohort for immunogenicity (Primary vaccination)
The According to protocol (ATP) cohort for immunogenicity will include all evaluable subjects (i.e. those meeting all eligibility criteria, complying with the procedures defined in the protocol, and with no elimination criteria during the study) from the ATP cohort for safety for whom assay results are available for antibodies against at least one study vaccine antigen component at Visit 4.

The Total vaccinated cohort (Booster vaccination)
The Total vaccinated cohort will include all subjects vaccinated in the booster phase of the study. For the Total analysis of safety, this will include all subjects vaccinated in the booster phase of the study with at least one vaccine administration documented in the booster phase. For the Total analysis of immunogenicity/efficacity, this will include subjects vaccinated in the booster phase of the study for whom data concerning immunogenicity / efficacy endpoint measures are available. The Total vaccinated cohort analysis will be performed per treatment actually administered. 759346

According To Protocol (ATP) cohort for safety (Booster vaccination)
The ATP cohort for safety will include all subjects from the ATP cohort (Primary vaccination) for safety • who have received the 3 doses in the primary vaccination course, • who have received the booster dose, • with documented safety follow-up, • for whom administration route of Mencevax ™ AC is known, • who have not received a vaccine not specified or forbidden in the protocol. This cohort will also be used for the analysis of persistence of the immune response.

According To Protocol (ATP) cohort for immune memory
The ATP cohort for immune memory will include all evaluable subjects (i.e., those meeting all eligibility criteria, complying with the procedures defined in the protocol, with no elimination criteria during the study) from the Booster ATP cohort for safety for whom assay results are available for antibodies against at least one study vaccine antigen component at Visit 7.

Derived and transformed data
Immunogenicity For a given subject cohort and the analysis of a given measurement, missing or unevaluable measurements will not be replaced. Therefore an analysis will exclude subjects with missing or unevaluable measurements.
• The cut-off value is defined by the laboratory before the analysis and is described in Section 5.5.2.
• A seronegative subject is a subject whose titer or concentration is below the cut-off value.
• A seropositive subject is a subject whose titer or concentration is greater than or equal to the cut-off value.
• Antibody concentrations or titers below the cut-off of the assay will be given an arbitrary value of half the cut-off for the purpose of GMC or GMT calculation. 759346

Other
Subjects who missed reporting symptoms (solicited or unsolicited or concomitant medications) will be treated as subjects without symptoms (solicited or unsolicited or concomitant medications, respectively).

Analysis of demographics/baseline characteristics
Demographic characteristics (age in weeks, gender, race) of each study cohort will be tabulated.
The mean age (plus range and standard deviation) by gender of the enrolled subjects, as a whole, and per group, will be calculated.
The distribution of subjects enrolled among the study sites will be tabulated as a whole and per group.

Analysis of Immunogenicity
The analysis of immunogenicity will be based on the ATP cohort for immunogenicity. If, for any vaccine group, the percentage of enrolled subjects with serological results excluded from this ATP cohort is more than 5%, a second analysis based on the Total vaccinated cohort will be performed to complement the ATP analysis.
Analysis of immunogenicity will be as follows:

Descriptive analysis
For each treatment group, one month after the third dose of the primary vaccination course, and in the mother prior to her child's vaccination, for each serological assay done: • Geometric Mean antibody Concentrations/Titers (GMCs/GMTs) with 95% confidence intervals (CIs) will be tabulated for each antibody. Calculation of the GMCs/GMTs is performed by taking the anti-log of the mean of the log concentration/titer transformations.
• Seropositivity / seroprotection rates with exact 95% CIs will be calculated. 759346 • Antibody concentrations/titers post-vaccination will also be investigated using reverse cumulative curves for each antigen and serogroup.

Inferential analysis
The inferential analysis will be done sequentially.
1. If the lower limit of the 95% CI for the percentage of subjects with SBA-MenC is above 80%, one will conclude that Tritanrix™-HepB/Hib-MenAC is immunogenic for MenC.
2. If, in addition, the lower limit of the 95% CI of subjects with SBA-MenA is above 80%, one will conclude that Tritanrix™-HepB/Hib-MenAC is immunogenic for MenA.
3. If, in addition, the upper limit of the 95% CI for the group difference for anti-PRP is below 10% one will conclude non-inferiority with respect to anti-PRP.
4. If, in addition, the upper limit of the 95% CI for the group difference for anti-Tetanus is below 10% one will conclude non-inferiority with respect to anti-Tetanus.

5.
If, in addition, the upper limit of the 95% CI for the group difference for anti-Diphtheria is below 10% one will conclude non-inferiority with respect to anti-Diphtheria.
6. If, in addition, the upper limit of the 95% CI for the anti-BP GMC ratio (control vaccine / new vaccine) is below 1.5 one will conclude non-inferiority with respect to anti-BP.
7. If, in addition, the upper limit of the 95% CI for the group difference for anti-HBs is below 10% one will conclude non-inferiority with respect to anti-HBs.

Analysis of Safety
The primary analysis will be based on the Total vaccinated cohort. If the percentage of enrolled subjects excluded from the ATP cohort for safety is more than 5%, a second analysis based on this ATP cohort will be performed to complement the Total vaccinated cohort analysis.
The percentage of subjects with at least one local adverse event (solicited and unsolicited), with at least one general adverse event (solicited and unsolicited) and with any adverse event during the 4-day solicited follow-up period will be tabulated with exact 759346 90 95% CI after each vaccine dose and overall. The percentage of doses followed by at least one local adverse event (solicited and unsolicited), by at least one general adverse event (solicited and unsolicited) and by any adverse event will be tabulated, overall vaccination course, with exact 95% CI.
The percentage of subjects reporting each individual solicited local and general adverse event during the 4-day solicited follow-up period will be tabulated with exact 95% CI. The percentage of doses followed by each individual solicited local and general adverse event will be tabulated, overall vaccination course, with exact 95% CI.
The same tabulation will be performed for grade 3 adverse events and for adverse events with relationship to vaccination.
The proportion of subjects with at least one report of unsolicited adverse event classified by the Medical Dictionary for Regulatory Activities (MedDRA), and reported up to 30 days after vaccination will be tabulated with exact 95% CI. The same tabulation will be performed for grade 3 unsolicited adverse events and for unsolicited adverse events with a relationship to vaccination.
Serious adverse events and withdrawal due to adverse event(s) will be described in detail.

Analysis of immunogenicity
The analysis of antibody persistence and the analysis of immune memory will be respectively based on the ATP cohort for safety and the ATP cohort for immune memory. If, for any vaccine group, the percentage of enrolled subjects with serological results excluded from an ATP cohort is more than 5%, a second analysis based on the Total vaccinated cohort will be performed to complement the ATP analysis.
The analysis of immunogenicity will be as follows:

Analysis of antibody persistence
For each treatment group, one month after the third dose of the primary vaccination course and just before the administration of one fifth of a dose of Mencevax ™ AC: • Geometric Mean antibody Concentrations/Titers (GMCs/GMTs) with 95% confidence intervals (CIs) will be tabulated for each antibody. Calculation of the 759346 GMCs/GMTs is performed by taking the anti-log of the mean of the log concentration/titer transformations.
• Antibody concentrations/titers pre-booster will also be investigated using reverse cumulative curves for each antigen and serotype.

Analysis of immune memory
For each treatment group, just before and one month after the administration of one fifth of a dose of Mencevax ™ AC: • Geometric Mean antibody Concentrations/Titers (GMCs/GMTs) with 95% confidence intervals (CIs) will be tabulated for each antibody. Calculation of the GMCs/GMTs is performed by taking the anti-log of the mean of the log concentration/titer transformations.
• Antibody concentrations/titers post-booster will also be investigated using reverse cumulative curves for each antigen and serotype.
The ratio of the GMCs/GMTs between the Tritanrix TM -HepB/Hib-MenAC group and the Tritanrix TM -HepB/Hiberix TM group one month after the administration of one fifth of a dose of Mencevax ™ AC, will be computed with its 95% CI. The ratio and its 95% CI will be computed using an ANOVA model on the log-transformed concentration/titer with group effect as regressor.

Analysis of safety
All analyses will be based on the Total vaccinated cohort. If the percentage of enrolled subjects excluded from the ATP cohort for safety is more than 5%, a second analysis based on this ATP cohort will be performed to complement the Total vaccinated cohort analysis.
The percentage of subjects with at least one local adverse event (solicited and unsolicited), with at least one general adverse event (solicited and unsolicited) and with any adverse event during the 4-day solicited follow-up period will be tabulated with exact 95% CI after each vaccine dose and overall. 759346 The percentage of subjects reporting each individual solicited local and general adverse event during the 4-day solicited follow-up period will be tabulated with exact 95% CI.
The same tabulation will be performed for grade 3 adverse events and for adverse events with relationship to vaccination.
The proportion of subjects with at least one report of unsolicited adverse event classified by the Medical Dictionary for Regulatory Activities (MedDRA), and reported up to 30 days after vaccination will be tabulated with exact 95% CI. The same tabulation will be performed for grade 3 unsolicited adverse events and for unsolicited adverse events with a relationship to vaccination.
Serious adverse events and withdrawal due to adverse event(s) will be described in detail.

Reporting of final analyses
A final analysis for the primary vaccination course will be carried out as soon as all data up to Visit 4 have been collected in order to analyse the immunogenicity endpoints (including the co-primary endpoints) and the analysis of safety/reactogenicity for the primary vaccination course (refer to Section 10.6 for details about this analysis). A report based on this analysis will be written and will be made available to the Principal Investigator and the sponsors involved in this study. The access to the individual treatment code will be limited to the statistician in charge of this analysis.
A final analysis for the booster vaccination will be carried out based on all data collected up to one month after the administration of one fifth of a dose of Mencevax™ AC. An annex report based on the final analysis for the booster vaccination will be provided.

Planned interim analysis
No interim analysis is planned. 759346

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

INTRODUCTION
It is the mission of the physician to safeguard the health of the people. His or her knowledge and conscience are dedicated to the fulfilment of this mission.
The Declaration of Geneva of the World Medical Association binds the physician with the words, "The health of my patient will be my first consideration," and the International Code of Medical Ethics declares that, "A physician shall act only in the patient's interest when providing medical care which might have the effect of weakening the physical and mental condition of the patient." The purpose of biomedical research involving human subjects must be to improve diagnostic, therapeutic and prophylactic procedures and the understanding of the etiology and pathogenesis of disease.
In current medical practice most diagnostic, therapeutic or prophylactic procedures involve hazards. This applies especially to biomedical research.
Medical progress is based on research which ultimately must rest in part on experimentation involving human subjects. 759346 In the field of biomedical research a fundamental distinction must be recognized between medical research in which the aim is essentially diagnostic or therapeutic for a patient, and medical research, the essential object of which is purely scientific and without implying direct diagnostic or therapeutic value to the person subjected to the research.
Special caution must be exercised in the conduct of research which may affect the environment, and the welfare of animals used for research must be respected.
Because it is essential that the results of laboratory experiments be applied to human beings to further scientific knowledge and to help suffering humanity, the World Medical Association has prepared the following recommendations as a guide to every physician in biomedical research involving human subjects. They should be kept under review in the future. It must be stressed that the standards as drafted are only a guide to physicians all over the world. Physicians are not relieved from criminal, civil and ethical responsibilities under the laws of their own countries.

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

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

III. NON-THERAPEUTIC BIOMEDICAL RESEARCH INVOLVING HUMAN SUBJECTS (Non-clinical biomedical research)
1. In the purely scientific application of medical research carried out on a human being, it is the duty of the physician to remain the protector of the life and health of that person on whom biomedical research is being carried out.
2. The subjects should be volunteers -either healthy persons or patients for whom the experimental design is not related to the patient's illness.
3. The investigator or the investigating team should discontinue the research if in his/her or their judgement it may, if continued, be harmful to the individual.

4.
In research on man, the interest of science and society should never take precedence over considerations related to the well being of the subject. 759346 To ensure that he/she has sufficient time to conduct and complete the study and has adequate staff and appropriate facilities and equipment which are available for the duration of the study and to ensure that other studies do not divert essential subjects or facilities away from the study at hand.

•
To submit an up-to-date curriculum vitae and other credentials (e.g., medical license number in the United States) to GSK Biologicals and-where required-to relevant authorities.
• To acquire the normal ranges for laboratory tests performed locally and, if required by local regulations, obtain the Laboratory License or Certification.
• To ensure that no clinical samples (including serum samples) are retained on site or elsewhere without the approval of GSK Biologicals and the express written informed consent of the subject and/or the subject's legally authorized representative.
• To perform no other biological assays at the investigator site except those described in the protocol or its amendment(s).

•
To prepare and maintain adequate case histories designed to record observations and other data pertinent to the study.
• To conduct the study in compliance with the protocol and appendices.
• To co-operate with a representative of GSK Biologicals in the monitoring process of the study and in resolution of queries about the data.

II. Protocol Amendments and Modifications
• No changes to the study protocol will be allowed unless discussed in detail with GSK Biologicals' Clinical Development Manager/Medical Monitor and filed as an amendment/modification to this protocol.
• Any amendment/modification to the protocol will be adhered to by the participating center(s) and will apply to all subjects. Written IRB/IEC approval of protocol amendments is required prior to implementation; modifications are submitted to IRBs/IECs for information only. 759346

III.
Sponsor's Termination of Study GSK Biologicals reserves the right to discontinue the clinical study at any time for medical or administrative reasons. When feasible, a 30-day written notification will be tendered.

IV. Case Report Form Instructions
Prior to screening the first potential participant, the investigator will provide the Site Monitor with a list (Site Staff Signature Sheet) showing the signature and hand-written initials of all individuals authorized to make or change entries on CRFs (already defined). If the authorized individuals should change during the study, the investigator is to inform GSK Biologicals of the specific change(s).
CRFs (and subject diary cards, if applicable), will be supplied by GSK Biologicals for recording all data. It is the responsibility of the investigator or co-investigator to ensure that CRFs (and subject diary cards) are legible and completely filled in with a black ink fountain or ballpoint pen.
Errors must be corrected by drawing a single line through the incorrect entry and writing in the new value/data positioned as close to the original as possible. The correction must then be initialled, dated and justified, where necessary, by the authorized individual making the change. The original entry must not be obliterated, overwritten or erased when a correction is made.
When a subject completes a visit, it is anticipated that relevant sections of the CRF will be completed by the investigator (or designated staff as documented in the Site Staff Signature Sheet) as soon as possible after the last data becoming available. Similarly, when a subject completes a study, it is anticipated that all relevant CRF pages will be completed promptly after the last data becoming available. This also applies to forms for potential study participants who were screened but not randomized to a study group.
As soon as the subject has completed/withdrawn from the study and the CRF is completed, the principal investigator or designated physician(s) under his/her supervision will sign the study conclusion pages of the CRF to confirm that they have reviewed the data and that the data are complete and accurate. In all cases the investigator remains accountable for the study data collected.
An original (top copy) CRF or log sheets must be submitted for all subjects who have undergone protocol specific procedures, whether or not the subject completed the study.
While completed CRFs are reviewed by a GSK Biologicals professional monitor at the study site, errors detected by subsequent in-house CRF review may necessitate 759346 clarification or correction of errors with documentation and approval by the investigator or appropriately qualified staff as documented on the Site Staff Signature Sheet. In all cases, the investigator remains accountable for the study data. Wherever possible the investigator should assist in the clarification or correction of errors detected after study finalization promptly after being brought to the attention of the investigator (preferably within 48 hours).
Any questions or comments related to the CRF should be directed to the assigned Site Monitor.

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

Monitoring by WHO
Independent WHO monitors will visit the study site to monitor procedures/data.

VI.
Archiving of Data A study report will be written after the statistical analysis. This report, including all individual data, will be given to the sponsors of the study and to the principal investigator.
Following closure of the study, the investigator must maintain all site study records in a safe and secure location. The records must be maintained to allow easy and timely retrieval, when needed (e.g., audit or inspection), and, whenever feasible, to allow any subsequent review of data in conjunction with assessment of the facility, supporting systems, and staff. Where permitted by applicable laws/regulations or institutional policy, some or all of these records can be maintained in a validated format other than 759346 hard copy (e.g., microfiche, scanned, electronic for studies with an eCRF, for example); however, caution needs to be exercised before such action is taken. The investigator must assure that all reproductions are legible and are a true and accurate copy of the original, and meet accessibility and retrieval standards, including re-generating a hard copy, if required. Furthermore, the investigator must ensure there is an acceptable back-up of these reproductions and that an acceptable quality control process exists for making these reproductions.
GSK will inform the investigator/ institution of the time period for retaining these records to comply with all applicable regulatory requirements. However, the investigator/ institution should seek the written approval of the sponsors before proceeding with the disposal of these records. The minimum retention time will meet the strictest standard applicable to that site for the study, as dictated by ICH GCP E6 Section 4.9, any institutional requirements or applicable laws or regulations, or GSK Biologicals standards/procedures; otherwise, the minimum retention period will default to 15 years.
The investigator/ institution must notify GSK Biologicals of any changes in the archival arrangements, including, but not limited to, the following: archival at an off-site facility, transfer of ownership of the records in the event the investigator leaves the site.

VII. Audits
For the purpose of compliance with Good Clinical Practice and Regulatory Agency Guidelines it may be necessary for GSK Biologicals or a Drug Regulatory Agency to conduct a site audit. This may occur at any time from start to after conclusion of the study.
When an investigator signs the protocol, he/she agrees to permit drug regulatory agencies and GSK Biologicals audits, providing direct access to source data/ documents. Furthermore, if an investigator refuses an inspection, his/her data will not be accepted in support of a New Drug Registration and/or Application, Biologics Licensing Application.

GSK Biologicals has made and is making a substantial investment in clinical studies.
Having the highest quality data and studies are essential aspects of vaccine development. GSK Biologicals has a Regulatory Compliance staff who audit investigational sites. Regulatory Compliance assesses the quality of data with regard to accuracy, adequacy and consistency. In addition, Regulatory Compliance assures that GSK Biologicals sponsored studies are in accordance with GCP and that relevant regulations/guidelines are being followed.
To accomplish these functions, Regulatory Compliance selects investigational sites to audit. These audits usually take 1 to 2 days. The GSK Biologicals' audits entail review of 759346 source documents supporting the adequacy and accuracy of CRFs, review of documentation required to be maintained, and checks on vaccine accountability. The GSK Biologicals' audit therefore helps prepare an investigator for a possible regulatory agency inspection as well as assuring GSK Biologicals of the validity of the database across investigational sites.
The Inspector will be especially interested in the following items:

VIII. Confidentiality and Publication
You, the investigator, agree that all information communicated to you by GSK Biologicals is the exclusive property of GSK Biologicals.You agree to keep such information strictly confidential and to restrict access to such information to the minimum number of persons connected with the work performed under this protocol on a need-toknow basis and you will ensure that such persons shall be made aware that the information is confidential and shall be bound by confidentiality obligations at least as strict as those contained herein. You will be responsible for any breach of the confidentiality by any of those persons to whom such information has been furnished. You and any such person agree that said information shall not be disclosed, either orally or in written form, by you or such person to any third party without the prior written consent of GSK Biologicals. You shall communicate the results of the work promptly to GSK Biologicals. Such results shall be the property of GSK Biologicals and shall be subject to the confidentiality obligations contained herein except that you shall be authorised to publish the results pursuant to the provisions below.
The results of the study covered by this protocol shall be the subject of a joint publication by the sponsors and you within six (6) months from the conclusion of the study and availability of a final study report. You and the sponsors will be expected to treat matters of authorship in a proper collaborative spirit, giving credit where it is due and proceeding in a manner that fosters cooperation and communication, but will not do anything in this regard that will jeopardize the issuance of a valid patent.

Collection
The whole blood (by capillary or venous route) should be collected observing appropriate aseptic conditions. It is recommended that Vacutainer tubes WITH integrated serum separator (e.g. Becton-Dickinson Vacutainer SST or Corvac Sherwood Medical) be used to minimize the risk of haemolysis and to avoid blood cell contamination of the serum when transferring to standard serum tubes.

Serum separation
These guidelines aim to ensure high quality serum by minimizing the risk of haemolysis, blood cell contamination of the serum or serum adverse cell toxicity at testing.
• For separation of serum using Vacutainer® tubes, the instructions provided by the manufacturer should be followed. Siliconized tubes should never be used (cell toxicity). Often the manufacturer's instruction states that the relative centrifugal acceleration known also as "G" must be "between 1000 and 1300 G" with tubes spinning for ten minutes. Error in calculation of centrifuge speed can occur when laboratory personnel confuse "G" acceleration with "RPM" (revolutions per minute). The speed of centrifugation must be calculated using the "G" rate provided in the manufacturer's instructions and the radius of the centrifuge head. After measuring the radius of the centrifuge machine, a speed/acceleration nomograph must be employed to determine the centrifuge speed in "RPM".

•
Following separation, the serum should be aseptically transferred to the appropriate standard tubes using a sterile disposable pipette. The serum should be transferred as gently as possible to avoid blood cell contamination.

•
The tube should not be overfilled (max. 3/4 of the total volume) to allow room for expansion upon freezing.

Labelling
• The standard labels provided by GSK Biologicals should be used to label each serum sample.
• If necessary, any hand-written additions to the labels should be made using indelible ink.
• The label should be attached to the tube as follows (see diagram): − first attach the paper part of the label to the tube − then wrap the label around the tube so that the transparent, plastic part of the label overlaps with the label text and bar code and shields them. This will ensure optimal label attachment.

Sorting and storage
• Tubes should be placed in the GSK Biologicals' carton in numerical order from left to right, starting from the lower left hand corner, beginning with the pre-vaccination samples series, then with the post-vaccination sample series.
• The tubes of serum should be stored in a vertical position at -20°C (alternatively at -70°/80°C is also acceptable) until shipment to GSK Biologicals. Wherever possible, a backup facility for storage of serum samples should be available. 759346 Serum samples should always be sent by air, preferably on a Monday, Tuesday or Wednesday, unless otherwise requested by the sponsor.
Serum samples must be placed with dry ice (-20°C) in a container complying with International Air Transport Association (IATA) requirements. The completed standard serum listing form should always accompany the shipment.
The container must be clearly identified with the labels provided by GSK Biologicals specifying the shipment address and the storage temperature (-20°C).
The airway bill should contain the instruction for storage of samples at -20°C.
A "proforma" invoice, stating a value for customs purposes only, should be prepared and attached to the container. This document should contain the instruction for storage of samples at -20°C.
Details of the shipment, including: * number of samples * airway bill * flight number * flight departure and arrival times should be sent by fax, two days before shipment, to: doses should match. All used vials should be placed in sealed envelopes by the vaccination team in order to maintain the blinding of the study. After the end of the study and unblinding, it must be possible to reconcile delivery records with those of used and unused stocks. An explanation must be given of any discrepancies.
After approval from GSK Biologicals, used vaccine vials should be destroyed at the study site using locally approved biosafety procedures and documentation unless otherwise described in the protocol. If no adequate biosafety procedures are available at the study site, the used vaccine vials are to be returned to an appropriate GSK Biologicals site for destruction in accordance with current GSK SOP NPD-112. Unused vaccine vials will be disposed at the local GSK Biologicals site in accordance with GSK SOP NPD-112. If no processes for destruction of unused vaccines are in place in the local GSK Biologicals site; the unused vials must be returned to GSK Biologicals in Rixensart, Belgium.

4.
Transfers of clinical vaccines or products from country medical department or dispatch center to study sites or between sites Storage temperatures must be maintained during transport and deviations must be reported to Logistics and Packaging for guidance. All transfers of clinical vaccines or products must be documented using the Clinical Supply Transfer Form. If the duration of the transfer is less than four hours, a transportable fridge or any suitable container (e.g. styrofoam container) with a maximum of eight refrigerated cold packs (cooling elements) must be used in order to maintain the vaccines at 2°-8°C during transport. If the duration is more than four hours, a transportable fridge or any suitable container (e.g. styrofoam container) with a minimum of eight cold packs (cooling elements) must be used as well as a temperature monitoring system that must be placed as close as possible to the doses and checked upon reception at the final destination. Never place frozen cold packs or dry ice inside vaccine/product boxes for vaccine that must be kept at +4°C in order to avoid cold-chain deviation (e.g. frozen vaccines). Exceptions to these instructions are detailed in product-specific transport guidelines.

Labels for sample identification
The investigator will receive labels from GSK Biologicals to identify samples taken from each subject at each timepoint. Each label will contain the following information: study number, subject number, sampling timepoint (e.g., post vacc 3), timing (e.g., study Month 3).

Other supplies provided by GSK Biologicals
In addition to the vaccines, the study documentation and the sample labels, the investigator will receive the following supplies:

1.
Purpose of the ancillary study Aim of the ancillary colonization study is to investigate how frequently and for how long infants <1 year are colonized by N. meningitidis and N. lactamica in the study area. This will provide important baseline data allowing sample size calculations in later studies accompanying the introduction of a meningococcal conjugate vaccine and addressing the issue of potential serogroup replacement by mass vaccination. The proposed ancillary study will also address the question, whether colonization by A meningococci is reduced following meningococcal conjugate vaccination.

2.
Background African countries, and more specifically the countries of the meningitis belt, face regular epidemics of meningococcal disease, including meningitis with an incidence as high as 1000/100,000. Except for an outbreak of serogroup W-135 that spread in Burkina Faso in 2002, serogroup A and to a lesser extent serogroup C are largely responsible for N. meningitidis epidemics in African countries (1). Children aged 5-14 years are the most 759346 affected. The existing meningococcal plain polysaccharide vaccines are less immunogenic in small children; also the duration of protection induced by these vaccines is short-lived, as polysaccharides vaccine do not induce immune memory. Therefore, there is a need to develop new generation meningococcal vaccines that protect against meningococcal disease in early childhood and induce immune memory (2,3). Recently, meningococcal serogroup C conjugate vaccines have been developed and have proven effective in small children and infants (4).
In order to protect children from early childhood against N. meningitidis of the two most prevalent serogroups in Africa, and to facilitate the incorporation of such a vaccine in the immunization calendar, GSK Biologicals is developing a combined Hib-MenAC conjugate vaccine to be extemporaneously mixed with the Tritanrix™-HepB (DTPw-HBV) vaccine for administration as a 3-dose primary vaccination course in infants aged 6 weeks and over. The new Tritanrix ™ -HepB/Hib-MenAC vaccine will first be evaluated in a feasibility trial outside Africa. Once feasibility of the new vaccine is shown, the obove mentioned core study plans to evaluate the use of GSK Biologicals' Tritanrix ™ -HepB/Hib-MenAC vaccine in a high endemicity region in Africa.
In 1998 an ongoing longitudinal study of the dynamics of carriage of Neisseria meningitidis and N. lactamica was started by STI and NHRC in the Kassena-Nankana District of Northern Ghana (5-9). In 6-monthly field surveys (once in the dry season and once in the wet season of each year) 37 compounds randomly selected from a complete listing of the district population (Navrongo Demographic Surveillance System) are visited and Neisseria carriage of the total population of these compounds is monitored. The studies have provided information on longitudinal patterns of colonisation with multiple types of N. meningitidis and N. lactamica, and spatio-temporal patterns of colonisation and disease during and before a potential new epidemic. The data indicate that in contrast to Europe and the USA, carriage of non-encapsulated meningococci and other nonpathogenic meningococci is infrequent in Africa. It is thought that carriage of nonserogroupable meningococci protects against meningococcal disease by eliciting crossreactive immunity against pathogenic strains. Therefore, in Africa, low levels of carriage may result in inadequate levels of naturally acquired immunity and thus an increased susceptibility of the population to meningococcal epidemics.
After a N. meningitidis serogroup A epidemic in the Kassena-Nankana district in 1997 serogroup A carriage decreased and for a period of about two years serogroup A meningococci were neither isolated from cases nor from carriers. However, after a period of only four years, a new serogroup A outbreak is now being observed, indicating that the decline in herd immunity may not be the crucial factor determining time intervals between epidemics. Molecular typing revealed that the current outbreak is caused by a different serogroup A clone (sequence type 7, a clone which was for the first time observed in 1995 in Africa) than the previous epidemic (sequence type 5, a clone which 759346 Mecca (1987) and subsequently in several nations of the meningitis belt). Between the two outbreaks, a carriage epidemic of serogroup X meningococci was observed, coinciding with a number of cases caused by serogroup X meningococci. Currently it is not clear whether X meningococci have an epidemic potential (like W-135 meningococci, which are the cause of a recent epidemic in Burkina Faso associated with an unusually high mortality). The recent increase in outbreaks caused by non-A non-C meningococci may reflect serogroup replacements caused by mass vaccination with serogroup A/C carbohydrate vaccine. Similarly, shifts in the serogroups of N. meningitidis causing disease could potentially arise as a result of immune selection resulting from mass-vaccination with conjugate vaccines. In view of recent outbreaks caused by W-135-and X meningococci this possibility is a real concern. Therefore, the database on longitudinal patterns of colonisation with multiple types of N. meningitidis, and the spatio-temporal patterns of colonisation and disease provides a unique baseline from which to carry out trials on the effects of vaccination on acquisition of meningococcal infection and on serogroup replacement. Number, gender, age, and race of subjects Target enrolment of the core vaccine study will be 280 healthy male and female infants (140 subjects per group) to provide 220 evaluable subjects (110 per group) for the core immunogenicity analysis.
For the ancillary colonization study, throat swabbing of all infants and their consenting parents/guardians will be done. Samples will be taken from all children enrolled in the core study.
The study will be conducted by the Navrongo Health Research Center in Ghana at the War Memorial Hospital (serving Navrongo central) and at other health centres in the Kassena-Nankana health district.
Enrolment will be terminated when 280 infants have been enrolled for the core vaccine study.
Currently there is hardly any information available on the frequency and duration of meningococcal colonization of infants <1 year in the study area. Since there is concern that mass vaccination with a meningococcal conjugate vaccine might result in serogroup replacement it is important to investigate this aspect in the target group of an EPIassociated meningococcal conjugate vaccine. The ancillary study is expected to give first insight, thus providing important baseline data allowing sample size calculations in later studies accompanying the introduction of a meningococcal conjugate vaccine. Our long term colonization studies indicate that meningococci spread very fast within individual compounds. A colonization analysis of parents or guardians will provide insight into the frequency of this spread to infants.
The intended racial and ethnic distribution is determined by the fact that the study should be performed in a population at high risk for epidemic meningococcal meningitis; i.e inhabitants of the African meningitis belt.

2.
Inclusion criteria All subjects must satisfy the following criteria at study entry: 759346 • Subjects for whom the investigator believes that their parents/guardians can and will comply with the requirements of the protocol (e.g., completion of the diary cards, return for follow-up visits) should be enrolled in the study.
• A male or female between, and including, 6 and 8 weeks of age at the time of the first vaccination.
• Written informed consent obtained from the parent or guardian of the subject for the participation of the subject and of the parent/guardian. • Free of obvious health problems as established by medical history and clinical examination before entering into the study.

3.
Exclusion criteria for enrolment The following criteria should be checked at the time of study entry. If any apply, the subject must not be included in the study: • Use of any investigational or non-registered drug or vaccine other than the study vaccine(s) within 30 days preceding the first dose of study vaccine, or planned use during the study period.
• Chronic administration (defined as more than 14 days) of immunosuppressants or other immune-modifying drugs since birth. (For corticosteroids, this will mean prednisone, or equivalent, ≥ 0.5 mg/kg/day. Inhaled and topical steroids are allowed.) • Planned administration/ administration of a vaccine not foreseen by the study protocol within 30 days before each dose of vaccine, with the exception of OPV.
• Hepatitis B and BCG vaccine given within two weeks prior to vaccination.
• Previous vaccination against diphtheria, tetanus, pertussis, Haemophilus influenzae type b or meningococcal serogroup A or C diseases.
• History of diphtheria, tetanus, pertussis, hepatitis B, Haemophilus influenzae type b or meningococcal serogroup A or C diseases. • Babies for which birth weight is <2 kg (if known), and/or malnutrition at visit 1.
• History of any neurologic disorders or seizures. 759346 • Acute disease at the time of enrolment. (Acute disease is defined as the presence of a moderate or severe illness with or without fever. All vaccines can be administered to persons with a minor illness such as diarrhea, mild upper respiratory infection with or without low-grade febrile illness, i.e., axillary temperature <37.5°C or rectal temperature <38.0°C). Note: Subjects with fever at the time of first vaccination (i.e., axillary temperature ≥ 37.5°C or rectal temperature ≥ 38.0°C) need not necessarily be excluded but can be vaccinated at a later date after the subject recovers and if the subject still meets all eligibility criteria.
• Administration of immunoglobulins and/or any blood products since birth or planned administration during the study period.
• Other conditions which in the opinion of the investigator may potentially interfere with interpretation of study outcomes.

4.
Elimination criteria during the study The following criteria should be checked at each visit subsequent to the first visit. If any become applicable during the study, it will not require withdrawal of the subject from the study but does determine a subject's evaluability in the according-to-protocol (ATP) analysis.
• Use of any investigational or non-registered drug or vaccine other than the study vaccine(s) during the study period.
• Chronic administration (defined as more than 14 days) of immunosuppressants or other immune-modifying drugs during the study period. (For corticosteroids, this will mean prednisone, or equivalent, ≥ 0.5 mg/kg/day. Inhaled and topical steroids are allowed).
• Administration of a vaccine not foreseen by the study protocol during the period starting from 30 days before the first dose of vaccine(s) and ending 30 days after the last dose of vaccine(s) with the exception of OPV.
• Administration of a meningococcal vaccine not foreseen by the study protocol during the entire study period.
• Administration of immunoglobulins and/or any blood products during the study period.

5.
Withdrawal of subject from core study If a study subject is withdrawn from the core study, that subject will also be withdrawn from the ancillary study.

III
METHODS AND PROCEDURES 1.
List of study procedures (core study) 759346

Study Conclusion
• • denotes a procedure which must be recorded in the individual CRF. ° denotes a procedure which need not be recorded in the individual CRF. # any parent/guardian accompanying the child at visits subsequent to the first visit who has not already signed an Informed Consent for this study will have to sign an Informed Consent prior to providing any throat swab .## Contraindications for measles and yellow fever vaccines. ** from the subject and subject's parents/guardians; ***Measles, yellow fever vaccine ****1/5 th of dose of Mencevax™ AC Note: BCG and OPV vaccines are given at birth (or at least 2 weeks before the subject's first visit) according to the nationally recommended immunization schedule. OPV vaccine can be administered to the subjects at 6, 10 and 14 weeks of age concomitantly with the study vaccines or at any time during the study, whichever is more appropriate.

2.
Methods and procedures specific for the ancillary study Throat swabbing of consenting parents/guardians and infant subjects will also be done at age 6w, 10w, 18w, 9m and 12m.
This non-invasive procedure is done using a sterile swab on stick. After consent is obtained from the subject (or his/her parent/guardian), the subject is asked to open the mouth as wide as possible and bend the head slightly backward. Children are assisted to bend their heads by an assistant. A trained investigator wearing disposable gloves depresses the tongue with a spatula in one hand. The subject is asked to say ah-ah (if an adult). The investigator picks the sterile swab with the other hand and passes it through the mouth gently rubs it against the posterior wall of the pharynx or behind the tonsils without touching the uvula and tongue. The swab stick is removed from the mouth and immediately inoculated directly on Thayer-Martin agar plates (Difco), which is labeled, and transported to the hospital laboratory within 2 hours for incubation in a candle jar at 37ºC for 24 hours.
Neisseria-like colonies will be picked and characterized by standard microbiological procedures in Navrongo. Isolates are then frozen and transported to Switzerland for detailed molecular characterization. N. meningitidis or N. lactamica are identified by Gram's stain morphology, cytochrome oxidase test using N,N,N',N'-tetramethyl-1,4phenylene-diammoniumdichloride as substrate, glucose, maltose and sucrose utilization in cystine-trypticase agar, ONPG discs and gamma-glutamyltransferase activity. All isolated meningococci will be serogrouped by slide agglutination with serogroup specific antisera and serotyped/subtyped with monoclonal antibodies by whole cell ELISA. Further analysis is carried out using pulsed-field gel electrophoresis and for a sub-set of isolates representing the different PFGE variants multi locus sequence typing. N. meningitidis isolates are tested for sensitivity to penicillin G, cefotaxime, ciprofloxacin, rifampicin, chloramphenicol, tetracycline and sulfadiazine by the E-test method (AB Biodiscs, Solna, Sweden) according to the manufacturer's instructions.

3.
Data analysis and data monitoring 3.1 Safety monitoring An independent committee consisting of up to five experts in meningococcal diseases, paediatrics, vaccines, statistics and other appropriate disciplines will be appointed to oversee ethical and safety aspects of the study conduct.
Terms of reference (operating procedures) of the IDMC will be written after the first meeting of the IDMC members, prior to study start.

Role of the IDMC
The role of the IDMC 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 IDMC will confer before the initiation of the study (pre-initiation review), during the study proper and at the close out of the study to review any relevant safety data and to review and approve the Report and Analysis Plan (RAP), where applicable. Other unscheduled meetings may be required. Meetings must be documented and minutes made available for the study files on site and to the sponsors. The IDMC may, if deemed necessary, convene a meeting with or request further information from the Principal Investigators, the Local Safety Monitor, GSK Biologicals and designated project representatives at any stage of the study.
The IDMC must be informed by the investigator of: • all subsequent protocol amendments, informed consent changes or revisions of other documents originally submitted for review • systemic grade 3 adverse events (AEs) related to vaccination, serious adverse events and adverse events suggesting a lack of efficacy of the vaccine occurring during the study (transmitted through GSK Biologicals for SAEs and through the Local safety Monitor for the other adverse events) 759346 • all subsequent protocol modifications (for information) • new information that may affect adversely the safety of the subjects or the conduct of the study.
The IDMC will be empowered to put the study on hold pending review of potential safety issues. All SAEs, including death, will be reported by the Principal Investigator to the local safety monitor, to the Manager of Clinical Safety Vaccines at GSK Biologicals, to WHO and to the WHO safety monitor. GSK Biologicals will be responsible to provide all information related to the SAEs to the IDMC according to the IDMC Charter.

3.3
Reporting to IDMC -Role of Local Safety Monitor

A Local Safety Monitor (LSM) will act independently from the investigational team.
The overall role of the Local Safety Monitor (LSM), who will be an experienced clinician based in-country, will be to support the clinical investigators and to act as a link between the investigators and the IDMC. All systemic grade 3 AEs probably or suspected to be related to vaccination, all SAEs and all AEs suggesting a lack of efficacy of the vaccine will be reported to him/her. His/her involvement will be particularly important when decisions have to be made quickly. In exceptional circumstances, for example a death possibly related to vaccination, he/she will have the authority to suspend the whole or any specific aspect of the trial pending discussion with the IDMC. The LSM's role will include: • acting as the study volunteer's advocate • provide the IDMC and the medical monitor of GSK Biologicals with a blinded listing of non-serious systemic grade 3 symptoms (solicited and unsolicited) that are related with vaccination, and of non-serious adverse events suggesting a lack of efficacy of the vaccine, this according to the IDMC Charter (note: GSK Biologicals will send all information related to serious adverse events to the IDMC according to the IDMC Charter). • reviewing blinded reactogenicity data according to IDMC charter after each dose and discussing with IDMC if necessary • promptly communicating relevant safety information to the IDMC • providing advice to the investigators on whether a set of clinical circumstances in a study warrants formal notification to the IDMC • providing clinical advice on any illness in study subjects especially in circumstances in which treatment might influence the course of the trial • Unblinding a subject if deemed necessary, upon request of the investigator/physician in charge of the subject, to allow for adequate treatment.
The LSM will liaise closely with the chair of the IDMC throughout the course of the trial.

3.4
Data analysis for ancillary study 759346 The ancillary study represents a pilot exploratory analyses that will provide important baseline data for later more extended studies. Only rough estimates can therefore be made at this stage about outcomes and types of statistical data analyses required.
It is expected that the proposed sampling of 280 infants at age 6w, 10w, 18w, 9m and 12m will provide much more detailed insight in the relative level of colonization and the duration of colonization in this age group than previous research. Results gained in this pilot study would therefore strongly facilitate the design of a more extended carriage study accompanying the large scale introduction of a conjugate vaccine. Furthermore, the sample size of 130 in each group will already give >80% power to detect a conjugate vaccine-associated 90% reduction in the number of children carrying A meningococci, assuming that in the control group approximately 5% of children carry these bacteria in one or more of the sequential samplings.

Data storage and confidentiality
Samples will be coded with running numbers. Records that correlate these numbers with the name of the patient will remain with the investigators of the core study and will not be provided to the researchers responsible for the ancillary study.
Electronic data will be stored in password-protected computers, and data in hardcopy form will be kept in a locked cabinet, accessible only to authorized study personnel.
Data from the ancillary study will be provided to the trial monitors and sponsors of the core study, upon request.
Publications of research results will not include any names or any personal information that could reasonably be associated with any research participant.
Risk category Risk associated with the throat swabbing procedure for the ancillary studiy is minimal.

2.
Potential risk and protection against risk Throat swabbing is considered a low risk procedure and is not known to produce substantial stress or discomfort.
The risk of stress or discomfort from the procedures will be further reduced, through the training of the investigators performing these procedures, which have been clearly described in writing (see herein).
In addition, there will be close monitoring for adverse events, largely because of potential 759346 Field workers will be trained on the definition of adverse events, including serious adverse events, under the supervision of the Principal Investigator. On the day of vaccination of any study vaccine and on each of the three subsequent days or up to resolution of symptoms, whichever is longer, field workers will visit each research participant at his/her home to record any adverse events. Field workers will also visit each research participants one month after the vaccination of any study vaccine and will observe the research participant for any adverse events.
The Principal Investigator will report each serious adverse event within twenty-four (24) hours of becoming aware of such event. The Principal Investigator will report such event to the Local Safety Monitor, the WHO monitor and GSK Biologicals safety department. GSK Biologicals safety department will transmit the information to the IDMC according to the operating procedures of the IDMC. In addition, the investigator will also provide a listing of all systemic grade 3 symptoms suspected to be related to vaccination and any case of diphtheria, tetanus, pertussis, hepatitis B, invasive diseases due to Haemophilus influenzae type b, measles, yellow fever or Neisseria meningitidis of serogroup A and C to the LSM. The Local Safety Monitor will send the listing and information related to the non-serious grade 3 systemic symptoms related with vaccination and non-serious adverse events suggesting a lack of efficacy of the vaccine to the IDMC and the GSK medical monitor (the GSK medical monitor will forward the information to the other members of the Steering Committee) according to the Charter for the IDMC.
The Principal Investigator will proactively follow each subject (a) with a serious adverse event or who has withdrawn from the study as a result of an adverse event, until the event has resolved, subsided, stabilized, disappeared, the event is otherwise explained, or the subject is lost to follow-up; (b) with a non-serious adverse event who has not withdrawn from the study, until the subject completes the study or is lost to follow-up; or (c) with clinically significant laboratory abnormalities, until the subject's conditions return to normal or a satisfactory explanation has been provided.
Updated information, including completed verbal autopsy questionnaire in case of death, will be recorded on each serious adverse event Report Form, and the Form will be signed and dated by the Principal Investigator. Updated information will be reported in the same manner as the original serious adverse event information.
After the study end, the Principal Investigator is not obliged to actively seek for adverse events or serious adverse events in former research participants. However, if the Principal Investigator learns of any serious adverse event, including a death, at any time after a research participant has been discharged from the study, and he considers the event

4.
Alternatives to participation After one parent/guardian has consented to participate in the ancillary study and core study, any other parent/guardian who accompanies the infant to a clinical trial site visit will have the option not to participate in the ancillary study. Subjects can withdraw from the core study and ancillary study at any time.

V
SUBJECT IDENTIFICATION, RECRUITMENT AND CONSENT/ASSENT 1.
Subject identification and recruitment 1.1.
General enrollment information The study will be conducted in the Kassena-Nankana district by the Navrongo Health Research Center in Ghana.
• All subjects should be enrolled within a period of 5 months.
• Anticipated recruitment rate is 56 subjects per month. Subjects will be recruited when they present themselves at the immunization clinics. • Enrolment will be terminated when 280 subjects are enrolled.
• The recruitment will be monitored by weekly update through regular contacts with study monitors.

1.2
A community information campaign will be conducted as detailed below: Prior to the start of the study, the Regional Director of Health Services will be briefed about the study and the start dates. The District Director of Health Services will be reminded of the start of the study and the use of the health facilities at the three sub districts during that period. The Medical Superintendent of the district hospital, War Memorial Hospital, will also be informed.
Meetings will be arranged with the District Chief Executive as well as paramount chiefs, sub chiefs and elders of the three sub districts separately at their convenience to explain the study to them and seek their consent. 759346 At Navrongo central, the Navropio, his sub chiefs and elders will be met at the Navropio's palace. The Kologo chief, his sub chiefs and elders will be met at Kologo whilst the Bui chief and his elders will be met at Bui.
In the East sub district, separate meetings will be held with the chiefs and elders of Sirigu, Yua, Kandiga, Nabogo, Mirigu, Natugnia and Amontanga as in the table below.
At these meetings, the study will be explained to them, questions related to the study answered and their consent solicited to start the study.

1.3
Recruitment through the Navrongo War Memorial Hospital: Mothers delivering at the Navrongo War Memorial Hospital will be informed about the study and permission solicited to contact them in future about their potential participation in the trial.

1.4
Recruitment through the NDSS Potential study participants will also be identified from the demographic surveillance system of the Kassena-Nankana health district, the Navrongo Demographic Surveillance System (NDSS). The NDSS was established by the Ghana Ministry of Health for the purpose of enhancing research in the Kassena-Nankana district. The NDSS serves as the platform from which studies draw potential research participants. At the start of the study, a request is made to the head of the NHRC for access to the NDSS data set; once he approves, the requested data can be accessed. The approval for this study is attached.

1.5
Recruitment by field workers Routine childhood immunizations are provided on fixed days at the Navrongo War Memorial Hospital and health clinics. One day before first immunization, the field workers will visit the potential study participants to invite them to attend the hospital/health centre the next day.

2.
Process and documentation of consent On the day of immunization, prior to any study procedure, the Parent Information Sheet and Informed Consent forms will be read and explained to the subject's parents/guardians in the local languages (Kassem and Nankam) and according to the local custom and questions will be answered. Literate parents/guardians will be given the Parent Information sheet and Informed Consent form to read.
When agreement is given by the parent/guardian, the IC is signed or thumbprinted (illiterates) by the parents/guardians of the subject, witnessed by an independent person.

Study Contact for Reporting of a Serious Adverse Event for WHO
TBD. The name, address, phone and fax numbers will be provided prior to study start.  17  Synopsis and Section 1.3 Rationale for the study (one paragraph was added at the end of this section)

The study will evaluate the immunogenicity induced by three doses of the vaccine given at 6, 10 and 14 weeks of age by measuring the immune response one month post-vaccination to ensure the vaccine is effective in the youngest children when the disease burden is highest. In order to assess whether the vaccine has induced long-term protection, the persistence of the immune response will be measured when the child is one year of age, and a small dose of plain polysaccharide (1/5 th of a dose of Mencevax™ AC, i.e.
10µ µ µ µg/polysaccharide) will be given to evaluate whether immune memory was induced by priming.

Section 1.4 Study site
The following paragraphs were added just before the sub-section "Health indicators":

Coordination with the EPI
The vaccine trial will be conducted at three EPI clinics in the Kassena-Nankana district where routine immunization is taking place:

Section 3 Study Design Overview
In the flow chart and in the 3 rd bullet point pluriel was used for measles and yellow fever vaccines.
Flow chart: -Measles & Yellow fever vaccines @ 9 months of age -At Visit 5, Month 7.5 Measles/ Yellow Fever Vaccines • To comply with the Ghanean Immunisation Calendar, at 9 months of age, all subjects will receive measles and yellow fever vaccines.

Synopsis and Section 3 Study Design Overview (one bullet point was deleted)
• Study start: The enrolment will preferably start at the onset of the rainy season (June) and should be completed prior to the onset of the dry season (October).

Section 5.3 Outline of study procedures
The following footnote was added to the informed consent row in the table:

Section 5.4 Detailed description of study stages/visits
The following note was added prior to the detailed description of Visits 3, 5, 6 and 7: Note: Any parent/guardian accompanying the child instead of the mother at visits subsequent to the first visit has to sign an Informed Consent prior to providing any saliva sample.

Section 6.5 Replacement of unusable vaccine doses (2 nd paragraph)
In addition to the vaccine doses provided for the planned number of subjects, at least 5% additional doses will be supplied… Section 8.3. Lack of efficacy (one sentence was added at the end of this section)

Any case of diphtheria, tetanus, pertussis, hepatitis B, invasive diseases due to Haemophilus influenzae type b, measles, yellow fever or Neisseria meningitidis of serogroup A and C will be reported to the Data and Safety Monitoring Board (DSMB).
In Section 8.7 Follow-up of adverse events and serious adverse events and assessment of outcome and Section 8.8.1 Time frames for submitting serious adverse event reports to GSK Biologicals and PATH the term "when appointed" was deleted for the WHO monitor as she is now appointed. The new combined Hib-MenAC (using TT as carrier protein) extemporaneously mixed with Tritanrix™-HepB was shown to be safe and immunogenic in preclinical studies. A phase II dose-range study in infants is ongoing outside the African continent and preliminary data will be available prior to starting this study.

In the first three-dose primary vaccination study performed in the Philippines, three formulations of the combined Hib-Men AC vaccine (using TT as carrier protein) each extemporaneously mixed with Tritanrix™-HepB were evaluated and the Tritanrix™-HepB/Hib-MenAC combination using the Hib-MenAC formulation with a dosage of 2.5 µg of each of the MenA, MenC and Hib conjugate antigens was selected for further clinical development: the selected formulation was well tolerated; it induced similar antibody levels against the diphtheria, tetanus, pertussis, hepatitis B, Hib and meningococcal serogroup C antigens as the benchmark vaccines (Tritanrix™-HepB/Hiberix™ and Meningitec™) and 97.7 % subjects had meningococcal serogroup A serum bactericidal antibody titres of at least 1:8. See Appendix I for a summary of clinical data of this study, including a preliminary report on the Serious
Adverse Events reported during that study 759346 The new Tritanrix ™ -HepB/Hib-MenAC vaccine is being has been evaluated in a feasibility trial outside Africa in the Philippines. The combination using the Hib-

MenAC formulation with a dosage of 2.5 µg of each of the MenA, MenC and Hib conjugate antigens has been selected for further clinical development: the selected formulation was well tolerated; it induced similar antibody levels against the diphtheria, tetanus, pertussis, hepatitis B, Hib and meningococcal serogroup C antigens as the benchmark vaccines (Tritanrix™-HepB/Hiberix™ and Meningitec™) and 97.7 % subjects had meningococcal serogroup A serum bactericidal antibody titres of at least 1:8. Once
As feasibility of the new vaccine is has now been shown, the present study plans to evaluate the use of GSK Biologicals' Tritanrix ™ -HepB/Hib-MenAC vaccine in a high endemicity region in Africa.

Section 2.2 Secondary objectives and Synopsis (Deletion of the first bullet)
Just prior to the administration of the first vaccine dose, to assess the serum bactericidal antibodies against N. meningitidis serogroups A and C in the mother of the child as an indirect measurement of the prevaccination status of her infant.

Section 3 Study design overview
In the flowchart, the number of subjects enrolled was amended from 130 per group to 140 per group and the Visit 1 "Pre blood sample in the child's mother" was changed to Blood sampling [of the child]. schedule. OPV can also be given at 6, 10, 14 weeks of age concomitantly with the study vaccines or at any time during the study, whichever is more appropriate.

Section 3 Study design overview and Synopsis
Experimental design: 2 parallel groups of 130 140 subjects each to receive either of the following vaccines: As the control group will not be properly immunised with 1/5 th of a dose of Mencevax™ AC, at study end, the control group Tritanrix ™ -HepB/Hiberix ™ will be offered one dose of Mencevax™-ACWY in order to protect the children against the meningococcal diseases after study end, one dose of Mencevax™ ACWY will be offered to the Tritanrix ™ -HepB/Hiberix ™ control group to protect them against meningococcal diseases of these serogroups:

Mencevax™ ACWY will be given when the child is two years of age, or, if a meningitis epidemic occurs, earlier in the second year of life.
• For all subjects: 3 4 blood samples (3.5 ml each): − immediately before the first dose of the primary vaccination course − one month after the third dose of the primary vaccination course − just prior to administration of the plain polysaccharides of serogroups A and C − one month after the administration of the plain polysaccharides of serogroups A and C.
For the mothers of all subjects, just prior to the administration of the first vaccine dose to their infants: one blood sample (3.5 ml). The Charter (terms of references) of the Terms of reference (operating procedures) of the IDMC will be written after the first meeting of the IDMC members, prior to study start.
Role of the IDMC (2 nd paragraph) The IDMC will confer before the initiation of the study (pre-initiation review), during the study proper and at the close out of the study to review any relevant safety data and to review and approve the Report and Analysis Plan (RAP), where applicable ; after study end, the IDMC will have a first assessment report… information sheet will be written and only one informed consent will be submitted to the approval of the parents/guardians of the child, however, any parent/guardian accompanying the child at visits subsequent to the first visit who has not already signed an Informed Consent for this study will also have to sign an Informed Consent prior to providing any throat swab or saliva sample).
This ancillary study will be reported separately by the investigators of the ancillary study. This ancillary study requires collection of throat swabs and saliva samples from the mothers parent/guardian and infants prior to the first dose (6-8 weeks) and at 10 weeks, 18 weeks, 9 months and 12 months of age. The collection of throat swab and saliva samples will be detailed in the study procedures (section 5. 3) and the subject information sheet.

Section 5.3 Outline of study procedures
In Note at end of footnotes (2 nd sentence amended): OPV vaccine can be administered to the subjects at 6, 10 and 14 weeks of age concomitantly with the study vaccines or at any time during the study, whichever is more appropriate.

Section 5.4 Detailed description of study stages/visits
(Visit 1 change of the following 2 bullet points, regarding informed consent and blood sampling) Written informed consent from the subject's mother parent/guardian.
Collection of 3.5 ml of whole blood from the mother. Collection of blood for serology: a minimum of 3.5 ml of whole blood to provide a minimum of 1.5 ml of serum according to instructions in Appendix D.
Collection of blood for serology: it is understood that collection of blood in infants aged 6 weeks may be technically difficult. Therefore a minimum of 2.0 ml of whole blood, which is the minimum volume of blood that will allow the essential serology to be undertaken, has to be taken according to Amendment

Pre-vaccination (Day 0) serum samples will be tested in all subjects for antibodies against SBA-MenA, SBA-MenC, anti-PSA, anti-PSC, diphtheria toxoid, pertussis antigen and recombinant DNA hepatitis B surface antigen (no pre-vaccination testing of anti-TT and anti-PRP is foreseen as immunogenicity results of the previous clinical study were highest for these two antibodies).
Post-vaccination III and pre-booster serum samples will be tested in all subjects for antibodies against all vaccine antigens (SBA-MenA, SBA-MenC, anti-PRP, anti-PSA, anti-PSC, diphtheria and tetanus toxoids, pertussis antigen and recombinant DNA hepatitis B surface antigen). 759346 The post-booster serum sample taken from all children and the prevaccination serum sample taken from their mothers will be tested for SBA-MenA and SBA MenC, anti-PSA and anti-PSC antibodies.
In case of insufficient blood sample volume to perform assays for all antibodies, they will be analyzed according to the following priority ranking:

Pre-booster) • anti-HBs • anti-tetanus, (only for Post vacc III and pre-booster)
• anti-BP • anti-PSA • anti-PSC Section 5.5.4 Endpoints for suboptimal response (suboptimal response for antidiphtheria concentration changed to Vero-cell cut-off and last sentence amended) anti-diphtheria concentration <0.1IU/ml < 0.016 IU/ml (Vero-cell assay) The Mencevax™ ACWY will be given when the child is two years of age or, if a meningitis epidemic occurs, already earlier in the second year of life.

Section 6.1.1 GSK Biologicals' Haemophilus influenzae type b meningococcal AC-TT (Hib-MenAC) conjugate vaccine (section amended as follows)
One lot of the vaccine will be used. The vaccine will be supplied as a white freeze dried pellet in monodose vials to be reconstituted with Tritanrix™-HepB before use. HepB before use (0.5 ml per vial). Only half of the dose (i.e. 0.5 ml will be administered. For details of the dose dilution see Section 6.1.7. One dose (0.5 ml) of the reconstituted vaccine contains the following components: 759346 Note: The dose of each component (2.5 µg or 5 µg for each polysaccharide) will be determined in a separate dose-range study, results of which will be available prior to study start. The vaccine will be supplied as a white freeze-dried pellet in monodose vials to be reconstituted with one 0.5 ml vial of Tritanrix   -HepB before use.

Section 6.1.7 Extemporaneous mixing of Tritanrix™-HepB and Hib-MenAC vaccines or Hiberix™
New subheading "Tritanrix™-HepB and Hiberix™" added and 2 nd sentence amended: The full content of the Tritanrix™-HepB vaccine vial should be extracted and injected into the vial containing the lyophilized Hib-MenAC or Hiberix™ vaccines.
Additional subsection with subheading added as follows:

Section 6.3 Treatment allocation and randomization (1 st sentence)
The target sample size is 260 280 enrolled subjects (220 evaluable for immunogenicity analysis, with 110 subjects in each group).

Section 6.4 Method of blinding and breaking the study blind (1 st paragraph)
The study will be double blind.

The study vaccine and the control vaccine will have the same whitish appearance after reconstitution. However, since the vaccines used in this study are different in their packaging volumes (Tritanrix™-HepB and Hiberix™ = 1 vial of 0.5ml of each vaccine, Tritanrix™-HepB and Hib-MenAC = 2 vials of 0.5 ml of Tritanrix™-HepB and 1 vial of 0.5 ml of Hib-MenAC), special precautions will be taken to ensure blinding. For each vaccination during the course of the study, a Syringe Filler and Vaccinator will work as a vaccination team: the Syringe Filler will prepare the vaccine for administration to a specific subject and the Vaccinator will administer the vaccine to each subject, according to the investigator's SOP. To ensure blinding, a vaccination team will perform no other function in the study.
The subject's parent/guardian as well as those responsible for the evaluation of safety or immunogenicity study parameters will all be unaware which vaccine preparation was administered to a particular subject. The subject's parents/guardians, the investigator and the study personnel will be blinded to the vaccines to be administered. In addition to the direct reporting of any SAE and any subsequent information by the investigator or its designate to GSK Biologicals and PATH, the investigator will also report the SAE and subsequent information to the local safety monitor for transmission to the IDMC (see section 5.1.3) and to the WHO monitor.

Completion and transmission of serious adverse events to GSK
Biologicals, WHO, and PATH and the local safety monitor Section 8.8.2 (1st paragraph and WHO study contact for reporting SAEs amended as follows) Once an investigator becomes aware that a SAE has occurred in a study subject, she/he will report the information to GSK Biologicals, WHO and PATH within 24 hours as outlined in Section 8.8.1. The SAE Report Form will always be completed as thoroughly as possible with all available details of the event, signed by the investigator (or designee), and forwarded to GSK Biologicals, WHO and PATH within the designated time frames. If the investigator does not have all information regarding an SAE, he/she will not wait to receive additional information before notifying GSK Biologicals, WHO and PATH of the event and completing the form. The form will be updated when additional information is received and forwarded to GSK Biologicals, WHO and PATH within 24 hours as outlined in Section 8.8.1.

IU/ml by Vero-cell (seroprotection)
Indirect pre vaccination status through assessment of the mother: Just prior to the administration of the first vaccine dose, in each mother.
• SBA-MenA titer ≥ 1:8 and titer • SBA-MenC titer ≥ 1:8 and titer • Anti-PSA and anti-PSC seropositivity and concentration Section 10.3 Estimated sample size (1 st sentence, Table 10-1 and Table 10-2) The target sample size is 260 280 enrolled subjects to reach 220 evaluable subjects in the ATP cohort for immunogenicity (110 evaluable subjects in each group).  A total of 6 cohorts are defined for the purpose of analysis.
• Three cohorts that will be used for the analysis of the primary vaccination course: ! the Primary Total vaccinated cohort ! the Primary ATP cohort for safety ! the Primary ATP cohort for immunogenicity (used for the analysis of the co-primary objectives of immunogenicity) • Three cohorts that will be used for the analysis of the booster phase: ! the Booster Total vaccinated cohort ! the Booster ATP cohort for safety ! the ATP cohort for immune memory

The Primary Total vaccinated cohort (Primary vaccination)
The primary total cohort will include all subjects vaccinated in the primary phase of the study. The analysis of an endpoint based on the primary total cohort will include all subjects vaccinated in the primary phase for whom data for the endpoint are available.

Primary According to protocol (ATP) cohort for safety (Primary vaccination)
The Primary According to protocol (ATP) cohort for safety will include all subjects:

Primary According to protocol (ATP) cohort for immunogenicity (Primary vaccination)
The Primary According to protocol (ATP) cohort for immunogenicity will include all evaluable subjects (i.e. those meeting all eligibility criteria, complying with the procedures defined in the protocol, and with no elimination criteria during the study) from the Primary ATP cohort for safety for whom assay results are available for antibodies against at least one study vaccine antigen component at Visit 4.

The Booster Total vaccinated Cohort cohort (Booster vaccination)
The Booster total cohort will include all subjects vaccinated in the booster phase of the study. The analysis of an endpoint based on the Booster total cohort will include all subjects vaccinated in the booster phase for whom data for the endpoint are available.

Booster According To Protocol (ATP) cohort for safety (Booster vaccination)
The Booster ATP cohort for safety will include all subjects from the Primary ATP cohort (primary vaccination) for safety Section 10.5 Derived and transformed data (whole section amended) For a given subject and cohort and the analysis of a given measurement, missing or non-evaluable measurements will not be replaced. Therefore, an analysis will exclude subjects with missing or non-evaluable measurements. 759346 For immunogenicity data, the antibody concentrations/titers below the cut-off of the assay will be given an arbitrary value of half the cut-off for the purpose of the GMC/GMT calculation. Immunogenicity For a given subject cohort and the analysis of a given measurement, missing or unevaluable measurements will not be replaced. Therefore an analysis will exclude subjects with missing or unevaluable measurements.

Subjects who missed reporting symptoms (solicited or unsolicited or concomitant medications) will be treated as subjects without symptoms (solicited or unsolicited or concomitant medications, respectively).
Section 10.6.2 Analysis of Immunogenicity (1 st paragraph) The analysis of immunogenicity will be based on the Primary ATP cohort for immunogenicity. If, for any vaccine group, the percentage of enrolled subjects with serological results excluded from this ATP cohort is more than 5%, a second analysis based on the Primary Total vaccinated cohort will be performed to complement the ATP analysis. The primary analysis will be based on the Primary Total vaccinated cohort. If the percentage of enrolled subjects excluded from the Primary ATP cohort for safety is more than 5%, a second analysis based on this ATP cohort will be performed to complement the Primary Total vaccinated cohort analysis. respectively based on the Booster ATP cohort for safety and the ATP cohort for immune memory. If, for any vaccine group, the percentage of enrolled subjects with serological results excluded from an ATP cohort is more than 5%, a second analysis based on the Booster Total vaccinated cohort will be performed to complement the ATP analysis. All analyses will be based on the Booster Total vaccinated cohort. If the percentage of enrolled subjects excluded from the Booster ATP cohort for safety is more than 5%, a second analysis based on this ATP cohort will be performed to complement the Booster Total vaccinated cohort analysis.

Section 10.8 Reporting of final analyses (2 nd paragraph)
A final analysis for the booster vaccination will be carried out based on all data collected up to one month after the administration of one fifth of a dose of Mencevax™ AC. An annex report based on the final analysis for the booster vaccination will be provided.