GSK Clinical Study Register

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1 In February 2013, GlaxoSmithKline (GSK) announced a commitment to further clinical transparency through the public disclosure of GSK Clinical Study Reports (CSRs) on the GSK Clinical Study Register. The following guiding principles have been applied to the disclosure: Information will be excluded in order to protect the privacy of patients and all named persons associated with the study Patient data listings will be completely removed* to protect patient privacy. Anonymized data from each patient may be made available subject to an approved research proposal. For further information please see the Patient Level Data section of the GSK Clinical Study Register. Aggregate data will be included; with any direct reference to individual patients excluded *Complete removal of patient data listings may mean that page numbers are no longer consecutively numbered

2 GlaxoSmithKline Biologicals Study title Complementary testing to further evaluate the immunogenicity of GSK Biologicals HPV vaccine (580299) in healthy female subjects aged over 26 years enrolled in study Study detailed title Complementary immunology testing in a phase III, double-blind, randomized, controlled study to evaluate the safety, immunogenicity and efficacy of GlaxoSmithKline Biologicals HPV-16/18 L1 AS04 vaccine administered intramuscularly according to a three-dose schedule (0, 1, 6 month) in healthy adult female subjects aged 26 years and above enrolled in study (HPV-015). Clinical Study Report for Study (Development Phase III) IND Number: BB-IND 7920 Indication Studied: For active immunization of girls and women from 10 years of age onwards for the prevention of persistent human papillomavirus (HPV) infections and related clinical outcomes (cytological abnormalities and pre-cancerous lesions) caused by oncogenic HPV types 16 and 18. Study initiation date: 05 April 2007 Study completion date: 02 January 2008 Data lock point: 07 October 2008 Date of Report: Amendment 1, Final: 04 December 2013 Earlier Study Reports: Clinical Study Report: 08 May 2009 Sponsor Signatory: MD, Director, Clinical Development, HPV Vaccines, Vaccines Discovery and Development, GlaxoSmithKline Biologicals (Amended: 04 December 2013) This study was performed in compliance with Good Clinical Practice including the archiving of essential documents. Copyright the GlaxoSmithKline group of companies. All rights reserved. Unauthorized copying or use of this information is prohibited. 9b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 1 1

3 Name of company: GlaxoSmithKline Biologicals, Rixensart, Belgium Name of finished product: GlaxoSmithKline Biologicals human papillomavirus (HPV) vaccine Name of active substance: HPV-16 L1 VLP protein HPV-18 L1 VLP protein SYNOPSIS TABULAR FORMAT REFERRING TO PART OF THE DOSSIER Volume: Page: (for national authority only) Title of the study: Complementary immunology testing in a phase III, double-blind, randomized, controlled study to evaluate the safety, immunogenicity and efficacy of GlaxoSmithKline Biologicals HPV-16/18 L1 AS04 vaccine administered intramuscularly according to a three-dose schedule (0, 1, 6 month) in healthy adult female subjects aged 26 years and above enrolled in study (HPV-015) Principal investigators: This study was conducted by two investigators in the Netherlands: MD, PhD and MD, PhD ( Study centers: This study was conducted at two sites in the Netherlands: Publication (reference): Not published as of 04 December Clinical Study Report revision history: GSK Biologicals investigated the quality of some serology assays used in clinical studies, including the HPV-16/18 Enzyme-Linked Immunosorbent Assay (ELISA) that is used in the present study. This clinical study report amendment describes the outcome of this investigation. (Amended: 04 December 2013) Study period: Clinical phase: III Study initiation date: 05 April 2007 Study completion date: 02 January 2008 Data lock point: 07 October 2008 Objectives: To measure T-cell-mediated immune responses specific to HPV virus-like particles (VLP) types 16 and 18 in peripheral blood cells. To measure memory B-cell immune responses specific to HPV VLP types 16 and 18 in peripheral blood cells. To evaluate anti-hpv-16 and anti-hpv-18 antibody responses in cervical samples. To evaluate the total level of IgG antibodies in cervical samples collected in this study, and in serum samples collected in study (HPV-015). To compare anti-hpv-16 and anti-hpv-18 antibody levels in cervical samples collected in this study with antibody levels in serum samples collected in study (HPV-015). Study design: A phase III, double-blind, controlled ancillary study performed as a supplement to study (HPV-015). The present study was conducted at pre-selected sites participating in study (HPV-015). Report Amendment 1 Synopsis page 1 of 5 2 9b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 2

4 Name of company: GlaxoSmithKline Biologicals, Rixensart, Belgium Name of finished product: GlaxoSmithKline Biologicals human papillomavirus (HPV) vaccine Name of active substance: HPV-16 L1 VLP protein HPV-18 L1 VLP protein TABULAR FORMAT REFERRING TO PART OF THE DOSSIER Volume: Page: (for national authority only) In study (HPV-015), subjects were randomized (1:1) to two parallel groups, the HPV group who received HPV-16/18 L1 AS04 vaccine and the Control group who received Al(OH) 3, and according to the following age stratification: 45% of the subjects in the years of age group, 45% of the subjects in the years of age group, and 10% of the subjects in the above 45 years of age group. Three doses of vaccine/control were administered intramuscularly according to a 0, 1, 6-month schedule. No additional doses of vaccine/control were administered in this ancillary study. Two scheduled visits: Visit 5 (Month 12) and Visit 6 (Month 18) after the first vaccine dose in study (HPV 015). At both visits, blood and Cervicovaginal (CVS)samples were collected from all subjects: 40 ml blood samples were collected for evaluation of cell-mediated immune responses. These samples were in addition to the 10 ml blood samples taken according to the protocol for study (HPV-015). Cervicovaginal secretion samples were collected for antibody detection. These samples were to be collected before the liquid-based cytology samples taken according to the protocol for study (HPV-015). Number of Subjects: Planned: 100 subjects (50 in the HPV group and 50 in the Control group). Enrolled: 100 subjects (53 in the HPV group and 47 in the Control group). Completed: 99 subjects (52 in the HPV group and 47 in the Control group). Immunogenicity: Total Vaccinated cohort was comprised of 100 subjects (53 in the HPV group and 47 in the Control group). Diagnosis and criteria for inclusion: Female subjects enrolled at pre-selected sites in study (HPV-015) who were part of the immunogenicity subset and had received all three doses of vaccine/control. Prior to the performance of any study procedure, written informed consent was obtained from the subject. Study vaccine, dose, mode of administration, lot no.: No vaccine was administered for the purpose of this ancillary study. Reference vaccine/comparator, dose, mode of administration, lot no.: No vaccine was administered for the purpose of this ancillary study. Duration of study: Duration of study was approximately six months for each subject. Criteria for evaluation of immunogenicity: Measurement of cell-mediated immune (CMI) responses in peripheral blood mononuclear cells (PBMC) and antibody titers against each vaccine antigen in CVS samples at approximately 12 and 18 months after the first dose of vaccine/control in study (HPV-015) in subjects from the two pre-selected study centers. Report Amendment 1 Synopsis page 2 of 5 3 9b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 3

5 Name of company: GlaxoSmithKline Biologicals, Rixensart, Belgium Name of finished product: GlaxoSmithKline Biologicals human papillomavirus (HPV) vaccine Name of active substance: HPV-16 L1 VLP protein HPV-18 L1 VLP protein Endpoints: TABULAR FORMAT REFERRING TO PART OF THE DOSSIER Volume: Page: (for national authority only) T-cell-mediated immune responses specific to HPV VLP types 16 and 18 (Month 12 and 18). Memory B-cell immune responses specific to HPV VLP types 16 and 18 (Month 12 and 18). HPV-16 and HPV-18 antibody titers assessed by ELISA in CVS samples at Month 12 and 18 in subjects who had CVS samples collected at Month 12 and 18 (seropositivity rates and geometric mean titers [GMTs]). Total IgG antibodies (by ELISA) in CVS samples collected in this study, and in serum samples collected in study (HPV-015) at Month 12 and Month 18. Correlation of anti-hpv-16 and anti-hpv-18 antibodies in serum collected in study (HPV-015) and in CVS samples collected in this study. Exploratory endpoints: At the discretion of GSK, all endpoints above could be expanded to HPV types not included in the vaccine. At the discretion of GSK, other immune parameters related to HPV or the HPV vaccine constituents could be tested. This may have included evaluation of neutralizing antibodies. No safety data were collected in this ancillary study. All safety data were collected in the context of study (HPV-015) and are not presented in this ancillary study report. Statistical methods: The primary analyses of immunogenicity were based on the Total Vaccinated cohort. Within group assessment The exact 95% CIs for proportion within a group were calculated from Proc StatXact 5.0 assuming independence between doses. The 95% CI for GMTs were obtained within each group separately. The 95% CI for the mean of log10- transformed titer were obtained assuming that log-transformed titers were normally distributed with unknown variance. The 95% CI for the GMT was then obtained by exponential transformation of the 95% CI for the mean of the log-transformed titers. Antibody responses in serum samples For each treatment group, at each blood sampling time point and for each antibody for which results were available: Seropositivity rates with 95% CIs were tabulated. GMTs with 95% CIs were tabulated. If the seropositivity rates were low, GMTs were to be calculated on seropositive subjects only. Antibody responses were also presented using Reverse Cumulative Curves. Report Amendment 1 Synopsis page 3 of 5 4 9b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 4

6 Name of company: GlaxoSmithKline Biologicals, Rixensart, Belgium Name of finished product: GlaxoSmithKline Biologicals human papillomavirus (HPV) vaccine Name of active substance: HPV-16 L1 VLP protein HPV-18 L1 VLP protein TABULAR FORMAT REFERRING TO PART OF THE DOSSIER Volume: Page: (for national authority only) Cervicovaginal secretion samples In order to obtain a valid evaluation of the antibody titer in CVS,, contamination with blood should be avoided. As a consequence, results of CVS with Hemastix test strip 200 erythrocytes/l were excluded from the analysis. For each antibody titer (HPV-16 and HPV-18) measured by ELISA at Month 12 and 18 in CVS, the following descriptive statistics were presented per group: Seroconversion and seropositivity rates (with exact 95% CI) were calculated, GMT with 95% CI and range of antibody titers were tabulated, Pearson coefficients of correlation between serum and CVS for anti-hpv-16 and anti-hpv-18 titers standardized for Total IgG were calculated and scatter plots were produced, and Correlations between serum and CVS for HPV-16 and HPV-18, standardized for Total IgG, by age strata, for subjects with Hemastix 200 erythrocytes/l were calculated. T-cell responses by Intracellular Cytokine Staining (ICS) ICS assay provided information on the frequency of CD4+ and CD8+ T-cells responding to a specified antigen and producing: at least CD40L and another cytokine (IFN, IL-2, TNF) (d-cd40l); at least IL-2 and another cytokine (CD40L, TNF, IFN) (d-il-2); at least TNF and another cytokine (CD40L, IL-2, IFN) (d-tnf); at least IFN and another cytokine (IL-2, TNF, CD40L) (d-ifn); at least two different cytokines (CD40L, IL-2, TNF, IFN) (all doubles). Frequency of cytokines-positive (d-cd40l, d-il2, d-tnf, d- IFN or all doubles) CD4+ or CD8+ T-cells, for each stimulant at each timing, were summarized for each group by the number of values (N), the number of missing values, minimum, 1st quartile, median, 3rd quartile, maximum and geometric mean (Gmean) with 95% CI. In addition, for each assay and for each stimulant at each timing, the number and percentage of subjects with 500 cells/million cells for CD4 T-cells and 200 cells/million cells for CD8 T-cells were calculated. Report Amendment 1 Synopsis page 4 of 5 5 9b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 5

7 Name of company: GlaxoSmithKline Biologicals, Rixensart, Belgium Name of finished product: GlaxoSmithKline Biologicals human papillomavirus (HPV) vaccine Name of active substance: HPV-16 L1 VLP protein HPV-18 L1 VLP protein TABULAR FORMAT REFERRING TO PART OF THE DOSSIER Volume: Page: (for national authority only) B-cell responses by ELISPOT ELISPOT assay provides information on the frequency of memory B-cells corresponding to a specified antigen. The results of each stimulant at each timing were summarized for each group by the number of values (N), the number of missing values, minimum, 1st quartile, median, 3rd quartile, maximum and geometric mean (Gmean) with 95%CI. Further, for each stimulant at each timing, the number and percentage of subjects above zero were calculated. Summary: Immunogenicity: In serum samples collected at Months 12 and 18, all subjects in the HPV group were seropositive for HPV-16 and HPV-18 antibodies by ELISA and were seropositive for anti-hpv-16 and anti- HPV-18 Pseudovirion-based Neutralizing Antibodies (PBNA). In CVS samples collected at Month 12, all subjects in the HPV group and none of the subjects in the Control group had detectable antibodies against HPV-16 and HPV-18. In CVS samples collected at Month 18, 96.2% and 88.5% of the subjects in the HPV group had detectable antibodies against HPV-16 and HPV-18, respectively, whereas none of the subjects in the Control group were seropositive for HPV-16 antibodies and 10% had detectable antibodies against HPV- 18. The correlation coefficients between serum and CVS for HPV-16 IgG titers were at Month 12 and at Month 18. The correlation coefficients between serum and CVS for HPV-18 IgG titers were at Month 12 and at Month 18. In the vaccine group, we observed robust specific CD4+ T-cell (55.8% increase in mean CD4+-cell response against HPV-16, and 44.2% increase for HPV-18 between Month 12 and Month 18) and memory B-cell responses (HPV-16 cell response in vaccine group vs. controls: 84.3% vs. 4.4% at Month 18; HPV-18 cell response in vaccine group vs. controls: 84.3% vs. 0.0% at Month 18) to HPV-16 and HPV-18. Conclusions: All subjects in the HPV group had detectable antibodies against HPV-16 and HPV-18 antibodies by ELISA and PBNA at the Month 12 and Month 18 time points. Comparing the HPV and Control groups, it can be seen that the HPV-16/18 L1 AS04 vaccine induced HPV-16 and HPV-18 specific cervicovaginal antibody responses. There was a good correlation between serum and CVS sample titers for both HPV-16 and HPV-18 IgG titers. The HPV-16/18 L1 AS04 vaccine induced immune memory for both HPV-16 and HPV-18. Date of Report: Amendment 1, Final: 04 December 2013 Report Amendment 1 Synopsis page 5 of 5 6 9b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 6

8 TABLE OF CONTENTS PAGE SYNOPSIS...2 LIST OF ABBREVIATIONS...13 GLOSSARY OF TERMS...15 TRADEMARKS ETHICS Independent Ethics Committee (IEC) or Institutional Review Board (IRB) Ethical conduct of the study Subject information and consent INVESTIGATORS AND STUDY ADMINISTRATIVE STRUCTURE Administrative structure Clinical Study Report revision history INTRODUCTION STUDY OBJECTIVES INVESTIGATIONAL PLAN Study design Overall study design Description Study procedures Outline of study procedures Intervals between study visits Selection of study population Inclusion criteria Exclusion criteria Elimination criteria Contraindications to subsequent doses of vaccine Subject completion and withdrawal from study Subject completion Subject withdrawal from the study Subject withdrawal from administration of the investigational product Composition and administration of vaccine Treatment allocation and randomization Blinding Prior and concomitant medication /vaccinations Laboratory assays and time points Serology ELISA Pseudovirion based neutralization assay (PBNA) IgG total b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 7

9 Cell mediated immune (CMI) T and B-cell responses Cervicovaginal secretion (CVS) samples for antibody detection Assessment of immunogenicity variables Assessment of safety variables Data quality assurance Statistical methods Endpoints Exploratory endpoints Determination of sample size Study cohorts analyzed Total Vaccinated cohort Modified Total Vaccinated cohort According to Protocol cohort Derived and transformed data Analysis of immunogenicity Within groups assessment Antibody responses in serum samples Cervicovaginal secretions (CVS) samples T-cell responses by Intracellular Cytokine Staining (ICS) B-cell responses by ELISPOT Interim analysis Changes in the conduct of the study or planned analyses Protocol amendments Other changes STUDY POPULATION RESULTS Study dates Subject eligibility and attrition from the study Number of subjects Study completion and withdrawal from study Demographic characteristics Total vaccinated cohort IMMUNOGENICITY RESULTS Data sets analyzed Total vaccinated cohort analysis Antibody responses in serum Antibody responses by ELISA Pseudovirion-based neutralizing antibodies (PBNA) assay Detection of antibodies in CVS samples Correlations between serum and CVS for HPV-16 and HPV-18 antibody responses T-cell mediated immune responses by ICS B-cell responses OVERALL CONCLUSIONS Overall conclusions b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 8

10 9. SUPPLEMENTS REFERENCES STUDY REPORT AUTHORS /CONTRIBUTING AUTHORS...71 MODULAR APPENDICES 9 9b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 9

11 LIST OF TABLES PAGE Table 1 Table 2 Table 3 Table 4 Outline of study procedures performed during ancillary study conducted at Visit 5 and Visit Laboratory assays...27 Summary of blood sampling time points and assays for the assessment of immunology variables...29 Number of subjects enrolled into the study as well as the number excluded from TVC analyses with reasons for exclusion...34 Table 5 Number of subjects completing each visit (TVC)...35 Table 6 Summary of demographic characteristics (TVC)...35 Table 7 Table 8 Table 9 Table 10 Table 11 Table 12 Table 13 Table 14 Table 15 Table 16 Table 17 DNA and serological status at baseline (TVC)...36 Seropositivity rates and GMTs by ELISA for HPV-16 L1 VLP IgG antibodies by pre-vaccination status (TVC)...38 Seropositivity rates and GMTs by ELISA for HPV-18 L1 VLP IgG antibodies by pre-vaccination status (TVC)...39 Seropositivity rates and GMTs for HPV-16 PSV neutralizing antibodies by pre-vaccination status (TVC)...40 Seropositivity rates and GMTs for HPV-18 PSV neutralizing antibodies by pre-vaccination status (TVC)...41 Seropositivity rates and GMTs for HPV-16 antibody (by ELISA) in CVS with Hemastix less than or equal to200 erythrocytes/microlitre (TVC)...42 Seropositivity rates and GMTs for HPV-18 antibody (by ELISA) in CVS with Hemastix less than or equal to200 erythrocytes/microlitre (TVC)...42 HPV-16 specific CD4 T-cell responses (by ICS): proportion of subjects above 500 cells/million cells (TVC)...45 HPV-18 specific CD4 T-cell responses (by ICS): proportion of subjects above 500 cells/million cells (TVC)...46 HPV-16 specific memory B-cell responses (by ELISPOT): proportion of subjects above zero (TVC)...47 HPV-18 specific memory B-cell responses (by ELISPOT): proportion of subjects above zero (TVC) b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 10

12 Table 18 HPV-16 specific memory B-cell responses (by ELISPOT) (TVC)...48 Table 19 HPV-18 specific memory B-cell responses (by ELISPOT) (TVC)...49 LIST OF FIGURES PAGE Figure 1 Figure 2 Correlation between serum and CVS for HPV-16 antibody response (by ELISA) (standardized for Total IgG) for subjects with Hemastix less than or equal to200 erythrocytes/microlitre...43 Correlation between serum and CVS for HPV-18 antibody response (by ELISA) (standardized for Total IgG) for subjects with Hemastix less than or equal to200 erythrocytes/microlitre b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 11

13 LIST OF SUPPLEMENTS PAGE Supplement 1 Number of subjects by center (TVC)...51 Supplement 2 Reverse cumulative distribution curve for HPV-16 IgG antibody titers (measured by ELISA) at Month 12 for seronegative subjects at baseline (TVC)...51 Supplement 3 Reverse cumulative distribution curve for HPV-16 IgG antibody titers (measured by ELISA) at Month 18 for seronegative subjects at baseline (TVC)...52 Supplement 4 Reverse cumulative distribution curve for HPV-18 IgG antibody titers (measured by ELISA) at Month 12 for seronegative subjects at baseline (TVC)...53 Supplement 5 Reverse cumulative distribution curve for HPV-18 IgG antibody titers (measured by ELISA) at Month 18 for seronegative subjects at baseline (TVC)...54 Supplement 6 Reverse cumulative distribution curve for HPV-16 PSV antibodies at Month 12 in seronegative subjects at baseline (TVC)...55 Supplement 7 Reverse cumulative distribution curve for HPV-16 PSV antibodies at Month 18 in seronegative subjects at baseline (TVC)...56 Supplement 8 Reverse cumulative distribution curve for HPV-18 PSV antibodies at Month 12 in seronegative subjects at baseline (TVC)...57 Supplement 9 Reverse cumulative distribution curve for HPV-18 PSV antibodies at Month 18 in seronegative subjects at baseline (TVC)...58 Supplement 10 Supplement 11 HPV-16 specific T-cell responses (by ICS) (TVC)...59 HPV-18 specific T-cell responses (by ICS) (TVC)...63 Supplement 12 HPV-16 specific CD8 T-cell responses (by ICS): proportion of subjects above 200 cells/million cells (TVC)...67 Supplement 13 HPV-18 specific CD8 T-cell responses (by ICS): proportion of subjects above 200 cells/million cells (TVC) b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 12

14 LIST OF ABBREVIATIONS Al(OH) 3 AS04 ATP Aluminum hydroxide GlaxoSmithKline Biologicals proprietary adjuvant AS04 containing aluminum salts (0.5 mg) and 3-O-desacyl-4 - monophosphoryl lipid A (MPL) (50 µg) According-To-Protocol CD4 Cluster of Differentiation 4 CD8 Cluster of Differentiation 8 CD40L CI CMI CpG CVS DNA ecrf ELISA EL.U/mL GCP Gmean GMT GSK HPV ICS IgG IEC CD40-ligand; a type II membrane protein that binds to CD40 which is a cell surface receptor of the tumor necrosis factor receptor family Confidence Interval Cell Mediated Immune Cytosine and Guanine separated by a phosphate, which links the two nucleosides together in DNA Cervicovaginal Secretions Deoxyribonucleic Acid electronic Case Report Form Enzyme-Linked Immunosorbent Assay ELISA Units per ml Good Clinical Practice Geometric Mean Geometric Mean Titer GlaxoSmithKline Human Papillomavirus Intracellular Cytokine Staining Immunoglobulin type G Independent Ethics Committee 13 9b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 13

15 IL-2 IFN IND IRB LBC LOQ ml Interleukin-2 Interferon-gamma Investigational New Drug Institutional Review Board Liquid-based Cytology Limit of Quantification Milliliter MTVC MPL NA NCI PBMC PBNA PCR PSV RAP RCC SOP TNF TVC VaIN VIN VLP Modified Total Vaccinated cohort 3-O-desacyl-4 -Monophosphoryl Lipid A Not Applicable National Cancer Institute Peripheral Blood Mononuclear Cells Pseudovirion-based Neutralizing Antibodies Polymerase Chain Reaction Pseudovirion Report Analysis Plan Reverse Cumulative Curve Standard operating Procedure Tumor Necrosis Factor-alpha Total Vaccinated cohort Vaginal Intraepithelial Neoplasma Vulvar Intraepithelial Neoplasma Virus-Like Particle 14 9b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 14

16 GLOSSARY OF TERMS Blinding: Completed: Eligible: Evaluable: Investigational product: Protocol amendment: Randomization: Subject(s): 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. 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. The level of blinding is maintained throughout the conduct of the trial, and only when the data are cleaned to an acceptable level of quality will appropriate personnel be unblinded or when required in case of a serious adverse event. Subjects who completed the last study visit. Qualified for enrolment into the study based upon strict adherence to inclusion/exclusion criteria. Meeting all eligibility criteria, complying with the procedures defined in the protocol, and, therefore, included in the analysis. 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. ICH defines a protocol amendment as: A written description of a change(s) to or formal clarification of a protocol. GSK Biologicals further details this to include a change to an approved protocol that affects the safety of subjects, scope of the investigation, study design, or scientific integrity of the study. Process of random attribution of treatment to subjects in order to reduce bias of selection Term used throughout the protocol to denote an individual who has been contacted in order to participate in the clinical study, either as a recipient of the investigational product(s) or as a control. 15 9b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 15

17 Treatment: Term used throughout the clinical study to denote a set of investigational product(s) or marketed product(s) or placebo intended to be administered to a subject, identified by a unique number, according to the study randomization or treatment allocation. 16 9b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 16

18 TRADEMARKS The following trademarks are used in the present report. Note: In the body of the CSR (including the synopsis), the names of the vaccines and/or medications will be written without the subscript symbol. Trademarks of the GlaxoSmithKline group of companies generic description Cervarix Human papillomavirus vaccine Types 16 and 18 (Recombinant, AS04 adjuvanted) 17 9b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 17

19 1. ETHICS 1.1. Independent Ethics Committee (IEC) or Institutional Review Board (IRB) The study protocol, the informed consent, and other information that required preapproval were reviewed and approved by a national IEC or IRB Ethical conduct of the study This study was conducted in accordance with "good clinical practice" (GCP) and all applicable regulatory requirements, including, where applicable, the Declaration of Helsinki Subject information and consent Written informed consent was obtained from each subject prior to the performance of any study-specific procedures. Case report forms were provided for each subject s data to be recorded. 2. INVESTIGATORS AND STUDY ADMINISTRATIVE STRUCTURE 2.1. Administrative structure This study was conducted by two investigators at two sites in the Netherlands: MD, PhD ( and MD, PhD 2.2. Clinical Study Report revision history GSK Biologicals investigated the quality of some serology assays used in clinical studies, including the HPV-16/18 Enzyme-Linked Immunosorbent Assay (ELISA) that is used in the present study. This clinical study report amendment describes the outcome of this investigation (Amended: 04 December 2013). 18 9b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 18

20 3. INTRODUCTION GlaxoSmithKline (GSK) Biologicals has developed a prophylactic HPV vaccine based on L1 proteins of HPV-16 and HPV-18 formulated with AS04 (comprised of aluminum hydroxide [Al(OH) 3 ] and 3-O-desacyl-4 -monophosphoryl lipid A [MPL]). To date, more than adolescent and adult females aged 10 years and above have received at least one dose of the vaccine in clinical studies. The vaccine was shown to be immunogenic and generally safe and well-tolerated [GlaxoSmithKline Biologicals, Data On File Summary]. When tested in young female adolescents, the vaccine induced immune responses that were approximately 2-fold higher than those elicited in women years of age [Pedersen, 2007]. Results of an interim efficacy analysis of a large multinational Phase III trial in women aged years demonstrated high vaccine efficacy against cervical intraepithelial neoplasia grade 2 and above (CIN2+), CIN1+ and persistent infection (6-month and 12-month definition) associated with HPV-16 and/or HPV-18, as well as significant vaccine efficacy against 6-month persistent infection with HPV types 45, 31 and 52 [Paavonen, 2007]. This study also demonstrated the vaccine to be generally well tolerated in a broad range of women including those of different nationalities and ethnicities. The results of a long-term efficacy follow-up study in women aged years at the time of first vaccination demonstrated high vaccine efficacy against incident and persistent HPV- 16/18 infections and their associated cytological cervical lesions with up to 6.4 years of follow-up [Gall, 2007; Harper, 2004; Harper 2006; Harper, 2008]. The first major market in which the HPV vaccine under evaluation in this study was licensed is Australia for use in 10 to 45 year old females. The vaccine is marketed under the name Cervarix. In September 2007, the vaccine was licensed in Europe for active immunization of girls and women from 10 years of age onwards for the prevention of persistent human papillomavirus (HPV) infections and related clinical outcomes (cytological abnormalities and pre-cancerous lesions) caused by oncogenic HPV types 16 and 18. The vaccine is currently licensed in over 90 countries worldwide. Study (HPV-015) is a phase III, controlled study to evaluate the safety, immunogenicity and efficacy of GSK Biologicals HPV-16/18 L1 VLP AS04 vaccine in women aged 26 years and above. The study includes two parallel groups receiving either three doses of HPV-16/18 L1 VLP AS04 vaccine or control (Al(OH) 3 ). To better characterize the immunological response elicited by vaccination with the HPV-16/18 L1 VLP AS04 vaccine, additional samples were collected at pre-selected sites from subjects participating in the study (HPV-015). Additional testing of peripheral blood mononuclear cells (PBMC)/cervicovaginal secretions (CVS) samples and serum samples was performed. This ancillary study consisted of supplemental testing of serological and cervical samples obtained at Visit 5 (Month 12) and Visit 6 (Month 18) of the primary study (HPV-015). This report presents the analyses of data collected at Month 12 and Month 18 ( HPV-028 ANC 015). 19 9b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 19

21 4. STUDY OBJECTIVES To measure T-cell-mediated immune responses specific to HPV virus-like particles (VLP) types 16 and 18 in peripheral blood cells. To measure memory B-cell immune responses specific to HPV VLP types 16 and 18 in peripheral blood cells. To evaluate anti-hpv-16 and anti-hpv-18 antibody responses in cervical samples. To evaluate the total level of immunoglobulin type G (IgG) antibodies in cervical samples collected in this study, and in serum samples collected in study (HPV-015). To compare anti-hpv-16 and anti-hpv-18 antibody levels in cervical samples collected in this study with antibody levels in serum samples collected in study (HPV-015). See Section 5.10 for details of the study endpoints. 5. INVESTIGATIONAL PLAN 5.1. Study design Overall study design Description Study (HPV -015) Randomization (1:1) Ancillary Study (HPV-028 ANC: 015) HPV -16/18 L1/AS04 vaccine (N=2700) Al(OH) 3 control (N=2700) Visit 1 Month 0 Visit 2 Month 1 Visit 3 Month 6 Visit 4 Month 7 Visit 5 Month 12 Visit 6 Month 18 Visit 7 Month 24 Visit 8 Month 30 Visit 9 Month 36 Vaccination I II III Blood sample I II III*^ IV*^ V VI Cervical sample I II III*^ IV*^ V VI VII * An additional 40mL blood sample ^ An additional CVS sample HPV-015 study conclusion Experimental design: a phase III, double-blind, controlled ancillary study performed as a supplement to study (HPV-015). The present study was conducted at pre-selected sites participating in study (HPV-015). 20 9b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 20

22 Study population: subjects who enrolled and received three doses of vaccine/control in study (HPV-015) from pre-selected sites were asked to participate in the study. In study (HPV-015), subjects were randomized (1:1) to two parallel groups, the HPV group who received HPV-16/18 L1 VLP AS04 vaccine and the Control group who received Al(OH) 3, and according to the following age stratification: 45% of the subjects in the years of age group, 45% of the subjects in the years of age group, and 10% of the subjects in the above 45 years of age group. Vaccination schedule: three doses of vaccine/control were administered intramuscularly according to a 0, 1, 6-month schedule in study (HPV-015). No additional doses of vaccine/control were administered in this ancillary study. Type of study: ancillary study to protocol (HPV-015). Two scheduled visits: Visit 5 (Month 12) and Visit 6 (Month 18) after the first vaccine dose in study (HPV-015). Blood samples: 40 ml blood samples were collected at Visit 5 (Month 12) and at Visit 6 (Month 18) for evaluation of cell-mediated immune responses in subjects recruited from pre-selected study sites. These blood samples were in addition to the 10 ml blood samples taken according to the protocol for study (HPV-015). Cervicovaginal samples: CVS samples were collected at Visit 5 (Month 12) and at Visit 6 (Month 18) for antibody detection. These samples were to be collected before the liquid-based cytology samples taken according to the protocol for study (HPV-015). Data collection: Remote Data Entry (RDE)/electronic Case Report Form (ecrf) Duration of the study: approximately six months for each subject. An interim immunogenicity analysis of the ancillary study was planned in the protocol when all subjects had completed Visit 5 (Month 12). Final analysis of the ancillary study was performed when all subjects had completed Visit 6 (Month 18). The current report presents the results of the final analysis of immunogenicity data collected at Visit 5 (Month 12) and Visit 6 (Month 18). No safety data were collected in this ancillary study. All safety data were collected in the context of study (HPV- 015) and are not presented in this ancillary study report. 21 9b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 21

23 5.2. Study procedures Outline of study procedures Table 1 provides an overview of the study procedures that were to be followed by the investigator at the identified study centers. Ancillary procedures were performed in addition to those already scheduled as a part of study HPV-015 (see HPV-015 (104820) Protocol Amendment 3 ). Table 1 Outline of study procedures performed during ancillary study conducted at Visit 5 and Visit 6 Visit VISIT 5 VISIT 6 Timing Month 12 Month 18 Sampling time point Post vacc III Post vacc III Main protocol procedures Check elimination criteria Record concomitant medication/vaccination Gynecological examination according to local practice 1,2,3 Blood sampling: for antibody determination (10 ml) Collection of cervical samples 1 Reporting of pregnancies and pregnancy outcomes Reporting of SAEs, new onset chronic diseases and other medically significant conditions Administration of behavioral questionnaire 4 Referral for colposcopy 3 Reporting of all colposcopy results 5 Counseling Additional ancillary protocol procedures Informed consent Check inclusion and exclusion criteria Cervical secretions sampling: for antibody response 6 Record time point of sample collection during subject s menstrual cycle Blood sampling: for CMI* response (40 ml) Ancillary Study Conclusion 7 Procedures of the main protocol are listed for information only. Procedures of the main protocol were to be recorded following instructions of the main protocol. indicates a study procedure that was to be documented in the subject s ancillary ecrf. indicates a study procedure that was to be recorded in the subject s ecrf in study (HPV-015). The double-line border after Visit 5 (Month 12) indicates the interim analysis that was planned on data obtained at that visit. * Cell Mediated Immunity was only measured in subjects enrolled in this ancillary study. 1 Gynecological examinations and collection of cervical specimens were to be suspended in women known to be pregnant and were to be resumed three months after resolution. Missing procedures did not need to be rescheduled. 2 Cervical samples were used for cervical cytology at Month 12 and HPV DNA typing at Month 12 and Month Women with abnormal cervical cytology were to be evaluated according to the cytology and colposcopy clinical management algorithms included in the protocol for study (HPV-015). 4 A questionnaire was to be administered to the subjects through an interview by study personnel to collect data about their current sexual behavior included as part of the protocol for study (HPV-015). 5 All colposcopy results, including any results obtained from outside the study, were to be reported in the ecrf. Treatment following Vulval Intraepithelial Neoplasia (VIN) or Vaginal Intraepithelial Neoplasia (VaIN) was to be recorded in the ecrf. 22 9b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 22

24 6 One cervical sample was to be collected at Visit 5 and at Visit 6: a cervicovaginal secretions sample for this ancillary protocol using Mercocel (or equivalent) sponges for the detection of HPV-16 and HPV-18 antibody responses; and a second liquid-based cytology (LBC) sample collected as already planned in the study (HPV-015). 7 For the interim analysis, an interim conclusion page was included in the ecrf Intervals between study visits It was the investigator s responsibility to ensure that the intervals between visits were strictly followed. The length of the interval for Visit 5 (Month 12) was between 301 and 480 days post Visit 1 (Month 0) with a recommended interval of 365 days post Visit 1. The length of the interval for Visit 6 (Month 18) was between 481 and 660 days post Visit 1 (Month 0) with a recommended interval of 545 days post Visit Selection of study population Approximately 100 subjects enrolled at pre-selected sites in study (HPV-015) who were included in the immunogenicity subset and had received all three doses of vaccine/control were asked to participate in the present ancillary study. This ancillary study was conducted in two centers located in the Netherlands Inclusion criteria All subjects had to satisfy the following criteria at study entry: A female enrolled in study (HPV-015) and who had received three doses of study vaccine/control. Subjects who the investigator believed that they could and would comply with the requirements of the protocol (e.g. return for follow-up visits). Written informed consent obtained from the subject prior to enrolment in this ancillary study Exclusion criteria The following criteria were to be checked at the time of ancillary study entry. If any applied, the subject was not to be included in the study: Pregnancy. Administration of any HPV vaccine other than that foreseen by the study protocol. Use of any investigational or non-registered product (drug or vaccine) other than the study vaccine since study start. Chronic administration (defined as more than 14 days) of immunosuppressants or other immune-modifying drugs since study start. (For corticosteroids, this meant prednisone, or equivalent, 0.5 mg/kg/day. Inhaled and topical steroids were allowed.) 23 9b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 23

25 Administration of immunoglobulins and/or any blood products within 90 days preceding a blood sampling Elimination criteria The following criteria were to be checked at each visit subsequent to the first visit. If any became applicable during the study, it was not required to withdraw the subject from the study but it determined a subject s evaluability in the ATP analysis. Pregnancy within the vaccination phase of the study, i.e. up to two months after completion of the vaccination series. Use of any investigational or non-registered product (drug or vaccine) other than the study vaccine during the study period (Month 0 to 36). Chronic administration (defined as more than 14 days) of immunosuppressants or other immune-modifying drugs occurring less than three months prior to the study visit. (For corticosteroids, this meant prednisone, or equivalent, 0.5 mg/kg/day. Inhaled and topical steroids were allowed.) Administration of a vaccine not foreseen by the study protocol during the period starting from 30 days before each dose of study vaccine and ending 30 days after (i.e. Days 0-29). Administration of routine meningococcal, hepatitis A or B, inactivated influenza, diphtheria/tetanus and/or diphtheria/tetanus-containing vaccine up to 8 days before each dose of study vaccine was allowed if the subject was outside of the 30 days follow-up period of the previous dose. Administration of any HPV vaccine other than that foreseen by the study protocol during the study period (Administration of any HPV vaccine other than that foreseen by the study protocol will also result in withdrawal from the study.). Administration of immunoglobulins and/or any blood products within 90 days preceding a vaccination visit or blood sampling. Subjects who had a colposcopy completed outside the study (after study entry) but whose biopsy sample was not available for study purposes Contraindications to subsequent doses of vaccine Not applicable to this ancillary study. No vaccine was administered Subject completion and withdrawal from study Subject completion A subject who returned for the concluding visit foreseen in the protocol (i.e. Visit 6 [Month 18]) was considered to have completed the ancillary study. 24 9b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 24

26 Subject withdrawal from the study From an analysis perspective, a withdrawal from the study was any subject who did not come back for the concluding visit foreseen by the protocol. A subject was qualified as a withdrawal from the study when no study procedure had occurred, no follow-up had been performed and no further information had been collected for this subject from the date of withdrawal/last contact. Investigators were to make an attempt to contact those subjects who did not return for scheduled visits or follow-up. All efforts were made to ensure active follow-up for women missing any study visit. Study personnel were to make at least three attempts to contact the subject by using established local communication methods. Each attempt was to be documented by study personnel and had to include any reason given by the subject for the missed study visit. Information relative to the withdrawal was to be documented on the Study Conclusion page of the ecrf. The investigator was to document whether the decision to withdraw from the study was made by the subject or the investigator and which of the following possible reasons was responsible for withdrawal: serious adverse event non-serious adverse event protocol violation (specify) consent withdrawal, not due to an adverse event administration of HPV vaccine other than that foreseen by the study protocol unblinding to allow the subject to decide if she would consider immunization with a licensed HPV vaccine moved from the study area lost to follow-up other (specify) Subject withdrawal from administration of the investigational product Not applicable to this ancillary study. No vaccine was administered Composition and administration of vaccine No additional vaccine dose was administered during the course of the ancillary protocol. 25 9b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 25

27 Treatment allocation and randomization Subjects were randomized at the time of enrolment in study (HPV-015). Please refer to Section 6.4 of the protocol for study (HPV-015) for a description of treatment allocation and randomization methods. Subject identification numbers allocated in study (HPV-015) were used in this ancillary study Blinding The double blind applied to this ancillary study. Please refer to Section 6.5 of the (HPV-015) protocol for the description of blinding methods and the procedure to be followed in case the investigator determined the need to break the study blind (see HPV- 015 (104820) Protocol Amendment 3 ). To maintain blinding of study (HPV-015), the analyses of study (HPV- 028 ANC 015) were done by an external statistician. Individual listings will be provided after completion of study (HPV-015) Prior and concomitant medication /vaccinations Please refer to Section 6.9 of the protocol for study (HPV-015) for a description of methods for concomitant medication/treatment reporting (see HPV-015 (104820) Protocol Amendment 3 ) Laboratory assays and time points The laboratory assays that were used during this ancillary study are summarized in Table b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 26

28 Table 2 Laboratory assays Assay type Marker Assay method Test kit/ Assay unit Assay Laboratory Manufacturer cut-off Quantitative Anti-HPV-16 ELISA (or multiplex) In-house assay EL.U/mL 8 EL.U/mL GSK Biologicals* Quantitative Anti-HPV-18 ELISA (or multiplex) In-house assay EL.U/mL 7 EL.U/mL GSK Biologicals* Quantitative Total IgG Commercial Commercial G/mL NA GSK Biologicals* Quantitative Anti-HPV-16 Anti-HPV-18 Cytokine flow cytometry assay GSK Biologicals Frequency of specific T-cells (cells per million NA GSK Biologicals* Quantitative Anti-HPV-16 Anti-HPV-18 Quantitative Anti-HPV-16 Pseudovirion based neutralization assay Quantitative Anti-HPV-18 Pseudovirion based neutralization assay cells) B-cell ELISPOT GSK Biologicals Frequency of specific memory B-cells (cells per million cells) NCI methodology modified by GSK 1 NCI methodology modified by GSK 1 NA GSK Biologicals* ED50 40 GSK Biologicals* ED50 40 GSK Biologicals* NA = not applicable * GSK Biologicals or designated laboratory 1 NCI = National Cancer Institute; The pseudovirion neutralization assay is based on a method developed by Pastrana, et al. (Pastrana 2004) at the NCI. Alternatively, an assay for detection of Total IgG may be developed by GSK. At the discretion of GSK, the protocol allowed for expansion of these assays to measure the immunological response of additional HPV type VLPs including HPV-31, 45, 52, 33, 58 and 59 during or after the final analysis. The GSK Biologicals clinical laboratories at Rixensart have established a Quality System supported by procedures. The activities of the clinical laboratories are audited regularly for quality assessment by an internal (sponsor-dependent) but laboratoryindependent Quality Department. The protocol and informed consent allowed for extra analyses (not described in the protocol) related to the immune response to HPV vaccination if deemed necessary Serology Serological assays were to be performed at GSK Biologicals laboratories, Rixensart, Belgium using standardized procedures with adequate controls. At Months 12 and 18 a 20 ml sample of whole blood (to provide a minimum of 10 ml of serum) was to be taken from each subject. 27 9b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 27

29 ELISA As part of the (HPV-015) protocol, anti-hpv-16 and anti-hpv-18 ELISA were performed on serological samples collected at Months 12 and 18. In addition, anti-hpv- 16 and anti-hpv-18 ELISA were performed on CVS samples collected at Months 12 and 18 in the ancillary study Pseudovirion based neutralization assay (PBNA) As part of the (HPV-015) protocol, anti-hpv-16 and anti-hpv-18 pseudovirion (PSV) neutralization assays were performed on serological samples collected at Months 12 and IgG total In order to account for the variation of the anti-hpv-16 and anti-hpv-18 titers and the Total IgG levels in the CVS during the menstrual cycle, Total IgG was also measured in the paired serum samples by an ELISA assay Cell mediated immune (CMI) T and B-cell responses At Months 12 and 18 an additional 40 ml blood sample was to be taken from all subjects. These blood samples were intended for the determination of CMI T and B-cell responses. Samples were to be stored in liquid nitrogen until shipment to GSK Biologicals Cervicovaginal secretion (CVS) samples for antibody detection At Months 12 and 18, CVS samples for evaluation of anti-hpv-16 and anti-hpv-18 antibodies were collected using Merocel ophthalmic sponges or equivalent by placing the sponge portion of the device in contact with the cervix for about one minute. Following collection, the sample was placed into a sterile cryovial and frozen at -20 C or colder (-70 C/-80 C was also acceptable) within a minute (in order to preserve the quality of the sample). All CVS samples were shipped to GSK Biologicals (Rixensart, Belgium) in a frozen state (dry ice [-70 C/-80 C]). Antibodies were extracted from the collected mucus by two successive washing steps, and the extracted samples were tested for their blood content using the Hemastix test. CVS extracted samples showing a concentration of 200 erythrocytes/µl were excluded from statistical analysis Assessment of immunogenicity variables Table 3 presents a summary of sampling time points and assays for the assessment of immunology variables in this ancillary study. 28 9b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 28

30 Table 3 Summary of blood sampling time points and assays for the assessment of immunology variables Blood/cervical sampling time point Marker No. subjects Timing Month Visit no. Post vacc III 12 5* HPV-16, HPV-18, IgG, CMI 100 Post vacc III 18 6* HPV-16, HPV-18, IgG, CMI 100 CMI = Cell Mediated Immune response Post vacc III: post vaccination III At the discretion of GSK, the protocol allowed for testing of additional HPV-type antibodies including HPV-31, 45, 52, 33, 58 and 59 antibodies during or after the final analysis. * Visit number according to HPV-015(104820) Protocol. In case of insufficient blood sample volume, the assays were to be performed according to the following priority ranking: 1. ELISA 2. PSV neutralization assays In case of insufficient sample volume to perform all assays for all antibodies, the assays were to be performed according to the following priority ranking: 1. HPV-16 antibody (by ELISA) 2. HPV-18 antibody (by ELISA) 5.8. Assessment of safety variables Safety data were not collected during this ancillary study. Please refer to Section 8 of the protocol for study (HPV-015) for the description of adverse event and serious adverse event reporting, which is part of study (HPV-015) (see HPV-015 (104820) Protocol Amendment 3 ) Data quality assurance To ensure that the study procedures conformed across all investigator sites, the protocol and case report form were reviewed with the investigators and their personnel responsible for the conduct of the study by the Company representative(s) prior to study start. Adherence to the protocol requirements and verification of data generation accuracy were achieved through monitoring visits to each investigator site. Computer checks and blinded review of subject tabulations were performed to ensure consistency of ecrf completion. All procedures were performed according to methodologies detailed in GlaxoSmithKline Biologicals Standard Operating Procedures (SOPs). GSK Biologicals investigated the quality of some serology assays used in clinical studies, including the HPV-16/18 ELISA that is used in the present study. As part of this investigation, the HPV-16/18 ELISA was found to be not in line with the quality standards defined in GSK Biologicals SOPs. 29 9b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 29

31 Therefore, the dataset and analyses performed in the present study were reviewed. It was concluded that, while the assay performance may have impacted the results for some individual subjects, the overall study conclusions pertaining to the HPV-16/18 ELISA remain unchanged (Amended: 04 December 2013). Independent Audit statement: No study specific audits were performed for this ancillary study Statistical methods The final analyses were performed as specified in the protocol dated 07 February 2007 and the Report Analysis Plan (RAP) dated 26 August This section summarizes the statistical analysis actually performed. Any changes to the planned analysis are described in Section The exact 95% CIs for proportion within a group was calculated from Proc StatXact 5.0 assuming independence between doses. The 95% CI for GMT was obtained within each group separately. The 95% CI for the mean of log-transformed titer was first obtained assuming that log-transformed titers were normally distributed with unknown variance. The 95% CI for the GMT was then obtained by exponential transformation of the 95% CI for the mean of the logtransformed titers Endpoints T-cell-mediated immune responses specific to HPV VLP types 16 and 18 (Month 12 and Month 18). Memory B-cell immune responses specific to HPV VLP types 16 and 18 (Month 12 and Month 18). HPV-16 and HPV-18 antibody titers assessed by ELISA in CVS samples at Month 12 and 18 in subjects who had CVS samples collected at Month 12 and 18 (seropositivity rates and geometric mean titers [GMTs]). Total IgG antibodies (ELISA) from CVS samples collected in this study, and serum samples collected in study (HPV-015) at Month 12 and Month 18. Correlation of anti-hpv-16 and anti-hpv-18 in serum collected in study (HPV-015) and in cervical responses collected in this study. 30 9b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 30

32 Exploratory endpoints At the discretion of GSK, all endpoints above could be expanded to HPV types not included in the vaccine. At the discretion of GSK, other immune parameters related to HPV or the HPV vaccine constituents could be tested. This may include evaluation of neutralizing antibodies Determination of sample size Approximately 100 subjects enrolled in pre-selected sites participating in study (HPV-015) were to be enrolled Study cohorts analyzed Total Vaccinated cohort The Total Vaccinated cohort (TVC) included all selected subjects who had received at least one vaccine dose in study (HPV-015) Modified Total Vaccinated cohort The Modified Total Vaccinated cohort (MTVC) included all vaccinated subjects from the two selected study sites who received three vaccine doses and for whom data were available. The analysis was planned to be performed on the MTVC. Since all subjects of the TVC received three doses of vaccine, the MTVC was equivalent to the TVC, and the population is called TVC throughout this ancillary study report According to Protocol cohort An According-to-Protocol (ATP) analysis was not planned [see Report Analysis Plan HPV-028 ANC 015] Derived and transformed data The assay cut-off value was defined by the laboratory before the analysis and is described in Section 5.6. A seronegative subject was a subject whose antibody titer was below the assay cutoff value. A seropositive subject was a subject whose antibody titer was greater than or equal to the assay cut-off value. The Geometric Mean Titers (GMTs) calculations were performed by taking the antilog of the mean of the log titer transformations. Antibody titers below the cut-off of 31 9b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 31

33 the assay were given an arbitrary value of half the cut-off for the purpose of GMT calculation. The Geometric Mean (GMean) of T-cell or B-cell frequencies calculations were performed by taking the anti-log of the mean of the log frequency transformations. A B-cell frequency equal to 0 was given an arbitrary value of 1 for the purpose of GMean calculation. Handling of missing data: for a given subject and a given immunogenicity measurement, missing or non-evaluable measurements were not replaced Analysis of immunogenicity All analyses were performed according to the RAP dated 26 August 2008 (Report Analysis Plan HPV-028 ANC 015) Within groups assessment Antibody responses in serum samples For each treatment group at each blood sampling time point and for each antibody for which results were available: Seropositivity rates with 95% CIs were tabulated. GMTs with 95% CIs were tabulated. If the seropositivity rates were low, GMTs were to be calculated on seropositive subjects only. Antibody responses were presented using Reverse Cumulative Curves (RCC) Cervicovaginal secretions (CVS) samples For each antibody titer (HPV-16 and HPV-18) measured by ELISA at Month 12 and 18 in CVS, the following descriptive statistics were presented per vaccine group: Seroconversion and seropositivity rates (with exact 95% CI) were calculated. GMT with 95% CI and range of antibody titers were tabulated. Pearson coefficients of correlation between serum and CVS for anti-hpv-16 and anti-hpv-18 titers standardized for Total IgG were calculated and scatter plots were produced. Correlations between serum and CVS samples for HPV-16 and HPV-18, standardized for Total IgG, by age strata, for subjects with Hemastix-values less than or equal to 200 erythrocytes/l were calculated. 32 9b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 32

34 T-cell responses by Intracellular Cytokine Staining (ICS) The ICS assay provides information on the frequency of CD4+ and CD8+ T-cells responding to a specific antigen and producing: at least CD40L and another cytokine (IFN, IL-2, TNF) (d-cd40l); at least IL-2 and another cytokine (CD40L, TNF, IFN) (d-il-2); at least TNF and another cytokine (CD40L, IL-2, IFN) (d-tnf); at least IFN and another cytokine (IL-2, TNF, CD40L) (d-ifn); at least two different cytokines (CD40L, IL-2, TNF, IFN) (all doubles). Frequency of cytokine-positive (d-cd40l, d-il2, d-tnf, d- IFN or all doubles) CD4+ or CD8+ T-cells, for each stimulant at each timing were summarized for each group by the number of values (N), the number of missing values, minimum, 1st quartile, median, 3rd quartile, maximum and geometric mean (Gmean) with 95% CI. Further, for each test and for each stimulant at each time point, the number and percentage of subjects with greater than or equal to500 cells/million cells for CD4 T-cells and greater than or equal to200 cells/million cells for CD8 T-cells were calculated B-cell responses by ELISPOT The results of each stimulant at each timing were summarized for each group by the number of values (N), the number of missing values, minimum, 1st quartile, median, 3rd quartile, maximum and geometric mean (Gmean) with 95%CI. Values of zero will be given an arbitrary value of one for the purpose of geometric mean calculation. Further, for each stimulant at each time point, the number and percentage of subjects above zero were calculated Interim analysis An immunogenicity interim analysis was planned in the protocol after all subjects had completed Visit 5 (Month 12). This analysis was not performed (see Section ) Changes in the conduct of the study or planned analyses Protocol amendments There was no amendment or modification to the protocol. 33 9b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 33

35 Other changes An interim analysis at Month 12 was initially planned but was not performed. As all results (Month 12 and Month 18) were available at the time of the first analysis, a single analysis was performed for both Month 12 and Month 18 data. 6. STUDY POPULATION RESULTS 6.1. Study dates The first volunteer was enrolled in the study on 05 April 2007 and the last study visit was on 02 January The data lock point for all immunogenicity data was 07 October Subject eligibility and attrition from the study Number of subjects A total of 100 subjects, 53 in the HPV group and 47 in the Control group, were enrolled in this study (see Table 4). All were included in the Total Vaccinated Cohort (TVC). Table 4 Number of subjects enrolled into the study as well as the number excluded from TVC analyses with reasons for exclusion Total HPV CNTRL Title n s % n s n s Total enrolled cohort 100 TVC HPV = HPV-16/18 L1 VLP AS04 vaccine CNTRL = Al(OH)3 Control Note: Subjects may have more than one elimination code assigned n = number of subjects with the elimination code assigned excluding subjects who have been assigned a lower elimination code number s = number of subjects with the elimination code assigned % = percentage of subjects in the considered ATP cohort relative to the TVC Study completion and withdrawal from study All enrolled subjects (n=100) completed Visit 5 (Month 12). All subjects (n = 99) except one completed Visit 6 (Month 18) (see Table 5). Refer to Supplement 1 for the number of subjects by study centre in the TVC. 34 9b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 34

36 Table 5 Number of subjects completing each visit (TVC) HPV N = 53 CNTRL N = 47 Total N = 100 Parameters or Value % Value % Value % Characteristics Categories or n or n or n Number completing Visit 5 Month Number completing Visit 6 Month Month 18 visit not done HPV = HPV-16/18 L1 VLP AS04 vaccine CNTRL = Al(OH)3 Control N = number of subjects n = number of subjects in a given category Value = value of the considered parameter % = n / Number of subjects with available results x Demographic characteristics Total vaccinated cohort A summary of demographic characteristics of the TVC is presented in Table 6. The demographic profiles of the HPV and Control groups were comparable with respect to mean age and menopausal status. The mean age was 40.9 years, and ranged from 26.0 to 56.0 years, in the HPV group and was 40.3 years, and ranged from 26.0 to 65.0 years, in the Control group. The majority of subjects in each group were pre-menopausal (66.0% in the HPV group and 70.2% in the Control group). Table 6 Summary of demographic characteristics (TVC) HPV N = 53 CNTRL N = 47 Total N = 100 Parameters or Value % Value % Value % Characteristics Categories or n or n or n Age (years) Mean SD Median Minimum Maximum Menopausal Status Pre-menopausal Peri-menopausal Post-menopausal Unknown HPV = HPV-16/18 L1 VLP AS04 vaccine CNTRL = Al(OH)3 Control N = number of subjects n = number of subjects in a given category Value = value of the considered parameter % = n / Number of subjects with available results x b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 35

37 The DNA and serological pre-vaccination status in study (HPV-015) for the TVC are presented in Table 7. The profiles of the Control and HPV groups are similar. In the Control group, no subjects were DNA positive for HPV-16 or HPV-18 subjects at Month 0, and 67.4% and 82.6% of the subjects were DNA negative and seronegative for HPV- 16 and HPV-18, respectively, at baseline. In the HPV group, 63.5% and 73.1% of the subjects were DNA negative and seronegative at baseline for HPV-16 and HPV-18, respectively. Also in the HPV group, 28.8% were DNA negative and seropositive, 1.9% were DNA positive and seronegative and 5.8% were DNA positive and seropositive for HPV-16; while 25.0% were DNA negative and seropositive, 1.9% were DNA positive and seronegative and no subjects were DNA positive and seropositive for HPV-18. Table 7 DNA and serological status at baseline (TVC) HPV N = 53 CNTRL N = 47 Total N = 100 Parameters or Value % Value % Value % Characteristics Categories or n or n or n HPV-16 DNA and sero (ELISA) DNA neg and Sero neg DNA neg and Sero pos DNA pos and Sero neg DNA pos and Sero pos Missing HPV-18 DNA and sero (ELISA) DNA neg and Sero neg DNA neg and Sero pos DNA pos and Sero neg DNA pos and Sero pos Missing HPV = HPV-16/18 L1 VLP AS04 vaccine CNTRL = Al(OH)3 Control N = number of subjects n = number of subjects in a given category Value = value of the considered parameter % = n / Number of subjects with available results x 100 neg = negative pos = positive sero = serological 7. IMMUNOGENICITY RESULTS 7.1. Data sets analyzed The analysis of immunogenicity was performed on the TVC (primary analysis). See Section for the definition of the cohorts identified for analyses and Section 6.2 for eligibility for analyses. The data lock point for all immunogenicity data except the neutralization data was 07 October b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 36

38 7.2. Total vaccinated cohort analysis Antibody responses in serum Antibody responses by ELISA The seropositivity rates and GMTs at Months 12 and 18 by pre-vaccination serostatus for anti-hpv-16 and anti-hpv-18 antibodies are presented in Table 8 and Table 9, respectively. At Month 12 and at Month 18 all subjects in the HPV group were seropositive for HPV-16 and HPV-18 antibodies. For subjects in the HPV group who were initially seronegative at baseline, the GMTs for anti-hpv-16 antibodies were EL.U/mL [95% CI: ; ] at Month 12 and EL.U/mL [95% CI: 891.2; ] at Month 18. The GMTs for anti-hpv-18 antibodies were EL.U/mL [95% CI: 437.7; 949.7] at Month 12 and EL.U/mL [95% CI: 317.5; 697.1] at Month 18. For subjects in the HPV group who were initially seropositive at baseline, the GMTs for anti-hpv-16 antibodies were EL.U/mL [95% CI: ; ] at Month 12 and EL.U/mL [95% CI: 934.6; ] at Month 18. The GMTs for anti-hpv-18 antibodies were EL.U/mL [95% CI: 566.1; ] at Month 12 and EL.U/mL [95% CI: 379.7; ] at Month 18. For subjects in the HPV group who were initially seronegative at baseline, the RCC for HPV-16 and HPV-18 IgG antibody titers at Month 12 and at Month 18 are shown in Supplement 2, Supplement 3, Supplement 4 and Supplement 5, respectively. 37 9b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 37

39 Table 8 Seropositivity rates and GMTs by ELISA for HPV-16 L1 VLP IgG antibodies by pre-vaccination status (TVC) Antibody Group HPV-16 VLP IgG Prevacc status 8 ELU/ML GMT 95% CI 95% CI Timing N n % LL UL value LL UL Min Max HPV S- PIII(M12) PIII(M18) S+ PIII(M12) PIII(M18) Total PIII(M12) PIII(M18) CNTRL S- PIII(M12) < PIII(M18) < S+ PIII(M12) < PIII(M18) < Total PIII(M12) < PIII(M18) < HPV = HPV-16/18 L1 VLP AS04 vaccine CNTRL = Al(OH)3 Control S- = seronegative subjects (antibody concentration < 8 ELU/ML) prior to vaccination S+ = seropositive subjects (antibody concentration 8 ELU/ML) prior to vaccination GMT = geometric mean titre N = number of subjects with pre-vaccination results available n/% = number/percentage of subjects with concentration within the specified range 95% CI = 95% confidence interval; LL = Lower Limit, UL = Upper Limit MIN/MAX = Minimum/Maximum PIII(M12)= Post-dose 3, Month 12 PIII(M18)= Post-dose 3, Month b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 38

40 Table 9 Seropositivity rates and GMTs by ELISA for HPV-18 L1 VLP IgG antibodies by pre-vaccination status (TVC) Antibody Group HPV-18 VLP IgG Prevacc status 7 ELU/ML GMT 95% CI 95% CI Timing N n % LL UL value LL UL Min Max HPV S- PIII(M12) PIII(M18) S+ PIII(M12) PIII(M18) Total PIII(M12) PIII(M18) CNTRL S- PIII(M12) <7.0 <7.0 PIII(M18) <7.0 <7.0 S+ PIII(M12) < PIII(M18) < Total PIII(M12) < PIII(M18) < HPV = HPV-16/18 L1 VLP AS04 vaccine CNTRL = Al(OH)3 Control S- = seronegative subjects (antibody concentration < 7 ELU/ML) prior to vaccination S+ = seropositive subjects (antibody concentration 7 ELU/ML) prior to vaccination GMT = geometric mean titre N = number of subjects with pre-vaccination results available n/% = number/percentage of subjects with concentration within the specified range 95% CI = 95% confidence interval; LL = Lower Limit, UL = Upper Limit MIN/MAX = Minimum/Maximum PIII(M12)= Post-dose 3, Month 12 PIII(M18)= Post-dose 3, Month Pseudovirion-based neutralizing antibodies (PBNA) assay PBNA responses at Months 12 and 18 by pre-vaccination serostatus to HPV-16 and HPV-18 are presented in Table 10 and Table 11. At Month 12 and at Month 18 all subjects in the HPV group were seropositive for anti- HPV-16 and anti-hpv-18 PSV neutralizing antibodies. For subjects in the HPV group who were initially seronegative at baseline, the GMTs for anti-hpv-16 PSV neutralizing antibodies were ED50 [95% CI: ; ] at Month 12 and EL.U/mL [95% CI: ; ] at Month 18. The GMTs for anti-hpv-18 PSV neutralizing antibodies were ED50 [95% CI: ; ] at Month 12 and EL.U/mL [95% CI: 856.4; ] at Month 18. For subjects in the HPV group who were initially seropositive at baseline, the GMTs for anti-hpv-16 PSV neutralizing antibodies were ED50 [95% CI: ; ] at Month 12 and EL.U/mL [95% CI: ; ] at Month 18. The GMTs for anti-hpv-18 PSV neutralizing antibodies were EL.U/mL [95% CI: ; ] at Month 12 and ED50 [95% CI: 896.2; ] at Month b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 39

41 For subjects in the HPV group who were initially seronegative at baseline, the RCC for HPV-16 and HPV-18 PSV neutralizing antibody titers at Month 12 and at Month 18 are shown in Supplement 6, Supplement 7, Supplement 8 and Supplement 9, respectively. Table 10 Seropositivity rates and GMTs for HPV-16 PSV neutralizing antibodies by pre-vaccination status (TVC) Antibody Group Pre-vacc status HPV-16 PSV AB 40 ED50 GMT 95% CI 95% CI Timing N n % LL UL value LL UL Min Max HPV S- PIII(M12) PIII(M18) S+ PIII(M12) PIII(M18) Total PIII(M12) PIII(M18) CNTRL S- PIII(M12) < PIII(M18) < S+ PIII(M12) PIII(M18) Total PIII(M12) < PIII(M18) < HPV = HPV-16/18 L1 VLP AS04 vaccine CNTRL = Al(OH)3 Control ED50 = effective dose 50 S- = seronegative subjects (antibody titre < 40 ED50) prior to vaccination S+ = seropositive subjects (antibody titre 40 ED50) prior to vaccination GMT = geometric mean antibody titre calculated on all subjects N = number of subjects with pre-vaccination results available n/% = number/percentage of subjects with titre within the specified range 95% CI = 95% confidence interval; LL = Lower Limit, UL = Upper Limit MIN/MAX = Minimum/Maximum PIII(M12)= Post-dose 3, Month 12 PIII(M18)= Post-dose 3, Month b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 40

42 Table 11 Seropositivity rates and GMTs for HPV-18 PSV neutralizing antibodies by pre-vaccination status (TVC) Antibody Group Pre-vacc status HPV-18 PSV AB 40 ED50 GMT 95% CI 95% CI Timing N n % LL UL value LL UL Min Max HPV S- PIII(M12) PIII(M18) S+ PIII(M12) PIII(M18) Total PIII(M12) PIII(M18) CNTRL S- PIII(M12) <40.0 <40.0 PIII(M18) <40.0 <40.0 S+ PIII(M12) < PIII(M18) < Total PIII(M12) < PIII(M18) < HPV = HPV-16/18 L1 VLP AS04 vaccine CNTRL = Al(OH)3 Control ED50 = effective dose 50 S- = seronegative subjects (antibody titre < 40 ED50) prior to vaccination S+ = seropositive subjects (antibody titre 40 ED50) prior to vaccination GMT = geometric mean antibody titre calculated on all subjects N = number of subjects with pre-vaccination results available n/% = number/percentage of subjects with titre within the specified range 95% CI = 95% confidence interval; LL = Lower Limit, UL = Upper Limit MIN/MAX = Minimum/Maximum PIII(M12)= Post-dose 3, Month 12 PIII(M18)= Post-dose 3, Month Detection of antibodies in CVS samples The seropositivity rates and GMTs at Month 12 and at Month 18 for HPV-16 and for HPV-18 antibodies by ELISA in CVS with Hemastix 200 erythrocytes/l are presented in Table 12 and Table 13. At Month 12 all subjects in the HPV group had detectable antibodies against HPV-16 and HPV-18 antigens. At Month 18, 96.2% and 88.5% of the subjects in the HPV group had detectable antibodies against HPV-16 and HPV-18 antigens, respectively. None of the subjects in the Control group had detectable antibodies against HPV-16 at Months 12 and 18; none of the subjects in the Control group had detectable antibodies against HPV-18 at Month 12 and 2 subjects at Month 18. For subjects in the HPV group, the GMTs for anti-hpv-16 antibodies were EL.U/mL [95% CI: 58.0; 289.2] at Month 12 and 99.9 EL.U/mL [95% CI: 48.9; 203.8] at Month 18. The GMTs for anti-hpv-18 antibodies were 67.2 EL.U/mL [95% CI: 28.6; 158.0] at Month 12 and 42.8 EL.U/mL [95% CI: 23.1; 79.6] at Month b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 41

43 Table 12 Seropositivity rates and GMTs for HPV-16 antibody (by ELISA) in CVS with Hemastix less than or equal to200 erythrocytes/microlitre (TVC) LOQ GMT 95% CI 95% CI Antibody Group Timing N n % LL UL value LL UL Min Max HPV-16 VLP IgG HPV PIII(M12) PIII(M18) CNTRL PIII(M12) PIII(M18) HPV = HPV-16/18 L1 VLP AS04 vaccine CNTRL = Al(OH)3 Control LOQ = Limit of quantification of secretion antibody test GMT = geometric mean titer calculated on subjects with result LOQ N = number of subjects with available results n/% = number/percentage of subjects with concentration within the specified range 95% CI = 95% confidence interval; LL = Lower Limit, UL = Upper Limit MIN/MAX = Minimum/Maximum PIII(M12)= Post-dose 3, Month 12 PIII(M18)= Post-dose 3, Month 18 Table 13 Seropositivity rates and GMTs for HPV-18 antibody (by ELISA) in CVS with Hemastix less than or equal to200 erythrocytes/microlitre (TVC) LOQ GMT 95% CI 95% CI Antibody Group Timing N n % LL UL value LL UL Min Max HPV-18 VLP IgG HPV PIII(M12) PIII(M18) CNTRL PIII(M12) PIII(M18) HPV = HPV-16/18 L1 VLP AS04 vaccine CNTRL = Al(OH)3 Control LOQ = Limit of quantification of secretion antibody test GMT = geometric mean titre on subjects with result LOQ N = number of subjects with available results n/% = number/percentage of subjects with concentration within the specified range 95% CI = 95% confidence interval; LL = Lower Limit, UL = Upper Limit MIN/MAX = Minimum/Maximum PIII(M12)= Post-dose 3, Month 12 PIII(M18)= Post-dose 3, Month b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 42

44 Correlations between serum and CVS for HPV-16 and HPV-18 antibody responses The correlations between serum and CVS for HPV-16 and HPV-18 antibody responses (by ELISA, standardized for Total IgG) on subjects with Hemastix 200 erythrocytes/l are shown in Figure 1 and Figure 2, respectively. The correlation coefficients between serum and CVS for HPV-16 IgG titers were at Month 12 and at Month 18. The correlation coefficients between serum and CVS for HPV-18 IgG titers were at Month 12 and at Month 18. Figure 1 Correlation between serum and CVS for HPV-16 antibody response (by ELISA) (standardized for Total IgG) for subjects with Hemastix less than or equal to200 erythrocytes/microlitre 43 9b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 43

45 Figure 2 Correlation between serum and CVS for HPV-18 antibody response (by ELISA) (standardized for Total IgG) for subjects with Hemastix less than or equal to200 erythrocytes/microlitre T-cell mediated immune responses by ICS The CMI response induced by the HPV-16/18 L1 VLP AS04 vaccine was characterized using ICS. HPV-16 and HPV-18 specific CD4 T-cell responses (proportion of subjects above 500 cells/million cells) are presented in Table 14 and Table 15, respectively. At Months 12 and 18, the proportions of subjects in the HPV group with HPV-16 specific CD4 T-cell responses were 55.8% and higher for all specified antigens except IFN (which was 21.2% at Month 12 and 26.9% at Month 18). The proportions of subjects in the HPV group with HPV-18 specific CD4 T-cell responses were 44.2% and higher for all specified antigens except IFN (which was 7.7% at Month 12 and 5.8% at Month 18). In the Control group, one subject showed an HPV-16 specific CD4 T-cell response at Month 12, and none of the subjects showed HPV-16 specific CD4 T-cell response at Month 18. None of the subjects in the Control group showed HPV-18 specific CD4 T-cell responses at either Month 12 or Month 18. HPV-16 and HPV-18 specific T-cell responses by ICS (descriptive statistics) are presented in Supplement 10 and Supplement 11, respectively. HPV-16 and HPV-18 specific CD8 T-cell responses (proportion of subjects above 200 cells/million cells) are presented in Supplement 12 and Supplement 13, respectively. 44 9b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 44

46 Table 14 HPV-16 specific CD4 T-cell responses (by ICS): proportion of subjects above 500 cells/million cells (TVC) HPV N = 53 CNTRL N = 47 Test Timing Parameters or Value % Value % Categories or n or n CD4-All doubles PIII(M12) N Below and above Missing PIII(M18) N Below and above Missing CD4-CD40L PIII(M12) N Below and above Missing PIII(M18) N Below and above Missing CD4-IFN PIII(M12) N Below and above Missing PIII(M18) N Below and above Missing CD4-IL2 PIII(M12) N Below and above Missing PIII(M18) N Below and above Missing CD4-TNF PIII(M12) N Below and above Missing PIII(M18) N Below and above Missing HPV = HPV-16/18 L1 VLP AS04 vaccine CNTRL = Al(OH)3 Control N = number of subjects n = number of subjects in a given category Value = value of the considered parameter % = n / Number of subjects with available results x b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 45

47 Table 15 HPV-18 specific CD4 T-cell responses (by ICS): proportion of subjects above 500 cells/million cells (TVC) HPV N = 53 CNTRL N = 47 Test Timing Parameters or Value % Value % Categories or n or n CD4-All doubles PIII(M12) N Below and above Missing PIII(M18) N Below and above Missing CD4-CD40L PIII(M12) N Below and above Missing PIII(M18) N Below and above Missing CD4-IFN PIII(M12) N Below and above Missing PIII(M18) N Below and above Missing CD4-IL2 PIII(M12) N Below and above Missing PIII(M18) N Below and above Missing CD4-TNF PIII(M12) N Below and above Missing PIII(M18) N Below and above Missing HPV = HPV-16/18 L1 VLP AS04 vaccine CNTRL = Al(OH)3 Control N = number of subjects n = number of subjects in a given category Value = value of the considered parameter % = n / Number of subjects with available results x b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 46

48 B-cell responses Memory B-cells specific to HPV-16 and HPV-18 were evaluated using the B-cell ELISPOT assay. HPV-16 and HPV-18 specific memory B-cell responses by ELISPOT (proportion of subjects above zero) are presented in Table 16 and Table 17, respectively. HPV-16 and HPV-18 specific memory B-cell responses by ELISPOT (descriptive statistics) are presented in Table 18 and Table 19, respectively. In the HPV group, 86.3% and 84.3% of subjects had a specific B-cell response to HPV-16 above zero at Month 12 and at Month 18, respectively. The median response was cells per million cells at Month 12 and cells per million cells at Month 18. In the HPV group, 84.3% of subjects had a specific B-cell response to HPV-18 above zero at both Month 12 and at Month 18. The median response was cells per million cells at Month 12 and cells per million cells at Month 18. In the Control group, 29.5% and 4.4% of subjects had a specific B-cell response to HPV-16 above zero at Month 12 and at Month 18, respectively. Also at Month 12 and at Month 18, 15.9% and 0.0% of the subjects, respectively, had a specific B-cell response to HPV-18 above zero. Table 16 HPV-16 specific memory B-cell responses (by ELISPOT): proportion of subjects above zero (TVC) HPV N = 53 CNTRL N = 47 Timing Parameters or Value % Value % Categories or n or n PIII(M12) N Above Missing PIII(M18) N Above Missing HPV = HPV-16/18 L1 VLP AS04 vaccine CNTRL = Al(OH)3 Control N = number of subjects n = number of subjects in a given category Value = value of the considered parameter % = n / Number of subjects with available results x b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 47

49 Table 17 HPV-18 specific memory B-cell responses (by ELISPOT): proportion of subjects above zero (TVC) HPV N = 53 CNTRL N = 47 Timing Parameters or Value % Value % Categories or n or n PIII(M12) N Above Missing PIII(M18) N Above Missing HPV = HPV-16/18 L1 VLP AS04 vaccine CNTRL = Al(OH)3 Control N = number of subjects n = number of subjects in a given category Value = value of the considered parameter % = n / Number of subjects with available results x 100 Table 18 HPV-16 specific memory B-cell responses (by ELISPOT) (TVC) HPV N = 53 CNTRL N = 47 95% CI 95% CI Test Timing Parameters or Categories Value or n LL UL Value or n LL UL B-cell PIII(M12) N Missing Minimum Q Median Q Maximum Gmean PIII(M18) N Missing Minimum Q Median Q Maximum Gmean HPV = HPV-16/18 L1 VLP AS04 vaccine CNTRL = Al(OH)3 Control N = number of subjects Q1 = 25% percentile; Q3 = 75% percentile Gmean = Geometric mean on subjects with a detectable B-cell response (frequency > 0) 95% CI = 95% Confidence Interval; LL = Lower Limit; UL = Upper Limit Value = value of the considered parameter PIII(M12) = Post Dose 3, Month 12 PIII(M18) = Post Dose 3, Month b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 48

50 Table 19 HPV-18 specific memory B-cell responses (by ELISPOT) (TVC) HPV N = 53 CNTRL N = 47 95% CI 95% CI Test Timing Parameters or Categories Value or n LL UL Value or n LL UL B-cell PIII(M12) N Missing Minimum Q Median Q Maximum Gmean PIII(M18) N Missing Minimum Q Median Q Maximum Gmean HPV = HPV-16/18 L1 VLP AS04 vaccine CNTRL = Al(OH)3 Control N = number of subjects Q1 = 25% percentile; Q3 = 75% percentile Gmean = Geometric mean on subjects with a detectable B-cell response (frequency > 0) 95% CI = 95% Confidence Interval; LL = Lower Limit; UL = Upper Limit Value = value of the considered parameter PIII(M12) = Post Dose 3, Month 12 PIII(M18) = Post Dose 3, Month b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 49

51 8. OVERALL CONCLUSIONS 8.1. Overall conclusions This ancillary study assessed the serum and CVS immunogenicity in a sub-population who received three doses of vaccine/control in study (HPV-015). All subjects in the HPV group were seropositive for HPV-16 and HPV-18 antibodies by ELISA and by PBNA at the Month 12 and Month 18 time points. Comparing the HPV and Control groups, it can be said that the HPV-16/18 L1 AS04 vaccine induced HPV-16 and HPV-18 specific cervicovaginal antibody responses. There was a good correlation between serum and CVS titers for both HPV-16 titers (r=0.903/0.898 for Month 12/Month 18, respectively) and HPV-18 IgG titers (r=0.901/0.876 for Month 12/Month 18, respectively). HPV-16/18-specific CD4 T-cell responses were higher in the HPV group than the vaccinated group (ICS 500 cells/million cells = 73.1 in HPV-group vs. 2.2 in Control group at Month 12; ICS 500 cells/million cells = 73.1 vs. 0.0 at Month 18) and HPV-18 (ICS 500 cells/million cells = 63.5 in HPV-group vs. 0.0 in Control group at Month 12; ICS 500 cells/million cells = 50.0 vs. 0.0 at Month 18) antibodies. The HPV-16/18 L1 AS04 vaccine induced immune memory for both HPV-16/18. ELISPOT results. 50 9b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 50

52 9. SUPPLEMENTS Supplement 1 Number of subjects by center (TVC) HPV CNTRL Total Center n n n % All HPV = HPV-16/18 L1 VLP AS04 vaccine CNTRL = Al(OH)3 Control n = number of subjects included in each group or in total for a given center or for all centers All = sum of all subjects in each group or in total (sum of all groups) % = n/all x 100 Center = GSK Biologicals assigned center number Supplement 2 Reverse cumulative distribution curve for HPV-16 IgG antibody titers (measured by ELISA) at Month 12 for seronegative subjects at baseline (TVC) 51 9b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 51

53 Supplement 3 Reverse cumulative distribution curve for HPV-16 IgG antibody titers (measured by ELISA) at Month 18 for seronegative subjects at baseline (TVC) 52 9b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 52

54 Supplement 4 Reverse cumulative distribution curve for HPV-18 IgG antibody titers (measured by ELISA) at Month 12 for seronegative subjects at baseline (TVC) 53 9b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 53

55 Supplement 5 Reverse cumulative distribution curve for HPV-18 IgG antibody titers (measured by ELISA) at Month 18 for seronegative subjects at baseline (TVC) 54 9b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 54

56 Supplement 6 Reverse cumulative distribution curve for HPV-16 PSV antibodies at Month 12 in seronegative subjects at baseline (TVC) 55 9b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 55

57 Supplement 7 Reverse cumulative distribution curve for HPV-16 PSV antibodies at Month 18 in seronegative subjects at baseline (TVC) 56 9b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 56

58 Supplement 8 Reverse cumulative distribution curve for HPV-18 PSV antibodies at Month 12 in seronegative subjects at baseline (TVC) 57 9b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 57

59 Supplement 9 Reverse cumulative distribution curve for HPV-18 PSV antibodies at Month 18 in seronegative subjects at baseline (TVC) 58 9b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 58

60 Supplement 10 HPV-16 specific T-cell responses (by ICS) (TVC) HPV N = 53 CNTRL N = 47 95% CI 95% CI Test Timing Parameters or Value LL UL Value LL UL Categories or n or n CD4-All doubles PIII(M12) N Missing Minimum Q Median Q Maximum Gmean PIII(M18) N Missing Minimum Q Median Q Maximum Gmean CD4-CD40L PIII(M12) N Missing Minimum Q Median Q Maximum Gmean PIII(M18) N Missing Minimum Q Median Q Maximum Gmean CD4-IFN PIII(M12) N Missing Minimum Q Median Q Maximum Gmean PIII(M18) N Missing Minimum Q Median Q Maximum Gmean b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 59

61 HPV CNTRL N = 53 N = 47 95% CI 95% CI Test Timing Parameters or Value LL UL Value LL UL Categories or n or n CD4-IL2 PIII(M12) N Missing Minimum Q Median Q Maximum Gmean PIII(M18) N Missing Minimum Q Median Q Maximum Gmean CD4-TNF PIII(M12) N Missing Minimum Q Median Q Maximum Gmean PIII(M18) N Missing Minimum Q Median Q Maximum Gmean CD8-All doubles PIII(M12) N Missing Minimum Q Median Q Maximum Gmean PIII(M18) N Missing Minimum Q Median Q Maximum Gmean b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 60

62 HPV CNTRL N = 53 N = 47 95% CI 95% CI Test Timing Parameters or Value LL UL Value LL UL Categories or n or n CD8-CD40L PIII(M12) N Missing Minimum Q Median Q Maximum Gmean PIII(M18) N Missing Minimum Q Median Q Maximum Gmean CD8-IFN PIII(M12) N Missing Minimum Q Median Q Maximum Gmean PIII(M18) N Missing Minimum Q Median Q Maximum Gmean CD8-IL2 PIII(M12) N Missing Minimum Q Median Q Maximum Gmean PIII(M18) N Missing Minimum Q Median Q Maximum Gmean b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 61

63 HPV CNTRL N = 53 N = 47 95% CI 95% CI Test Timing Parameters or Value LL UL Value LL UL Categories or n or n CD8-TNF PIII(M12) N Missing Minimum Q Median Q Maximum Gmean PIII(M18) N Missing Minimum Q Median Q Maximum Gmean HPV = HPV-16/18 L1 VLP AS04 vaccine CNTRL = Al(OH)3 Control N = number of subjects Q1 = 25% percentile; Q3 = 75% percentile Gmean = Geometric mean 95% CI = 95% Confidence Interval; LL = Lower Limit; UL = Upper Limit Value = value of the considered parameter PIII(M12) = Post Dose 3, Month 12 PIII(M18) = Post Dose 3, Month b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 62

64 Supplement 11 HPV-18 specific T-cell responses (by ICS) (TVC) HPV N = 53 CNTRL N = 47 95% CI 95% CI Test Timing Parameters or Value LL UL Value LL UL Categories or n or n CD4-All doubles PIII(M12) N Missing Minimum Q Median Q Maximum Gmean PIII(M18) N Missing Minimum Q Median Q Maximum Gmean CD4-CD40L PIII(M12) N Missing Minimum Q Median Q Maximum Gmean PIII(M18) N Missing Minimum Q Median Q Maximum Gmean CD4-IFN PIII(M12) N Missing Minimum Q Median Q Maximum Gmean PIII(M18) N Missing Minimum Q Median Q Maximum Gmean b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 63

65 HPV CNTRL N = 53 N = 47 95% CI 95% CI Test Timing Parameters or Value LL UL Value LL UL Categories or n or n CD4-IL2 PIII(M12) N Missing Minimum Q Median Q Maximum Gmean PIII(M18) N Missing Minimum Q Median Q Maximum Gmean CD4-TNF PIII(M12) N Missing Minimum Q Median Q Maximum Gmean PIII(M18) N Missing Minimum Q Median Q Maximum Gmean CD8-All doubles PIII(M12) N Missing Minimum Q Median Q Maximum Gmean PIII(M18) N Missing Minimum Q Median Q Maximum Gmean b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 64

66 HPV CNTRL N = 53 N = 47 95% CI 95% CI Test Timing Parameters or Value LL UL Value LL UL Categories or n or n CD8-CD40L PIII(M12) N Missing Minimum Q Median Q Maximum Gmean PIII(M18) N Missing Minimum Q Median Q Maximum Gmean CD8-IFN PIII(M12) N Missing Minimum Q Median Q Maximum Gmean PIII(M18) N Missing Minimum Q Median Q Maximum Gmean CD8-IL2 PIII(M12) N Missing Minimum Q Median Q Maximum Gmean PIII(M18) N Missing Minimum Q Median Q Maximum Gmean b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 65

67 HPV CNTRL N = 53 N = 47 95% CI 95% CI Test Timing Parameters or Value LL UL Value LL UL Categories or n or n CD8-TNF PIII(M12) N Missing Minimum Q Median Q Maximum Gmean PIII(M18) N Missing Minimum Q Median Q Maximum Gmean HPV = HPV-16/18 L1 VLP AS04 vaccine CNTRL = Al(OH)3 Control N = number of subjects Q1 = 25% percentile; Q3 = 75% percentile Gmean = Geometric mean 95% CI = 95% Confidence Interval; LL = Lower Limit; UL = Upper Limit Value = value of the considered parameter PIII(M12) = Post Dose 3, Month 12 PIII(M18) = Post Dose 3, Month b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 66

68 Supplement 12 HPV-16 specific CD8 T-cell responses (by ICS): proportion of subjects above 200 cells/million cells (TVC) HPV N = 53 CNTRL N = 47 Test Timing Parameters or Value % Value % Categories or n or n CD8-All doubles PIII(M12) N Below and above Missing PIII(M18) N Below and above Missing CD8-CD40L PIII(M12) N Below and above Missing PIII(M18) N Below and above Missing CD8-IFN PIII(M12) N Below and above Missing PIII(M18) N Below and above Missing CD8-IL2 PIII(M12) N Below and above Missing PIII(M18) N Below and above Missing CD8-TNF PIII(M12) N Below and above Missing PIII(M18) N Below and above Missing HPV = HPV-16/18 L1 VLP AS04 vaccine CNTRL = Al(OH)3 Control N = number of subjects n = number of subjects in a given category Value = value of the considered parameter % = n / Number of subjects with available results x b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 67

69 Supplement 13 HPV-18 specific CD8 T-cell responses (by ICS): proportion of subjects above 200 cells/million cells (TVC) HPV N = 53 CNTRL N = 47 Test Timing Parameters or Value % Value % Categories or n or n CD8-All doubles PIII(M12) N Below and above Missing PIII(M18) N Below and above Missing CD8-CD40L PIII(M12) N Below and above Missing PIII(M18) N Below and above Missing CD8-IFN PIII(M12) N Below and above Missing PIII(M18) N Below and above Missing CD8-IL2 PIII(M12) N Below and above Missing PIII(M18) N Below and above Missing CD8-TNF PIII(M12) N Below and above Missing PIII(M18) N Below and above Missing HPV = HPV-16/18 L1 VLP AS04 vaccine CNTRL = Al(OH)3 Control N = number of subjects n = number of subjects in a given category Value = value of the considered parameter % = n / Number of subjects with available results x b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 68

70 10. REFERENCES Gall SA, Teixeira J, Wheeler CM, Naud P, Harper DM, Franco EL et al. Substantial impact on precancerous lesions and HPV infections through 5.5 years in women vaccinated with the HPV-16/18 L1 VLP AS04 candidate vaccine. In: American Association for Cancer Research Annual Meeting: Proceedings; 2007 Apr 14-18; Los Angeles, CA. Philadelphia (PA): AACR; Abstract nr:4900. GlaxoSmithKline Biologicals, HPV-015 (104820) Protocol Amendment 3 (23-OCT- 2008), A phase III, double-blind, randomized, controlled study to evaluate the safety, immunogenicity and efficacy of GlaxoSmithKline Biologicals HPV-16/18 L1/AS04 vaccine administered intramuscularly according to a three-dose schedule (0, 1, 6 month) in healthy adult female subjects aged 26 years and above. GlaxoSmithKline Biologicals, Data On File Summary: Number of subjects (doses) of AS04 containing vaccines administered in 3 major GSK clinical programs. March (Available upon request). GlaxoSmithKline Biologicals, Report Analysis Plan HPV-028 ANC 015, Complementary testing to further evaluate the immunogenicity of GSK Biologicals HPV vaccine (580299) in healthy female subjects aged over 26 years Harper DM, Franco EL, Wheeler C, Ferris DG, Jenkins D, Schuind A, Zahaf T, Innis B, Naud P, De Carvalho NS, Roteli-Martins CM, Teixeira J, Blatter MM, Korn AP, Quint W and Dubin G. Efficacy of a bivalent L1 virus-like particle vaccine in prevention of infection with human papillomavirus types 16 and 18 in young women: a randomised controlled trial. The Lancet. 2004;364: Harper DM, Franco EL, Wheeler CM, Moscicki AB, Romanowski B, Roteli-Martins CM, Jenkins D, Schuind A, Costa Clemens SA & Dubin G. Sustained efficacy up to 4.5 years of a bivalent L1 virus-like particle vaccine against human papillomavirus types 16 and 18: follow-up from a randomised control trial. The Lancet. 2006;367: Harper DM, Gall S, Naud P, Quint W, Dubin G, Jenkins D and Schuind A. Sustained immunogenicity and high efficacy against HPV-16/18 related cervical neoplasia: Longterm follow up through 6.4 years in women vaccinated with Cervarix (GSK s HPV 16/18 AS04 candidate vaccine). Presented at: Thirty-Ninth Annual Meeting of the Society of Gynecologic Oncologists; 2008 Mar 10; Florida, USA: Gynecologic Oncology. 2008;109: ; Abstract nr:1. Paavonen J, Jenkins D, Bosch FX, Naud P, Salmeron J, Wheeler CM, Chow SN, Apter DL, Kitchener HC, Castellsague X, De Carvalho NS, Skinner SR, Harper DM, Hedrick JA, Jaisamrarn U, Limson GA, Dionne M, Quint W, Spiessens B, Peeters P, Struyf F, Wieting SL, Lehtinen MO, Dubin G. Efficacy of a prophylactic adjuvanted bivalent L1 virus-like-particle vaccine against infection with human papillomavirus types 16 and 18 in young women: an interim analysis of a phase III double-blind, randomised controlled trial. Lancet. 2007;369: b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 69

71 Pastrana DV, Buck CB, Pang YY, Thompson CD, Castle PE, FitzGerald PC, Kruger KS, Lowy DR, Schiller JT. Reactivity of human sera in a sensitive, high-throughput pseudovirus-based papillomavirus neutralization assay for HPV16 and HPV18. Virology. 2004; 321: Pedersen C, Petaja T, Strauss G, et al. Immunization of adolescent females with human papillomavirus type 16 and 18 L1 virus-like particle vaccine containing AS04 adjuvant. J Adolesc Health 2007; 40: b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 70

72 11. STUDY REPORT AUTHORS /CONTRIBUTING AUTHORS 2009 Scientific Writers: / Scientific Writers, Global Clinical R&D Project Statistician: Biostatistician, Global Clinical R&D Global Study Manager: Global Study Manager, Global Clinical R&D Clinical Development Manager: Director, Global Clinical R&D Regulatory Affairs representative: Expert Scientist, Technical/Clinical Regulatory Affairs N + 1 of CDM: Director, Global Clinical R&D 2013 Scientific Writer: Scientific Writing Manager: Emtex, contractor for GSK Biologicals Senior Manager, VVHS Sponsor Signatory: MD, Director, Clinical Development, HPV Vaccines, Vaccines Discovery and Development, GlaxoSmithKline Biologicals (Amended: 04 December 2013) 71 9b15d717b0599cec59924ca1bb dd10fd ceed72e452f1b0921dbf2f6d67545a4 71

73 MODULAR APPENDICES (HPV-028 ANC: 015) List of modular appendices available for the study report and ICH-specific appendices - Study Information equivalent numbering Modular appendices ICH numbering Sponsor information - Protocol and protocol amendments Sample Case Report form (unique pages only) List of IECs or IRBs (plus name of committee chair if required by regulatory authority) Representative written information for patient and sample consent forms List of investigators and other important participants in the study Investigator CVs or equivalent summaries of training and experience relevant to the performance of the clinical study Signatures of principal or coordinating investigator(s) or sponsor s responsible medical officer, depending on the regulatory authority s requirement Listings of patients receiving test drug(s) /investigational product(s) from specific batches, where more than one batch was used (if applicable) Randomization list (patient identification and treatment assigned) Audit certificates (if available) Documentation of statistical methods Documentation of inter-laboratory standardization methods and quality assurance procedures, if used Publications based on the study Important publications referenced in the report Individual listings 16.2 Case report forms (CRFs /ecrfs) CRFs /ecrfs for deaths, other SAEs and withdrawals due to adverse events e32cc5f78b3db e0e2ef66d56 1beb12c7289f0ba36767fc0c9d1790c225f1bd77 MAY

74 Sponsor Information efa77d688c374c6dd623b60c d0d17 90be55dd014299fbbd49da28af1bfe4bdbec4ecc 73 1

75 GlaxoSmithKline Biologicals Rue de l Institut 89, B-1330 Rixensart, Belgium Sponsor Information Study title Complementary immunology testing in a phase III, double-blind, randomized, controlled study to evaluate the safety, immunogenicity and efficacy of GlaxoSmithKline Biologicals HPV-16/18 L1 AS04 vaccine administered intramuscularly according to a three-dose schedule (0, 1, 6 month) in healthy adult female subjects aged 26 years and above enrolled in study HPV-015 (104820). etrack study number etrack abbreviated title HPV-028 ANC:015 IND number BB-IND 7920 EudraCT number Date of document May 14, (Principal) Investigator Principal investigators MD, PhD The Netherlands MD, PhD The Netherlands MD, PhD The Netherlands 1 68b020e57f7342ea7ce608c04c2372d6 90be55dd014299fbbd49da28af1bfe4bdbec4ecc 74 2

76 (HPV-015) Sponsor Information 2. Medical Monitor Until 1 June 2007: MD GlaxoSmithKline Huis ter Heideweg LZ ZEIST The Netherlands Tel: From 1 june 2007 Medical head: Vaccines, HIV and CNS GlaxoSmithKline Huis ter Heideweg LZ ZEIST The Netherlands Tel: 3. Study Monitor 3.1. Central GlaxoSmithKline Biologicals Rue de l Institut 89 B-1330 Rixensart, Belgium Tel: Fax: 2 68b020e57f7342ea7ce608c04c2372d6 90be55dd014299fbbd49da28af1bfe4bdbec4ecc 75 3

77 (HPV-015) Sponsor Information 3.2. Local Clinical Trial Monitor GlaxoSmithKline Huis ter Heideweg LZ ZEIST The Netherlands Tel: Fax Mobile: Country Study Manager Vaccines GlaxoSmithKline Huis ter Heideweg LZ Zeist The Netherlands Tel: Fax: Mobile: 4. Study Contact for Reporting of a Serious Adverse Event GlaxoSmithKline Huis ter Heideweg 62 NL-3705 LZ ZEIST The Netherlands Tel: Fax Mobile: for 7/7 day availability: 5. Study Contact for Emergency Code Break (Head SERM Adult/Adolescent/ED - Pediaetric is using it until arrival of Head SERM 6. Study Centres (Head BCSP - for US/Canada (Head SERM North America - The Netherlands 3 68b020e57f7342ea7ce608c04c2372d6 90be55dd014299fbbd49da28af1bfe4bdbec4ecc 76 4

78 (HPV-015) Sponsor Information The Netherlands The Netherlands 4 68b020e57f7342ea7ce608c04c2372d6 90be55dd014299fbbd49da28af1bfe4bdbec4ecc 77 5

79 Protocol and Protocol Amendments d1880f4bab9905da5f557c701c29b0a577af64f0 1d073efcfea318b54b165ad19806b01d1175fa

80 Study vaccine etrack study number and abbreviated title Study vaccine number Sponsor: GlaxoSmithKline Biologicals Rue de l Institut 89, B-1330 Rixensart Belgium GlaxoSmithKline Biologicals candidate human papillomavirus (HPV) vaccine containing HPV-16/18 L1 proteins and AS04 adjuvant (HPV-028 ANC: 015) Investigational New Drug BB-IND 7920 (IND) number EudraCT number Date of approval Final 07 February 2007 Title Complementary testing to further evaluate the immunogenicity of GSK Biologicals HPV vaccine (580299) in healthy female subjects aged over 26 years enrolled in study Detailed title Complementary immunology testing in a phase III, double-blind, randomized, controlled study to evaluate the safety, immunogenicity and efficacy of GlaxoSmithKline Biologicals HPV-16/18 L1 AS04 vaccine administered intramuscularly according to a three-dose schedule (0, 1, 6 month) in healthy adult female subjects aged 26 years and above enrolled in study (HPV-015) Co-ordinating author PhD, Scientific Writer Contributing authors MD, Director, Clinical Development PhD, Clinical Development Manager PhD, Central Study Coordinator PhD, Biostatistician PhD, Clinical Immunology Clinical Laboratories Scientific Writer 1 GSK Biologicals Protocol DS V 12.3 Copyright 2006 the GlaxoSmithKline group of companies. All rights reserved. Unauthorized copying or use of this information is prohibited. CARS Id : CLIN_200611_2714/ Version : 1.18,Admin. QC/ Modify Date : 07/02/2007 fc4511a37499ec c76c32be9dca75c110f 1d073efcfea318b54b165ad19806b01d1175fa

81 (HPV-028 ANC: 015) etrack study number and abbreviated title Investigational New Drug (IND) number (HPV-028 ANC: 015) BB-IND 7920 EudraCT number Date of approval Final 07 February 2007 Detailed Title Sponsor signatory approval Complementary immunology testing in a phase III, double-blind, randomized, controlled study to evaluate the safety, immunogenicity and efficacy of GlaxoSmithKline Biologicals HPV-16/18 L1 AS04 vaccine administered intramuscularly according to a three-dose schedule (0, 1, 6 month) in healthy adult female subjects aged 26 years and above enrolled in study (HPV-015) Sponsor signatory: MD Director, Clinical Development HPV Vaccines GlaxoSmithKline Biologicals Rue de l Institut 89, B-1330 Rixensart, Belgium Tel: Fax: Signature: Date: Final 07 February CARS Id : CLIN_200611_2714/ Version : 1.18,Admin. QC/ Modify Date : 07/02/2007 fc4511a37499ec c76c32be9dca75c110f 1d073efcfea318b54b165ad19806b01d1175fa

82 (HPV-028 ANC: 015) Investigator Agreement etrack study number and abbreviated title Investigational New Drug (IND) number (HPV-028 ANC: 015) BB-IND 7920 Date of approval Final 07 February 2007 Detailed Title Complementary immunology testing in a phase III, double-blind, randomized, controlled study to evaluate the safety, immunogenicity and efficacy of GlaxoSmithKline Biologicals HPV-16/18 L1 AS04 vaccine administered intramuscularly according to a three-dose schedule (0, 1, 6 month) in healthy adult female subjects aged 26 years and above enrolled in study (HPV-015) I agree: To assume responsibility for the proper conduct of the study at this site. To conduct the study in compliance with this protocol, any mutually agreed future protocol amendments, and with any other study conduct procedures provided by GlaxoSmithKline Biologicals (GSK Biologicals). To ensure that all persons assisting me with the study are adequately informed about the GSK Biologicals investigational product(s) and other study-related duties and functions as described in the protocol. Not to implement any changes to the protocol without agreement from the sponsor and prior review and written approval from the Institutional Review Board (IRB) or Independent Ethics Committee (IEC), except where necessary to eliminate an immediate hazard to the subjects, or where permitted by all applicable regulatory requirements (for example, for administrative aspects of the study). That I am thoroughly familiar with the appropriate use of the vaccine(s), as described in this protocol, and any other information provided by the sponsor, including, but not limited to, the following: the current Investigator s Brochure (IB) or equivalent document, IB supplement (if applicable) and/or prescribing information (in the case of a marketed vaccine). That I am aware of, and will comply with, Good Clinical Practice (GCP) and all applicable regulatory requirements. That I have been informed that certain regulatory authorities require the sponsor to obtain and supply, as necessary, details about the investigator s ownership interest in the sponsor or the investigational product, and more generally about his/her financial ties with the sponsor. GSK Biologicals will use and disclose the information solely for the purpose of complying with regulatory requirements. Final 07 February CARS Id : CLIN_200611_2714/ Version : 1.18,Admin. QC/ Modify Date : 07/02/2007 fc4511a37499ec c76c32be9dca75c110f 1d073efcfea318b54b165ad19806b01d1175fa

83 (HPV-028 ANC: 015) Hence I: Agree to supply GSK Biologicals with any necessary information regarding ownership interest and financial ties (including those of my spouse and dependent children). Agree to promptly update this information if any relevant changes occur during the course of the study and for 1 year following completion of the study. Agree that GSK Biologicals may disclose any information it has about such ownership interests and financial ties to regulatory authorities. Agree to provide GSK Biologicals with an updated Curriculum Vitae and other FDA required documents. Investigator name: Investigator signature Date Final 07 February CARS Id : CLIN_200611_2714/ Version : 1.18,Admin. QC/ Modify Date : 07/02/2007 1d073efcfea318b54b165ad19806b01d1175fa68 fc4511a37499ec c76c32be9dca75c110f 82 5

84 (HPV-028 ANC: 015) etrack study number and abbreviated title Detailed Title Indication Study Population Rationale Synopsis (HPV-028 ANC: 015) Complementary immunology testing in a phase III, doubleblind, randomized, controlled study to evaluate the safety, immunogenicity and efficacy of GlaxoSmithKline Biologicals HPV-16/18 L1 AS04 vaccine administered intramuscularly according to a three-dose schedule (0, 1, 6 month) in healthy adult female subjects aged 26 years and above enrolled in study (HPV-015) For active immunization of girls and women from 10 years of age onwards for the prevention of persistent human papillomavirus (HPV) infections and related clinical outcomes (cytological abnormalities and pre-cancerous lesions) caused by oncogenic HPV types 16 and 18. Healthy adult women aged 26 years and above enrolled from pre-selected sites and participating in study HPV-015 (104820). To better characterize the immunological response elicited by vaccination with the HPV16/18 L1 AS04 vaccine, additional samples will be collected at pre-selected sites from subjects participating in the HPV-015 study. Additional testing of peripheral blood mononuclear cells [PBMC]/cervical samples will be performed. Objectives To measure T cell-mediated immune responses specific to HPV virus-like particles (VLP) types 16 and 18 in peripheral blood cells. To measure memory B cell immune responses specific to HPV VLP types 16 and 18 in peripheral blood cells. To evaluate anti-hpv-16 and anti-hpv-18 antibody responses in cervical samples. To evaluate the total level of IgG antibodies in cervical samples collected in this study, and in serum samples collected in study (HPV-015). To compare anti-hpv-16 and anti-hpv-18 antibody levels in cervical samples collected in this study with antibody levels in serum samples collected in study (HPV-015). Final 07 February CARS Id : CLIN_200611_2714/ Version : 1.18,Admin. QC/ Modify Date : 07/02/2007 fc4511a37499ec c76c32be9dca75c110f 1d073efcfea318b54b165ad19806b01d1175fa

85 (HPV-028 ANC: 015) Study design primary study (HPV-015) Ancillary study design Experimental design: A phase III, controlled study with two parallel groups: - HPV vaccine group (received HPV-16/18 L1 AS04) - Control group (received Al(OH) 3 ) Treatment allocation in study HPV-015: subjects were recruited in three age strata (26-35 years, years and 46+ years), taking into account their previous HPV history, and randomized. Blinding: double-blind. Vaccination schedule: three doses of vaccine/control were administered intramuscularly according to a 0, 1, 6-month schedule under the (HPV-015) protocol. Blood sampling in the immunogenicity subset: six blood samples will be collected in a subset of subjects at selected study sites at Month 0, 7, 12, 18, 24 and 36. This ancillary study will consist of supplemental testing of serological and cervical samples obtained at Visit 5 (Month 12) and Visit 6 (Month 18) of the primary study HPV-015 from pre-selected sites. Number of subjects: Approximately 100 subjects enrolled at pre-selected sites in the primary study (HPV-015) who are in the immunogenicity subset and have received all three doses of vaccine/control will be asked to participate in the present ancillary study. Blinding: The double blind in the primary study will be maintained. Data collection: Remote Data Entry (RDE). Duration of the study: approximately six months for each subject. Two scheduled visits: at Month 12 and 18 after the first vaccine dose received in the context of protocol (HPV-015). Blood samples: A 40 ml blood sample will be collected at Visit 5 (Month 12) and Visit 6 (Month 18) for evaluation of cell mediated immune response in subjects recruited from pre-selected sites. Final 07 February CARS Id : CLIN_200611_2714/ Version : 1.18,Admin. QC/ Modify Date : 07/02/2007 fc4511a37499ec c76c32be9dca75c110f 1d073efcfea318b54b165ad19806b01d1175fa

86 (HPV-028 ANC: 015) Cervical samples: One cervical secretions sample will be collected at Visit 5 (Month 12) and Visit 6 (Month 18) for antibody detection. An interim immunogenicity analysis will be performed when all subjects have completed Visit 5 (Month 12). Final analysis will be performed when all subjects have completed Visit 6 (Month 18). Endpoints T cell-mediated immune responses specific to HPV VLP types 16 and 18 (Month 12 and Month 18). Memory B cell immune responses specific to HPV VLP types 16 and 18 (Month 12 and Month 18). HPV-16 and HPV-18 antibody titres assessed by ELISA in cervical samples at Month 12 and 18 (seropositivity rates and GMTs). Total IgG antibodies (ELISA) from cervical samples collected in this study, and serum samples collected in study (HPV-015) at Month 12 and Month 18. Correlation of anti-hpv-16 and anti-hpv-18 in serum collected in study (HPV-015) and in cervical samples collected in this study. Exploratory endpoints At the discretion of GSK, all the endpoints above may be expanded to HPV types not included in the vaccine. At the discretion of GSK, other immune parameters related to HPV or the HPV vaccine may be tested. This may include evaluation of neutralizing antibodies. Final 07 February CARS Id : CLIN_200611_2714/ Version : 1.18,Admin. QC/ Modify Date : 07/02/2007 fc4511a37499ec c76c32be9dca75c110f 1d073efcfea318b54b165ad19806b01d1175fa

87 (HPV-028 ANC: 015) TABLE OF CONTENTS PAGE SYNOPSIS...5 LIST OF ABBREVIATIONS...10 GLOSSARY OF TERMS INTRODUCTION OBJECTIVES STUDY DESIGN OVERVIEW STUDY COHORT Number of subjects / centers Inclusion criteria Exclusion criteria for enrolment in the ancillary study Elimination criteria during the study Contraindications to subsequent vaccination CONDUCT OF STUDY Ethics and regulatory considerations Institutional Review Board/Independent Ethics Committee (IRB/IEC) Informed consent General study aspects Subject identification Outline of study procedures Detailed description of study stages/visits Visit 5 (Month 12) Visit 6 (Month 18) Sample handling and analysis Treatment and storage of biological samples Laboratory assays Serology Cell mediated immune (CMI) T and B-cell responses Cervical samples for antibody detection Immunological read-outs INVESTIGATIONAL PRODUCTS AND ADMINISTRATION Treatment allocation and randomization Method of blinding and breaking the study blind Concomitant medication/treatment ADVERSE EVENTS AND SERIOUS ADVERSE EVENTS Definition of an adverse event Definition of a serious adverse event Lack of efficacy...25 Final 07 February CARS Id : CLIN_200611_2714/ Version : 1.18,Admin. QC/ Modify Date : 07/02/2007 fc4511a37499ec c76c32be9dca75c110f 1d073efcfea318b54b165ad19806b01d1175fa

88 (HPV-028 ANC: 015) 7.4. Clinical laboratory parameters and other abnormal assessments qualifying as serious adverse events Time period, frequency, and method of detecting serious adverse events Solicited adverse events Evaluating adverse events and serious adverse events Follow-up of adverse events and serious adverse events and assessment of outcome Prompt reporting of serious adverse events to GSK Biologicals Regulatory reporting requirements for serious adverse events Post-study adverse events and serious adverse events Pregnancy Treatment of adverse events SUBJECT COMPLETION AND WITHDRAWAL Subject completion Subject withdrawal Subject withdrawal from the study Subject withdrawal from investigational product DATA EVALUATION: CRITERIA FOR EVALUATION OF OBJECTIVES Endpoints Exploratory endpoints Estimated sample size Study cohorts to be evaluated Derived and transformed data Final analyses Planned interim analysis ADMINISTRATIVE MATTERS REFERENCES...30 APPENDICES Appendix A World Medical Association Declaration of Helsinki...31 Appendix B Appendix C Appendix D Appendix E Administrative Matters...35 Overview of the Recruitment Plan for the Ancillary Study...41 Handling of Biological Samples Collected by the Investigator...42 Shipment of Biological Samples...44 Appendix F Laboratory Assays...45 Appendix G Vaccine Supplies, Packaging and Accountability...48 Final 07 February CARS Id : CLIN_200611_2714/ Version : 1.18,Admin. QC/ Modify Date : 07/02/2007 fc4511a37499ec c76c32be9dca75c110f 1d073efcfea318b54b165ad19806b01d1175fa

89 (HPV-028 ANC: 015) List of Abbreviations Al(OH) 3 AS04 Aluminium hydroxide Adjuvant containing aluminium salts and MPL CD4 Cluster of differentiation 4 CD8 Cluster of differentiation 8 CI CMI CpG CRF/eCRF CRA CSC ELISA EL.U/mL FDA GCP GMT GSK HPV ICF IgG IEC IL-2 IFN IND IRB Confidence Interval Cell mediated immune (response) Cytosine and Guanine separated by a phosphate, which links the two nucleosides together in DNA Case Report Form/electronic Case Report Form Clinical Research Associate Central Study Coordinator Enzyme-linked immunosorbent assay ELISA units per ml Food and Drug Administration, United States Good Clinical Practice Geometric Mean Titre GlaxoSmithKline Human papillomavirus Informed Consent Form Immunoglobulin type G Independent Ethics Committee Interleukin-2 Interferon-gamma Investigational New Drug Institutional Review Board Final 07 February CARS Id : CLIN_200611_2714/ Version : 1.18,Admin. QC/ Modify Date : 07/02/2007 fc4511a37499ec c76c32be9dca75c110f 1d073efcfea318b54b165ad19806b01d1175fa

90 (HPV-028 ANC: 015) PBMC NA RAP RDE SOP TBD TNF VLP Peripheral blood mononuclear cells Not applicable Report Analysis Plan Remote Data Entry Standard Operating Procedure To be determined Tumor necrosis factor Virus-like particle(s) Glossary of Terms Please refer to the Glossary of Terms protocol (HPV-015). Final 07 February CARS Id : CLIN_200611_2714/ Version : 1.18,Admin. QC/ Modify Date : 07/02/2007 fc4511a37499ec c76c32be9dca75c110f 1d073efcfea318b54b165ad19806b01d1175fa

91 (HPV-028 ANC: 015) 1. INTRODUCTION To better characterize the immunological response elicited by vaccination with the HPV16/18 L1 AS04 vaccine, additional samples will be collected at pre-selected sites from subjects participating in the study (HPV-015). Additional testing of peripheral blood mononuclear cells [PBMC]/cervical samples will be performed. This ancillary study will consist of supplemental testing of serological and cervical samples obtained at Visit 5 (Month 12) and Visit 6 (Month 18) of the primary study (HPV-015). 2. OBJECTIVES To measure T cell-mediated immune responses specific to HPV virus-like particles (VLP) types 16 and 18 in peripheral blood cells. To measure memory B cell immune responses specific to HPV VLP types 16 and 18 in peripheral blood cells. To evaluate anti-hpv-16 and anti-hpv-18 antibody responses in cervical samples. To evaluate the total level of IgG antibodies in cervical samples collected in this study, and in serum samples collected in study (HPV-015). To compare anti-hpv-16 and anti-hpv-18 antibody levels in cervical samples collected in this study with antibody levels in serum samples collected in study (HPV-015). Refer to Section 10.1 for definitions of endpoints. Final 07 February CARS Id : CLIN_200611_2714/ Version : 1.18,Admin. QC/ Modify Date : 07/02/2007 fc4511a37499ec c76c32be9dca75c110f 1d073efcfea318b54b165ad19806b01d1175fa

92 (HPV-028 ANC: 015) 3. STUDY DESIGN OVERVIEW Study (HPV-015) Randomization (1:1) Ancillary Study (HPV-028 ANC: 015) HPV-16/18 L1/AS04 vaccine (N=2700) Al(OH) 3 control (N=2700) Visit 1 Month 0 Visit 2 Month 1 Visit 3 Month 6 Visit 4 Month 7 Visit 5 Month 12 Visit 6 Month 18 Visit 7 Month 24 Visit 8 Month 30 Visit 9 Month 36 Vaccination I II III Blood sample I II III*^ IV*^ V VI Cervical sample I II III*^ IV*^ V VI VII * An additional 40mL blood sample HPV-015 study conclusion ^ An additional cervical sample Experimental design: a phase III, double-blind, controlled ancillary study performed as a supplement to study (HPV-015). The present study will be conducted at pre-selected sites participating in study (HPV-015). Study population: subjects enrolled and who received 3 doses of vaccine/control in study (HPV-015) from pre-selected sites will be asked to participate in the study. Subjects were randomized 1:1 to two parallel treatment groups in the primary study: o HPV vaccine group received HPV-16/18 L1 AS04 o control group received Al(OH) 3 Vaccination schedule: Three doses of vaccine/control were administered intramuscularly according to a 0, 1, 6-month schedule. No additional doses of vaccine/control will be administered in this ancillary study. Blinding: The double-blind will be applied to this ancillary study. Type of study: ancillary study for protocol (HPV-015). Two scheduled visits: at Visit 5 (Month 12) and Visit 6 (Month 18) after the first vaccine dose. Final 07 February CARS Id : CLIN_200611_2714/ Version : 1.18,Admin. QC/ Modify Date : 07/02/2007 1d073efcfea318b54b165ad19806b01d1175fa68 fc4511a37499ec c76c32be9dca75c110f 91 14

93 (HPV-028 ANC: 015) Blood samples: A 40 ml blood sample will be collected at Visit 5 (Month 12) and Visit 6 (Month 18) for evaluation of cell mediated immune response in subjects recruited from pre-selected study sites. This is in addition to the 10 ml blood sample which is taken as part of the primary study protocol (HPV-015). Cervical samples: One cervical secretions sample will be collected at Visit 5 (Month 12) and Visit 6 (Month 18) for antibody detection. The sample must be collected before the liquid-based cytology sample which is taken as part of the primary study protocol (HPV-015). Data collection: Remote Data Entry (RDE)/electronic Case Report Form (ecrf) Duration of the study: approximately six months for each subject. An interim immunogenicity analysis of the ancillary study will be performed when all subjects have completed Visit 5 (Month 12). Final analysis of the ancillary study will be performed when all subjects have completed Visit 6 (Month 18). 4. STUDY COHORT 4.1. Number of subjects / centers Approximately 100 subjects enrolled at pre-selected sites in the primary study (HPV-015) who are in the immunogenicity subset and have received all three doses of vaccine/control will be asked to participate in the present ancillary study Inclusion criteria All subjects must satisfy the following criteria at study entry: A female enrolled in study (HPV-015) and who received three doses of study vaccine/control. Subjects who the investigator believes that they can and will comply with the requirements of the protocol (e.g. return for follow-up visits) should be enrolled in the study. Written informed consent obtained from the subject prior to enrolment in this ancillary study Exclusion criteria for enrolment in the ancillary study The following criteria should be checked at the time of ancillary study entry. If any apply, the subject must not be included in the study: Pregnancy. Final 07 February CARS Id : CLIN_200611_2714/ Version : 1.18,Admin. QC/ Modify Date : 07/02/2007 fc4511a37499ec c76c32be9dca75c110f 1d073efcfea318b54b165ad19806b01d1175fa

94 (HPV-028 ANC: 015) Administration of any HPV vaccine other than that foreseen by the study protocol. Use of any investigational or non-registered product (drug or vaccine) other than the study vaccine since study start. Chronic administration (defined as more than 14 days) of immunosuppressants or other immune-modifying drugs since study start. (For corticosteroids, this will mean prednisone, or equivalent, 0.5 mg/kg/day. Inhaled and topical steroids are allowed.) Administration of immunoglobulins and/or any blood products within 90 days preceding a blood sampling Elimination criteria during the study Please refer to the protocol for study (HPV-015) Contraindications to subsequent vaccination Not applicable to this ancillary study. No vaccine will be administered. 5. CONDUCT OF STUDY 5.1. 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), {US 21 CFR Part 50 Protection of Human Subjects, and Part 56 Institutional Review Boards} and local rules and regulations of the country. Submission of the protocol and any protocol amendments to regulatory agencies will occur in accordance with local regulatory requirements. For some countries, submission to the local regulatory authority may not be required. When submission to the local regulatory authority is required, the timing of the submission relative to IEC/IRB submission or approval and whether or not the authority will provide their approval of or favourable opinion on the protocol or amendment before it can be implemented will depend on local regulatory requirements Institutional Review Board/Independent Ethics Committee (IRB/IEC) The IRB/IEC must be constituted according to the local laws/customs of each participating country. The ICH Harmonized Tripartite Guideline for Good Clinical Practice recommends that the IRB/IEC should include: a. At least five members. b. At least one member whose primary area of interest is in a non-scientific area. c. At least one member who is independent of the institution/ study site. Final 07 February CARS Id : CLIN_200611_2714/ Version : 1.18,Admin. QC/ Modify Date : 07/02/2007 fc4511a37499ec c76c32be9dca75c110f 1d073efcfea318b54b165ad19806b01d1175fa

95 (HPV-028 ANC: 015) 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 or designate.a list of the professions of the IRB/IEC members should be obtained by the investigator or designate 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 or designate. Written and dated unconditional approval/favourable opinion from the IRB/IEC of the protocol and amendment (if any and applicable), written informed consent form, consent form updates (if any), subject recruitment procedure(s) (e.g. advertisements), and any other written information to be provided to subjects must be in the possession of the investigator and GSK before commencement of the study. This approval/favourable opinion must refer to the study by study title and number with exact protocol version and date, and should identify the documents reviewed and state the date of review. Relevant GSK Biologicals data will be supplied by the investigator or designate to the hospital/ university/ independent IRB/IEC for review and approval of the protocol. Verification of the unconditional approval/favourable opinion of the IRB/IEC will be transmitted by the local CRA to the Central Study Coordinator prior to shipment of vaccine supplies and CRFs to the site. No deviations from, or changes to, the protocol should be initiated without prior written sponsor and IRB/IEC approval/ favourable opinion of an appropriate amendment or administrative change, except when necessary to eliminate immediate hazards to the subjects or where permitted by all applicable regulatory requirements or when the change(s) involves only logistical or administrative aspects of the study (e.g., change of monitor[s], telephone number[s].) Except for the US, administrative changes and amendments not submitted for approval are submitted to the IRB/IEC for information only. However, written verification that such documents were submitted should be obtained. Approvals/ verifications must be transmitted in writing to the Central Study Coordinator by the local CRA. The IRB/IEC must be informed by the investigator of: all subsequent protocol amendments, informed consent changes or revisions of other documents originally submitted for review, serious and/or unexpected adverse events occurring during the study, where required, all subsequent protocol administrative changes (for information, except for US studies), new information that may affect adversely the safety of the subjects or the conduct of the study, an annual update and/or request for re-approval, where required, when the study has been completed, where required. Final 07 February CARS Id : CLIN_200611_2714/ Version : 1.18,Admin. QC/ Modify Date : 07/02/2007 fc4511a37499ec c76c32be9dca75c110f 1d073efcfea318b54b165ad19806b01d1175fa

96 (HPV-028 ANC: 015) If a trial is prematurely terminated or suspended for reasons including, but not limited to, safety or ethical issues or severe non-compliance, the sponsor will promptly inform the regulatory authorities of the termination or suspension and the reason(s) for the termination or suspension. If required by applicable regulations, the investigator must inform the IEC/IRB promptly and provide the reason for the suspension or termination (see Appendix B for further details) 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. Informed consent will be obtained in accordance with 21 CFR Freely given informed consent should be obtained from every subjects prior to clinical trial participation. Information should be given in both oral and written form whenever possible and as deemed appropriate by the IRB/IEC. An investigator or designate will describe the protocol to potential subjects face to face. The Informed Consent Form may be read to the subjects but, in any event, the investigator or designate shall give the subjects ample opportunity to inquire about details of the study and ask any questions before dating and signing the Informed Consent Form. While informed consent information can be presented to groups at an initial information session, each subject must be given the opportunity to individually pose questions to the investigator or designate prior to the subject dating and signing the Informed Consent Form. Informed Consent Form must be in a language fully comprehensible to the prospective subjects. Informed consent shall be documented by the use of a written consent form approved by the IRB/IEC and signed and dated by the subjects and by the person who conducted the informed consent discussion. The signature confirms the consent is based on information that has been understood. All illiterate individuals will have the study, the Informed 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 subject s incapacity precludes this and provided that the local legal obligations are fulfilled. Each subject's signed informed consent form must be kept on file by the investigator for possible inspection by Regulatory Authorities and/or GSK Biologicals professional and Regulatory Compliance persons. The subjects should receive a copy of the signed and dated written informed consent form and any other written information provided to the Final 07 February CARS Id : CLIN_200611_2714/ Version : 1.18,Admin. QC/ Modify Date : 07/02/2007 fc4511a37499ec c76c32be9dca75c110f 1d073efcfea318b54b165ad19806b01d1175fa

97 (HPV-028 ANC: 015) subjects, and should receive copies of any signed and dated consent form updates. Any amendments to the written information will be provided to subjects. Both the informed consent discussion and the written informed consent form and any other written information to be provided to the subjects should include explanations of the following: a. That the trial involves research. b. The purpose of the trial. c. The trial treatment(s) and the probability for random assignment to each treatment. d. The trial procedures to be followed, including all invasive procedures. e. The subject s 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 should be made aware of this. i. The alternative procedure(s) or course(s) of treatment/ methods of prevention that may be available to subjects, and their important potential benefits and risks. j. The compensation and/or treatment available to subjects in the event of trial-related injury. k. The anticipated prorated payment, if any, to subjects for participating in the trial. l. The anticipated expenses, if any, to subjects for participating in the trial. m. That the subjects participation in the trial is voluntary and subjects may refuse to participate or withdraw from the trial, at any time, without penalty or loss of benefits to which subjects are otherwise entitled. n. That the monitor(s), the auditor(s), the IRB/IEC, and the regulatory authority(ies) will be granted direct access to the subject s original medical records for verification of clinical trial procedures and/or data, without violating the confidentiality of subjects, to the extent permitted by the applicable laws and regulations and that, by signing a written informed consent, the subject is authorizing such access. o. That records identifying subjects will be kept confidential and, to the extent permitted by the applicable laws and/or regulations, will not be made publicly available. If the results of the trial are published, subjects identity will remain confidential. p. That the subjects will be informed in a timely manner if information becomes available that may be relevant to the subjects willingness for continued participation in the trial. q. The person(s) to contact for further information regarding the trial and the rights of trial subjects, and who to contact in the event of trial-related injury. Final 07 February CARS Id : CLIN_200611_2714/ Version : 1.18,Admin. QC/ Modify Date : 07/02/2007 fc4511a37499ec c76c32be9dca75c110f 1d073efcfea318b54b165ad19806b01d1175fa

98 (HPV-028 ANC: 015) r. The foreseeable circumstances and/or reasons under which a subject s participation in the trial may be terminated. s. The expected duration of a subject s participation in the trial. t. The approximate number of subjects involved in the trial. GSK Biologicals will prepare a model Informed Consent Form which will embody all the elements described above. While it is strongly recommended that this model document be followed as closely as possible, the informed consent requirements given in this document are not intended to pre-empt any local regulations which require additional information to be disclosed for informed consent to be legally effective. Clinical 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 Informed Consent Form, respecting the mandatory requirements of local regulations. The consent form generated by the investigator with the assistance of the sponsor s representative, must be approved (along with the protocol, and any other necessary documentation) by the IRB/IEC and be acceptable to GSK Biologicals General study aspects This is an ancillary study conducted as part of study (HPV-015) that will be performed at scheduled Visit 5 and Visit 6 of study (HPV-015). The protocol for study (HPV-015) included the possibility of ancillary studies to be conducted as a part of the primary study Subject identification For the purpose of this ancillary study, the subject identification number allocated in study (HPV-015) will be used. The double-blind in study (HPV-015) will be retained Outline of study procedures The following section describes the study procedures to be followed by the investigator at the identified study centers. See Table 1 for an overview of study procedures. Ancillary procedures will be performed in addition to those already scheduled as a part of the primary study. Final 07 February CARS Id : CLIN_200611_2714/ Version : 1.18,Admin. QC/ Modify Date : 07/02/2007 fc4511a37499ec c76c32be9dca75c110f 1d073efcfea318b54b165ad19806b01d1175fa

99 (HPV-028 ANC: 015) Table 1 List of study procedures to be performed during ancillary study (HPV-028 ANC: 015) conducted at Visit 5 and Visit 6 Visit VISIT 5 VISIT 6 Timing Month 12 Month 18 Sampling timepoint Post vacc III Post vacc III Main protocol procedures Check elimination criteria Record concomitant medication/vaccination Gynaecological examination according to local practice 1,2,3 Blood sampling: for antibody determination (10 ml) Collection of cervical samples 1 Reporting of pregnancies and pregnancy outcomes Reporting of SAEs, new onset chronic diseases and other medically significant conditions Administration of behavioural questionnaire 4 Referral for colposcopy 3 Reporting of all colposcopy results 5 Counselling Additional ancillary protocol procedures Informed consent Check inclusion and exclusion criteria Cervical secretions sampling: for antibody response 6 Record time point of sample collection during subject s menstrual cycle Blood sampling: for CMI* response (40 ml) Ancillary Study Conclusion 7 Procedures of the main protocol are listed for information only. Procedures of the main protocol are to be recordedfollowing instructions of the main protocol. indicates a study procedure that must be documented in the subject s ancillary ecrf. indicates a study procedure that will be recorded in the subject s ecrf in study (HPV-015). The double-line border after Visit 1 (Month 12) indicates the interim analysis to be performed on data obtained at that visit. * Cell Mediated Immunity will only be measured in subjects enrolled in this ancillary study. 1 Gynaecological examinations and collection of cervical specimens will be suspended in women known to be pregnant and will resume three months after resolution. Missing procedures will not need to be rescheduled. 2 Cervical samples will be used for cervical cytology at Month 12 and HPV DNA typing at Month 12 and Month Women with abnormal cervical cytology will be evaluated according to the cytology and colposcopy clinical management algorithms included in the protocol for study (HPV-015). 4 A questionnaire will be administered to the subjects through an interview by study personnel to collect data about their current sexual behaviour included as part of the protocol for study (HPV-015). 5 All colposcopy results, including any results obtained from outside the study, must be reported in the ecrf. Treatment following Vulval Intraepithelial Neoplasia (VIN) or Vaginal Intraepithelial Neoplasia (VaIN) must be recorded in the ecrf. 6 Two cervical samples will be collected at Visit 5 and 6: a cervicovaginal secretions sample for this ancillary protocol using Mercocel (or equivalent) sponges for the detection of HPV-16 and HPV-18 antibody responses; and a second liquid-based cytology (LBC) sample collected as already planned in the study (HPV-015). 7 For the interim analysis, there will be an interim conclusion page included in the ecrf. It is the investigator s responsibility to ensure that the intervals between visits/contacts are strictly followed. Please refer to the main protocol for the indicated intervals between study visits. Final 07 February CARS Id : CLIN_200611_2714/ Version : 1.18,Admin. QC/ Modify Date : 07/02/2007 1d073efcfea318b54b165ad19806b01d1175fa68 fc4511a37499ec c76c32be9dca75c110f 98 21

100 (HPV-028 ANC: 015) 5.5. Detailed description of study stages/visits Please refer to the protocol for the primary study (HPV-015) for the description of the procedures to be performed in the context of the main protocol. The following describes the procedures to be performed in the context of the ancillary protocol. Use of specified study materials 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. Refer to Appendix D and Appendix E Visit 5 (Month 12) Ancillary study procedures Obtain written informed consent. (All subjects who agree to participate in the ancillary study must sign an original informed consent form prior to enrolment/study procedures). Check inclusion and exclusion criteria for the ancillary study (see Sections 4.2 and 4.3). Collect a minimum of 40 ml of whole blood from subjects recruited at the preselected study sites. This blood will be used for the determination of CMI T and B- cell responses (as described in Section 5.6.2). Collection of a cervicovaginal secretions sample for antibody detection according to instructions in Appendix D. Note: This sample should be collected prior to collection of the cervical sample for cytology and HPV DNA polymerase chain reaction (PCR) testing, which is part of the main study [HPV-015]. Record the time point at which the cervical sample was collected during the subject s menstrual cycle (duration of the subject s cycle and the last menstruation date). Study conclusion visit for interim analysis for ancillary protocol to be performed on data obtained at this visit. Subjects will continue in the study. Final 07 February CARS Id : CLIN_200611_2714/ Version : 1.18,Admin. QC/ Modify Date : 07/02/2007 fc4511a37499ec c76c32be9dca75c110f 1d073efcfea318b54b165ad19806b01d1175fa

101 (HPV-028 ANC: 015) Visit 6 (Month 18) Ancillary study procedures Collect a minimum of 40 ml of whole blood from subjects recruited at the preselected sites. This blood will be used for the determination of CMI T and B-cell responses (as described in Section 5.6.2). Collection of a cervicovaginal secretions sample for antibody detection according to instructions in Appendix D. Note: This sample should be collected prior to collection of the cervical sample for cytology and HPV DNA polymerase chain reaction (PCR) testing, which is part of the main study [HPV-015]. Record the time point at which the cervical sample was collected during the subject s menstrual cycle (duration of the subject s cycle and the last menstruation date). Study conclusion visit for the final analysis of this protocol. Subjects will continue in study (HPV-015) Sample handling and analysis Please refer to the protocol for the primary study (HPV-015) for the description of the handling of samples collected in the context of the main protocol. The following sections will describe the handling and analysis of samples collected in the context of the ancillary protocol Treatment and storage of biological samples See Appendix D of the protocol for details of treatment and storage of biological samples. See Appendix E for instructions for shipment of biological samples Laboratory assays Serology Please refer to Section of the (HPV-015) protocol for details of the serology assays to be performed at Month 12 and Cell mediated immune (CMI) T and B-cell responses During both visits (Months 12 and 18 of study protocol [HPV-015]), an additional 40 ml blood sample will be taken from 100 subjects recruited at the sites. This blood will be used for the determination of CMI T and B-cell responses at GSK Biologicals laboratories (or a designated laboratory). Samples should be stored in liquid nitrogen until shipment to GSK Biologicals, if needed. Final 07 February CARS Id : CLIN_200611_2714/ Version : 1.18,Admin. QC/ Modify Date : 07/02/2007 fc4511a37499ec c76c32be9dca75c110f 1d073efcfea318b54b165ad19806b01d1175fa

102 (HPV-028 ANC: 015) Cervical samples for antibody detection During both visits (Months 12 and 18 of study protocol [HPV-015]), cervical secretions will be collected using Merocel ophthalmic sponges or equivalent for evaluation of anti-hpv-16 and anti-hpv-18 antibodies by placing the sponge portion of the device in contact with the cervix for about one minute. Following collection, the sample will be placed into a sterile cryovial and frozen at -20 C or colder (-70 C/-80 C is also acceptable) within a minute (in order to preserve the quality of the sample). All cervical samples are to be shipped to GSK Biologicals (Rixensart, Belgium) in a frozen state (dry ice [-70 C/-80 C]). The laboratory assays to be used during the study are summarized in Table 2 below, see Appendix F for details of these assays. Table 2 Laboratory assays Assay type Marker Assay Test kit/ Assay unit Assay Laboratory method Manufacturer cut-off Quantitative anti-hpv-16 ELISA (or In-house assay EL.U/mL 8 EL.U/mL* GSK Biologicals** multiplex) Quantitative anti-hpv-18 ELISA (or In-house assay EL.U/mL 7 EL.U/mL* GSK Biologicals** multiplex) Quantitative Total IgG Commercial Commercial TBD TBD GSK Biologicals** Quantitative Cytokine positive CD4 or CD8 T cells Cytokine Flow cytometry GSK Biologicals frequency of specific CD4/CD8 NA GSK Biologicals** Quantitative Specific memory B cell B cell ELISPOT GSK Biologicals frequency of specific memory B cells NA GSK Biologicals** TBD=to be determined, NA=not applicable *assay cut-off for serum samples; assay cut-off for cervical samples is TBD. ** GSK Biologicals or designated laboratory Alternatively, an assay for detection of total IgG may be developed by GSK. At the discretion of GSK, these assays can also be expanded to measure the immunological response of additional HPV type VLPs including HPV-31, 45, 52, 33, 58 and 59 during or after the final analysis. The GSK Biologicals clinical laboratories at Rixensart have established a Quality System supported by procedures. The activities of the clinical laboratories are audited regularly for quality assessment by an internal (sponsor-dependent) but laboratory-independent Quality Department Immunological read-outs Serological and cervical assays for HPV-16/18 serology testing and total IgG testing will be performed at GSK Biologicals laboratories, Rixensart, Belgium. CMI assays will be performed at GSK Biologicals laboratories, Rixensart, Belgium or at a GSK Biologicals designated laboratory. Final 07 February CARS Id : CLIN_200611_2714/ Version : 1.18,Admin. QC/ Modify Date : 07/02/2007 1d073efcfea318b54b165ad19806b01d1175fa68 fc4511a37499ec c76c32be9dca75c110f

103 (HPV-028 ANC: 015) Table 3 Immunological read-outs Blood/cervical sampling time point Marker No. subjects Timing Month Visit no. Post vacc III 12 5 HPV-16, HPV-18, IgG, (CMI) 100 Post vacc III 18 6 HPV-16, HPV-18, IgG, (CMI) 100 CMI = Cell Mediated Immune response At the discretion of GSK, additional HPV-type antibodies including HPV-31, 45, 52, 33, 58 and 59 antibodies can also be tested during or after the final analysis. In case of insufficient blood sample volume to perform all assays for all antibodies, the assays will be performed according to the following priority ranking: HPV-16 antibody (by ELISA) HPV-18 antibody (by ELISA) Samples will not be labelled with information that directly identifies the subjects but will be coded with the identification number for the subject. Collected samples may be used for purposes related to the quality assurance of the laboratory tests described in this protocol. This may include the management of the quality of these current tests, the maintenance or improvement of these current tests, the development of new test methods for the markers described in this protocol, as well as making sure that new tests are comparable to previous methods and work reliably. It may be that any findings in the present or in other studies necessitate further investigation by GSK Biologicals into the efficacy or immunogenicity of the HPV vaccine and its constituents under study or further research in the HPV disease under study. Under these circumstances, additional testing on the samples may be performed by GSK Biologicals outside the scope of this protocol. Refer also to protocol Appendix B, where it is noted that the Investigator cannot perform any other biological assays except those described in the protocol or its amendment(s). Collected samples will be stored for up to 15 years (counting from when the last subject performed the last study visit), unless local rules, regulations or guidelines require different timeframes or different procedures, which will then be in line with the subject consent. These extra requirements need to be communicated formally to and discussed and agreed with GSK Biologicals. 6. INVESTIGATIONAL PRODUCTS AND ADMINISTRATION No additional vaccine dose(s) will be administered during the course of the ancillary protocol Treatment allocation and randomization Subjects were randomized at the time of enrolment in the study (HPV-015). Please refer to Section 6.4 of the protocol for study (HPV-015) for a description of treatment allocation and randomization methods. Final 07 February CARS Id : CLIN_200611_2714/ Version : 1.18,Admin. QC/ Modify Date : 07/02/2007 fc4511a37499ec c76c32be9dca75c110f 1d073efcfea318b54b165ad19806b01d1175fa

104 (HPV-028 ANC: 015) Subject identification numbers allocated in study (HPV-015) will be used in this ancillary study Method of blinding and breaking the study blind The double blind applies to this ancillary study. Please refer to Section 6.5 of the (HPV-015) protocol for the description of blinding methods and procedure to follow should the investigator determine the need to break the study blind Concomitant medication/treatment Please refer to Section 6.9 of the protocol for study (HPV-015) for a description of methods for concomitant medication/treatment reporting. 7. HEALTH ECONOMICS Please refer to Section 7 of the protocol for study (HPV-015). 8. ADVERSE EVENTS AND SERIOUS ADVERSE EVENTS Please refer to the primary study. Please refer to Section 8 of the (HPV-015) protocol for the description of adverse event and serious adverse event reporting which is part of the primary study Definition of an adverse event Please refer to Section 8.1 of the (HPV-015) protocol for the definition of an adverse event Definition of a serious adverse event Please refer to Section 8.2 of the (HPV-015) protocol for the definition of a serious adverse event Lack of efficacy Please refer to Section 8.3 of the (HPV-015) protocol for the definition of lack of vaccine protective efficacy Clinical laboratory parameters and other abnormal assessments qualifying as serious adverse events Please refer to Section 8.4 of the (HPV-015) protocol for the description of reporting abnormal laboratory findings or other abnormal assessments which is part of the primary study. Final 07 February CARS Id : CLIN_200611_2714/ Version : 1.18,Admin. QC/ Modify Date : 07/02/2007 fc4511a37499ec c76c32be9dca75c110f 1d073efcfea318b54b165ad19806b01d1175fa

105 (HPV-028 ANC: 015) 8.5. Time period, frequency, and method of detecting serious adverse events Please refer to Section 8.5 of the (HPV-015) protocol for the description of the time period, frequency, and method of detecting serious adverse events and adverse events related to new onset chronic diseases which are part of the primary study Solicited adverse events Please refer to Section of the (HPV-015) protocol for the description of the time period, frequency, and method of detecting solicited adverse events which are part of the primary study Evaluating adverse events and serious adverse events Please refer to Section 8.6 of the (HPV-015) protocol for the description of methods of evaluating adverse events and serious adverse events (including the assessment of intensity, causality and medically attended events) which are part of the primary study Follow-up of adverse events and serious adverse events and assessment of outcome Please refer to Section 8.7 of the (HPV-015) protocol for the description of methods of follow-up and assessment of outcome of adverse events and serious adverse events which are part of the primary study Prompt reporting of serious adverse events to GSK Biologicals Please refer to Section 8.8 of the (HPV-015) protocol for the description of time frames for submitting, completion and transmission of serious adverse event reports which are part of the primary study. A list of Study Contacts for Reporting SAEs in the different regions is provided as a separate attachment to the (HPV-015) protocol. Back-up Study Contact for Reporting SAEs GSK Biologicals Clinical Safety Physician Tel: Fax: or Mobile phone for 7/7 day availability: Back-up mobile phone contact: 24/24 hour and 7/7 day availability Final 07 February CARS Id : CLIN_200611_2714/ Version : 1.18,Admin. QC/ Modify Date : 07/02/2007 fc4511a37499ec c76c32be9dca75c110f 1d073efcfea318b54b165ad19806b01d1175fa

106 (HPV-028 ANC: 015) 8.9. Regulatory reporting requirements for serious adverse events Please refer to Section 8.9 of the (HPV-015) protocol for the description of regulatory reporting requirements for serious adverse events which are part of the primary study Post-study adverse events and serious adverse events Please refer to Section 8.10 of the (HPV-015) protocol for the definition and reporting of post-study adverse events and serious adverse events which are part of the primary study Pregnancy Please refer to Section 8.11 of the (HPV-015) protocol for the description of reporting and follow-up of pregnancy which are part of the primary study Treatment of adverse events Please refer to Section 8.12 of the (HPV-015) protocol for instructions regarding the treatment of adverse events and the reporting of any medication administered for the treatment of a serious adverse event which are part of the primary study. 9. SUBJECT COMPLETION AND WITHDRAWAL 9.1. Subject completion A subject who returns for the concluding visit foreseen in the protocol is considered to have completed the ancillary study Subject withdrawal Not applicable to this ancillary study. Please refer to Section 9.2 of the main study (HPV- 015) protocol Subject withdrawal from the study Not applicable to this ancillary study. Please refer to Section of the main study (HPV-015) protocol Subject withdrawal from investigational product Not applicable to this ancillary study. Final 07 February CARS Id : CLIN_200611_2714/ Version : 1.18,Admin. QC/ Modify Date : 07/02/2007 fc4511a37499ec c76c32be9dca75c110f 1d073efcfea318b54b165ad19806b01d1175fa

107 (HPV-028 ANC: 015) 10. DATA EVALUATION: CRITERIA FOR EVALUATION OF OBJECTIVES Endpoints T cell-mediated immune responses specific to HPV VLP types 16 and 18 (Month 12 and Month 18). Memory B cell immune responses specific to HPV VLP types 16 and 18 (Month 12 and Month 18). HPV-16 and HPV-18 antibody titres assessed by ELISA in cervical samples at Month 12 and 18 in subjects who have cervical samples collected at Month 12 and 18 (seropositivity rates and GMTs). Total IgG antibodies (ELISA) from cervical samples collected in this study, and serum samples collected in study (HPV-015) at Month 12 and Month 18. Correlation of anti-hpv-16 and anti-hpv-18 in serum collected in study (HPV-015) and in cervical responses collected in this study Exploratory endpoints At the discretion of GSK, all endpoints above may be expanded to HPV types not included in the vaccine. At the discretion of GSK, other immune parameters related to HPV or the HPV vaccine constituents may be tested. This may include evaluation of neutralizing antibodies Estimated sample size Approximately 100 subjects enrolled in pre-selected sites participating in study (HPV-015) will be enrolled Study cohorts to be evaluated Modified total vaccinated cohort The modified total vaccinated cohort will include all vaccinated subjects from the specific study sites that have received three vaccine doses and for whom data are available Derived and transformed data The assay cut-off value is defined by the laboratory before the analysis and is described in Section A seronegative subject is a subject whose antibody titre is below the assay cut-off value. Final 07 February CARS Id : CLIN_200611_2714/ Version : 1.18,Admin. QC/ Modify Date : 07/02/2007 fc4511a37499ec c76c32be9dca75c110f 1d073efcfea318b54b165ad19806b01d1175fa

108 (HPV-028 ANC: 015) A seropositive subject is a subject whose antibody titre is greater than or equal to the assay cut-off value. Seroconversion is defined as the appearance of antibodies (i.e. titre greater than or equal to the assay cut-off value) in the serum of subjects seronegative before vaccination. The Geometric Mean Titres (GMTs) calculations will be performed by taking the anti-log of the mean of the log titre transformations. Antibody titres below the cut-off of the assay will be given an arbitrary value of half the cut-off for the purpose of GMT calculation. For a given subject and a given immunogenicity measurement, missing or non-evaluable measurements will not be replaced Final analyses The primary study will be ongoing at the time of final analyses. This analysis will be performed on the modified total vaccinated cohort by an external statistician to safeguard the blinding in the remaining of the trial. No stopping rules will be applied. For each treatment group at each time point that a blood sample result is available in study HPV-015 the following analyses will be performed: calculation of seropositivity rates for HPV-16 and HPV-18 (with exact 95% confidence intervals [CI]) tabulation of GMT with 95% CI and range for antibodies for each antigen comparison with serum antibody titres of study (HPV-015) and other studies of the HPV development program Cell mediated immunity The following analysis will be performed: evaluation of cell mediated immune response (exploratory analysis) The analysis of the cell-mediated immune (CMI) response will be described in the report analysis plan (RAP). Cervicovaginal secretions The analysis of antibody titres measured by binding ELISA at Month 12 and Month 18 in cervicovaginal secretions will be described in the RAP Planned interim immunogenicity analysis An immunogenicity interim analysis is planned after all subjects have completed Visit 5 (Month 12). This analysis will be performed on the modified total vaccinated cohort by an external statistician to safeguard the blinding of the trial. No stopping rules will be applied. Final 07 February CARS Id : CLIN_200611_2714/ Version : 1.18,Admin. QC/ Modify Date : 07/02/2007 fc4511a37499ec c76c32be9dca75c110f 1d073efcfea318b54b165ad19806b01d1175fa

109 (HPV-028 ANC: 015) 11. 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. 12. REFERENCES GlaxoSmithKline Biologicals Protocol (HPV-015). Final 07 February CARS Id : CLIN_200611_2714/ Version : 1.18,Admin. QC/ Modify Date : 07/02/2007 fc4511a37499ec c76c32be9dca75c110f 1d073efcfea318b54b165ad19806b01d1175fa

110 (HPV-028 ANC: 015) Appendix A World Medical Association Declaration of Helsinki INTRODUCTION Recommendations guiding physicians in biomedical research involving human subjects Adopted by the 18 th World Medical Assembly Helsinki, Finland, June 1964 and amended by the 29 th World Medical Assembly Tokyo, Japan, October th World Medical Assembly Venice, Italy, October st World Medical Assembly Hong Kong, September 1989 and the 48 th General Assembly Somerset West, Republic of South Africa, October 1996 It is the mission of the physician to safeguard the health of the people. His or her knowledge and conscience are dedicated to the fulfilment of this mission. The Declaration of Geneva of the World Medical Association binds the physician with the words, "The health of my patient will be my first consideration," and the International Code of Medical Ethics declares that, "A physician shall act only in the patient's interest when providing medical care which might have the effect of weakening the physical and mental condition of the patient." The purpose of biomedical research involving human subjects must be to improve diagnostic, therapeutic and prophylactic procedures and the understanding of the etiology and pathogenesis of disease. In current medical practice most diagnostic, therapeutic or prophylactic procedures involve hazards. This applies especially to biomedical research. Medical progress is based on research which ultimately must rest in part on experimentation involving human subjects. In the field of biomedical research a fundamental distinction must be recognized between medical research in which the aim is essentially diagnostic or therapeutic for a patient, and medical research, the essential object of which is purely scientific and without implying direct diagnostic or therapeutic value to the person subjected to the research. Special caution must be exercised in the conduct of research which may affect the environment, and the welfare of animals used for research must be respected. Final 07 February CARS Id : CLIN_200611_2714/ Version : 1.18,Admin. QC/ Modify Date : 07/02/2007 fc4511a37499ec c76c32be9dca75c110f 1d073efcfea318b54b165ad19806b01d1175fa

111 (HPV-028 ANC: 015) Because it is essential that the results of laboratory experiments be applied to human beings to further scientific knowledge and to help suffering humanity, the World Medical Association has prepared the following recommendations as a guide to every physician in biomedical research involving human subjects. They should be kept under review in the future. It must be stressed that the standards as drafted are only a guide to physicians all over the world. Physicians are not relieved from criminal, civil and ethical responsibilities under the laws of their own countries. I. BASIC PRINCIPLES 1. Biomedical research involving human subjects must conform to generally accepted scientific principles and should be based on adequately performed laboratory and animal experimentation and on a thorough knowledge of the scientific literature. 2. The design and performance of each experimental procedure involving human subjects should be clearly formulated in an experimental protocol which should be transmitted for consideration, comment and guidance to a specially appointed committee independent of the investigator and the sponsor provided that this independent committee is in conformity with the laws and regulations of the country in which the research experiment is performed. 3. Biomedical research involving human subjects should be conducted only by scientifically qualified persons and under the supervision of a clinically competent medical person. The responsibility for the human subject must always rest with a medically qualified person and never rest on the subject of research, even though the subject has given his or her consent. 4. Biomedical research involving human subjects cannot legitimately be carried out unless the importance of the objective is in proportion to the inherent risk to the subject. 5. Every biomedical research project involving human subjects should be preceded by careful assessment of predictable risks in comparison with foreseeable benefits to the subject or to others. Concern for the interests of the subject must always prevail over the interests of science and society. 6. The right of the research subject to safeguard his or her integrity must always be respected. Every precaution should be taken to respect the privacy of the subject and to minimize the impact of the study on the subject's physical and mental integrity and on the personality of the subject. 7. Physicians should abstain from engaging in research projects involving human subjects unless they are satisfied that the hazards involved are believed to be predictable. Physicians should cease any investigation if the hazards are found to outweigh the potential benefits. 8. In publication of the results of his or her research, the physician is obliged to preserve the accuracy of the results. Reports of experimentation not in accordance with the principles laid down in this Declaration should not be accepted for publication. 9. In any research on human beings, each potential subject must be adequately informed of the aims, methods, anticipated benefits and potential hazards of the Final 07 February CARS Id : CLIN_200611_2714/ Version : 1.18,Admin. QC/ Modify Date : 07/02/2007 fc4511a37499ec c76c32be9dca75c110f 1d073efcfea318b54b165ad19806b01d1175fa

112 (HPV-028 ANC: 015) study and the discomfort it may entail. He or she should be informed that he or she is at liberty to abstain from participation in the study and that he or she is free to withdraw his or her consent to participation at any time. The physician should then obtain the subject's freely-given informed consent, preferably in writing. 10. When obtaining informed consent for the research project the physician should be particularly cautious if the subject is in a dependent relationship to him or her or may consent under duress. In that case the informed consent should be obtained by a physician who is not engaged in the investigation and who is completely independent of this official relationship. 11. In case of legal incompetence, informed consent should be obtained from the legal guardian in accordance with national legislation. Where physical or mental incapacity makes it impossible to obtain informed consent, or when the subject is a minor, permission from the responsible relative replaces that of the subject in accordance with national legislation. Whenever the minor child is in fact able to give a consent, the minor's consent must be obtained in addition to the consent of the minor's legal guardian. 12. The research protocol should always contain a statement of the ethical considerations involved and should indicate that the principles enunciated in the present Declaration are complied with. II. MEDICAL RESEARCH COMBINED WITH PROFESSIONAL CARE (Clinical research) 1. In the treatment of the sick person, the physician must be free to use a new diagnostic and therapeutic measure, if in his or her judgement it offers hope of saving life, re-establishing health or alleviating suffering. 2. The potential benefits, hazards and discomfort of a new method should be weighed against the advantages of the best current diagnostic and therapeutic methods. 3. In any medical study, every patient - including those of a control group, if any - should 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. Final 07 February CARS Id : CLIN_200611_2714/ Version : 1.18,Admin. QC/ Modify Date : 07/02/2007 fc4511a37499ec c76c32be9dca75c110f 1d073efcfea318b54b165ad19806b01d1175fa

113 (HPV-028 ANC: 015) 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. In research on man, the interest of science and society should never take precedence over considerations related to the well being of the subject. Final 07 February CARS Id : CLIN_200611_2714/ Version : 1.18,Admin. QC/ Modify Date : 07/02/2007 fc4511a37499ec c76c32be9dca75c110f 1d073efcfea318b54b165ad19806b01d1175fa

114 (HPV-028 ANC: 015) Appendix B Administrative Matters I. Responsibilities of the Investigator II. 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 or Investigator Biography and other credentials (e.g., medical license number in the United States) to GSK and where required to relevant authorities. It is recommended that this documentation indicates any previous clinical research experience and history of training in GCP. To acquire the reference ranges for laboratory tests performed locally and, if required by local regulations, obtain the laboratory s current certification or Quality Assurance procedure manual. 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 on the clinical samples except those described in the protocol or its amendment(s). To prepare and maintain adequate subject source data or raw data designed to record observations, and other data pertinent to the study. To conduct the study in compliance with the protocol any amendment and Good Clinical Practice (GCP) and all applicable regulatory requirements. To co-operate with a representative of GSK Biologicals in the monitoring process of the study and in resolution of queries about the data. To permit drug regulatory agencies and GSK audits. Protocol Amendments and Administrative changes No changes to the study protocol will be allowed unless discussed in detail with the GSK Biologicals' Clinical Development Manager/Medical Monitor and filed as an amendment/administrative change to this protocol. Any amendment/administrative change 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, except where permitted by all applicable regulatory requirements; administrative changes and amendments not submitted for approval are submitted to IRBs/IECs for information only. Any amendment/administrative change 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/administrative changes is required prior to implementation for US studies only. Final 07 February CARS Id : CLIN_200611_2714/ Version : 1.18,Admin. QC/ Modify Date : 07/02/2007 fc4511a37499ec c76c32be9dca75c110f 1d073efcfea318b54b165ad19806b01d1175fa

115 (HPV-028 ANC: 015) III. Submission of protocol amendments to regulatory agencies will occur in accordance with local regulatory requirements. For some countries, submission to the local regulatory authority may not be required. When submission to the local regulatory authority is required, the timing of the submission relative to IEC/IRB submission or approval and whether or not the authority will provide their approval of or favorable opinion on the amendment before it can be implemented will depend on local regulatory requirements. Sponsor s Termination of Study GSK Biologicals reserves the right to temporarily suspend or prematurely discontinue this study either at a single site or at all sites at any time for reasons including, but not limited to, safety or ethical issues or severe non-compliance. Reasons for suspension or early termination will be documented in the study file at GSK Biologicals. If GSK Biologicals determines that suspension or early termination is needed, GSK Biologicals will discuss this with the Investigator (including the reasons for taking such action). When feasible, GSK Biologicals will provide advance notification to the investigator of the impending action prior to it taking effect. GSK Biologicals will promptly inform, via written communication, all investigators and/or institutions conducting the study, if the study is suspended or terminated for safety reasons, and will also inform the regulatory authorities of the suspension or termination of the study and the reason(s) for the action. If required by applicable regulations, the investigator must inform the IEC/IRB promptly and provide the reason for the suspension or termination. If the study is prematurely discontinued, all study data must be returned to GSK. In addition, arrangements will be made for all unused investigational product(s) in accordance with the applicable GSK procedures for the study. Financial compensation to investigators and/or institutions will be in accordance with the agreement established between the investigator and/or institutions and GSK. IV. Remote Data Entry Instructions Remote Data Entry (RDE) will be used as the method for data collection, which means that subject information will be entered into a computer at the investigational site preferably within 5 working days of becoming available. The site will be capable of modifying the data to assure accuracy with source documentation. All new/updated information will be reviewed and verified by a GSK Biologicals' representative. This information will finally be stored in a central database maintained by GSK Biologicals. At the conclusion of the study, GSK Biologicals will archive the study data in accordance with internal procedures. In addition, the investigator will be provided with a CD-ROM of the final version of the data generated at the investigational site. Specific instructions for use of RDE will be included in the training material provided to the investigational site. Final 07 February CARS Id : CLIN_200611_2714/ Version : 1.18,Admin. QC/ Modify Date : 07/02/2007 fc4511a37499ec c76c32be9dca75c110f 1d073efcfea318b54b165ad19806b01d1175fa

116 (HPV-028 ANC: 015) V. Monitoring by GSK Biologicals To ensure compliance with the protocol, monitoring visits by a professional representative of the sponsor will be scheduled to take place early in the study, 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 and communicated to the investigator before study start. These visits are for the purpose of confirming that GSK Biologicals sponsored studies are being conducted in accordance with the ethical principles that have their origins in the Declaration of Helsinki and that are consistent with Good Clinical practice (GCP) and the applicable regulatory requirement(s) (verifying continuing compliance with the protocol, amendment(s), reviewing the investigational product accountability records, verifying that the site staff and facilities continue to be adequate to conduct the study. Direct access to all study-related site and source data/ documents is mandatory for the purpose of monitoring review. The monitor will perform a CRF review and a Source Document verification (verifying CRF/ RDE entries by comparing them with the source data/documents that will be made available by the investigator for this purpose: any data item for which the CRF will serve as the source must be identified, agreed and documented. Data to be recorded directly into the CRF pages/rde screens will be specified in writing preferably in the source documentation agreement form that is contained in both the monitor s and investigator s study file. For RDE, the monitor will mark completed and approved screens at each visit. The investigator must ensure provision of reasonable time, space and adequate qualified personnel for monitoring visits. Source data verification (SDV) must be conducted using a GSK standard SDV sampling strategy (as defined at the study start in the study specific monitoring guidelines) in which monitors will perform partial SDV for all subjects and full SDV for selected subjects. VI. Archiving of Data Following closure of the study, the investigator must maintain all site study records in a safe and secure location. The records must be maintained to allow easy and timely retrieval, when needed (e.g., audit or inspection), and, whenever feasible, to allow any subsequent review of data in conjunction with assessment of the facility, supporting systems, and staff. Where permitted by applicable laws/regulations or institutional policy, some or all of these records can be maintained in a validated format other than hard copy (e.g., microfiche, scanned, electronic for studies with an ecrf, for example); however, caution needs to be exercised before such action is taken. The investigator must assure that all reproductions are legible and are a true and accurate copy of the original, and meet accessibility and retrieval standards, including re-generating a hard copy, if required. Furthermore, the investigator must ensure there is an acceptable back-up of these reproductions and that an acceptable quality control process exists for making these reproductions. GSK will inform the investigator/ institution of the time period for retaining these records to comply with all applicable regulatory requirements. However, the investigator/ institution should seek the written approval of the sponsor before proceeding with the Final 07 February CARS Id : CLIN_200611_2714/ Version : 1.18,Admin. QC/ Modify Date : 07/02/2007 fc4511a37499ec c76c32be9dca75c110f 1d073efcfea318b54b165ad19806b01d1175fa

117 (HPV-028 ANC: 015) disposal of these records. The minimum retention time will meet the strictest standard applicable to that site for the study, as dictated by ICH GCP E6 Section 4.9, any institutional requirements or applicable laws or regulations, or GSK standards/procedures; otherwise, the minimum retention period will default to 15 years. The investigator/ institution must notify GSK of any changes in the archival arrangements, including, but not limited to, the following: archival at an off-site facility, transfer of ownership of the records in the event the investigator leaves the site. VII. Audits For the purpose of compliance with Good Clinical Practice and Regulatory Agency Guidelines it may be necessary for GSK or a Drug Regulatory Agency to conduct a site audit. This may occur at any time from start to after conclusion of the study. When an investigator signs the protocol, he agrees to permit drug regulatory agencies and GSK audits, providing direct access to source data/ documents. Furthermore, if an investigator refuses an inspection, his data will not be accepted in support of a New Drug Registration and/or Application, Biologics Licensing Application. Having the highest quality data and studies are essential aspects of vaccine development. GSK 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. GSK s audits entail review of source documents supporting the adequacy and accuracy of CRFs, review of documentation required to be maintained, and checks on vaccine accountability. GSK s 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: Log of visits from the sponsor s representatives Study personnel Study file Safety reporting IRB/IEC and regulatory authority approvals Facilities monitoring Vaccine accountability Approved study protocol and amendments and investigator brochure Final 07 February CARS Id : CLIN_200611_2714/ Version : 1.18,Admin. QC/ Modify Date : 07/02/2007 fc4511a37499ec c76c32be9dca75c110f 1d073efcfea318b54b165ad19806b01d1175fa

118 (HPV-028 ANC: 015) Informed consent of the subjects (written consent or witnessed oral if applicable ) Medical records and other source documents supportive of CRF data Reports to the IRB/IEC and the sponsor Record retention. GSK Biologicals will gladly help investigators prepare for an inspection. VIII. Ownership, Confidentiality and Publication Ownership: All information provided by GSK and all data and information generated by the site as part of the study (other than a subject s medical records) are the sole property of GSK. All rights, title, and interests in any inventions, know-how or other intellectual or industrial property rights which are conceived or reduced to practice by site staff during the course of or as a result of the study are the sole property of GSK, and are hereby assigned to GSK. If a written contract for the conduct of the study which includes ownership provisions inconsistent with this statement is executed between GSK and the study site, that contract s ownership provisions shall apply rather than this statement. Confidentiality: Documented evidence that a potential investigator is aware and agrees to the confidential nature of the information related to the study must be obtained by means of a confidentiality agreement. All information provided by GSK and all data and information generated by the site as part of the study (other than a subject s medical records) will be kept confidential by the investigator and other site staff. This information and data will not be used by the investigator or other site personnel for any purpose other than conducting the study. These restrictions do not apply to: (1) information which becomes publicly available through no fault of the investigator or site staff; (2) information which it is necessary to disclose in confidence to an IEC or IRB solely for the evaluation of the study; (3) information which it is necessary to disclose in order to provide appropriate medical care to a study subject; or (4) study results which may be published as described in the next paragraph. If a written contract for the conduct of the study which includes confidentiality provisions inconsistent with this statement is executed, that contract s confidentiality provisions shall apply rather than this statement. Publication: For multicenter studies, the first publication or disclosure of study results shall be a complete, joint multicenter publication or disclosure coordinated by GSK. Thereafter, any secondary publications will reference the original publication(s). Prior to submitting for publication, presentation, use for instructional purposes, or otherwise disclosing the study results generated by the site (collectively, a Publication ), the investigator shall provide GSK with a copy of the proposed Publication and allow Final 07 February CARS Id : CLIN_200611_2714/ Version : 1.18,Admin. QC/ Modify Date : 07/02/2007 fc4511a37499ec c76c32be9dca75c110f 1d073efcfea318b54b165ad19806b01d1175fa

119 (HPV-028 ANC: 015) GSK a period to review the proposed Publication (at least 21 (twenty-one) days [or at least 15 (fifteen) working days for abstracts/posters/presentations]. Proposed Publications shall not include either GSK confidential information other than the study results or personal data on any subject, such as name or initials. At GSK s request, the submission or other disclosure of a proposed Publication will be delayed a sufficient time to allow GSK to seek patent or similar protection of any inventions, know-how or other intellectual or industrial property rights disclosed in the proposed Publication. If a written contract for the conduct of the study, which includes publication provisions inconsistent with this statement is executed, that contract s publication provisions shall apply rather than this statement. Final 07 February CARS Id : CLIN_200611_2714/ Version : 1.18,Admin. QC/ Modify Date : 07/02/2007 fc4511a37499ec c76c32be9dca75c110f 1d073efcfea318b54b165ad19806b01d1175fa

120 (HPV-028 ANC: 015) Appendix C Overview of the Recruitment Plan for the Ancillary Study A total of approximately 100 subjects in the immunogenicity subset who have received the three doses of study vaccine/control at pre-selected sites in study (HPV-015) will be asked to participate in the present study. Final 07 February CARS Id : CLIN_200611_2714/ Version : 1.18,Admin. QC/ Modify Date : 07/02/2007 fc4511a37499ec c76c32be9dca75c110f 1d073efcfea318b54b165ad19806b01d1175fa

121 (HPV-028 ANC: 015) Appendix D Handling of Biological Samples Collected by the Investigator When materials are provided by GSK Biologicals or Quest Diagnostics, it is mandatory that all clinical 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 analysis. The investigator must ensure that his/her personnel and the laboratory(ies) under his/her supervision comply with this requirement. However, when GSK Biologicals or Quest Diagnostics do not provide material for collecting and storing clinical samples, then appropriate materials from the investigator s site are to be used. Instructions for Handling Cells for Cell-Mediated Immunity Assay 1. Collection of whole blood Collect blood by venipuncture in Terumo tubes with heparin (or equivalent) and record time of collection. The tubes should be kept at room temperature and shipped to a designated clinical site for separation of peripheral blood mononuclear cells (PBMCs). The shipment must be timed to ensure that PBMC separation will be performed within 24 hours. Use well closed styrofoam boxes of 5 cm thickness for blood samples transport (see current version of GSK Biologicals SOP RD_HCI_001 for guidance). Detailed instructions for handling and shipment of whole blood samples will be provided in a separate manual. 2. Separation and freezing of PBMCs PBMCs will be separated on a density gradient, aliquoted and frozen at 80 C for 24 hours and further cryopreserved in liquid nitrogen until testing (see GSK Biologicals SOP: RD_HCI_007 for guidance). 3. Labelling of cryotubes for PBMC samples If labels are provided by GSK, it is mandatory to use them. If necessary, any hand-written additions to the labels should be made using indelible ink. 4. Sorting and storage of PBMC samples Samples should be stored in liquid nitrogen until shipment to GSK Biologicals, if needed. Wherever possible, a backup facility for storage of samples should be available. A standard Cryotube Listing Form (see current version of GSK Biologicals SOP RD_HCI_009 for guidance), specifying the samples being shipped for individual subjects at each timepoint, should be prepared for each shipment. A copy of this list should be retained at the study site, while the original should be sealed in a plastic envelope and shipped with the PBMC samples. Final 07 February CARS Id : CLIN_200611_2714/ Version : 1.18,Admin. QC/ Modify Date : 07/02/2007 fc4511a37499ec c76c32be9dca75c110f 1d073efcfea318b54b165ad19806b01d1175fa

122 (HPV-028 ANC: 015) 5. Instructions for Handling Cervical Samples Cervical secretions will be collected using Merocel ophthalmic sponges for evaluation of anti-hpv-16 and anti-hpv-18 antibodies by placing the sponge portion of the device in contact with the cervix for about one minute. Following collection, the sample is placed into a sterile cryovials and frozen at -70 C/-80 C (-20 C is also acceptable) within a minute (in order to preserve the quality of the sample). All cervical samples are to be shipped to GSK Biologicals (Rixensart, Belgium) in a frozen state (dry ice [-70 C/-80 C]). Final 07 February CARS Id : CLIN_200611_2714/ Version : 1.18,Admin. QC/ Modify Date : 07/02/2007 fc4511a37499ec c76c32be9dca75c110f 1d073efcfea318b54b165ad19806b01d1175fa

123 (HPV-028 ANC: 015) Appendix E Shipment of Biological Samples Instructions for Shipment of PBMC Samples Frozen PBMCs must be transported in liquid nitrogen in an appropriate dry shipper (ideally CP400 or equivalent containing up to 4 boxes of 100 cryovials). The dry shipper must be previously filled with liquid nitrogen as described on the lid of the shipper, as follows: 1. Remove the unit from its shipping enclosure. 2. Fill the refrigerator unit completely with liquid nitrogen (do not overfill). 3. Let the unit stand undisturbed while the refrigerant is being absorbed, for 24 hours. 4. Repeat refills three times. 5. Pour out the remaining liquid standing in the central cavity of the refrigerator to prevent spillage during shipment. When completely filled, the CP400 should weigh 25 kg. When the refrigerator is charged, transfer the material from the liquid nitrogen storage tank into the dry shipper as quickly as possible (see GSK Biologicals SOP: RD_HCI_009_E for guidance). Details of the shipment, including number of samples and date of transfer should be sent by fax, two days before shipment, to: GLAXOSMITHKLINE BIOLOGICALS Attention Biospecimen Reception Clinical Immunology R & D Department/Building 44 Rue de l'institut, 89 B-1330 Rixensart Belgium Telephone: or or or Fax: Instructions for Shipment of Cervical Samples Cervical samples should be sent to GSK Biologicals at regular intervals. The frequency of shipment of samples should be decided upon by the Site Monitor, Central Study Coordinator and the investigator prior to the study start. The container must be clearly identified with the labels provided by GSK Biologicals specifying the shipment address and the storage temperature. Cervical samples should always be sent by air, preferably on a Monday, Tuesday or Wednesday, unless otherwise requested by the sponsor. Final 07 February CARS Id : CLIN_200611_2714/ Version : 1.18,Admin. QC/ Modify Date : 07/02/2007 fc4511a37499ec c76c32be9dca75c110f 1d073efcfea318b54b165ad19806b01d1175fa

124 (HPV-028 ANC: 015) Cervical samples must be placed with dry ice (maximum -20 C) in a container complying with International Air Transport Association (IATA) requirements. The completed standard Biological Specimen Listing Form and the completed Biological Specimen Transfer Form should always accompany the shipment. A standard Biological Specimen Listing Form, specifying the cervical samples being shipped for individual subjects at each time point, should be prepared for each shipment. A copy of this list should be retained at the study site, while the original should be sealed in a plastic envelope and shipped with the cervical samples. Once shipment details are known (number samples, airway bill, flight number and flight departure and arrival times), a standard Biological Specimen Transfer Form must be completed and faxed to GSK Biologicals two days before shipment to the number provided below. A copy of the Biological Specimen Transfer Form must be in the parcel. 1 The airway bill should contain the instruction for storage of samples at maximum -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 maximum -20 C. The shipment address is the following: GLAXOSMITHKLINE BIOLOGICALS Attention Biospecimen Reception Clinical Immunology R & D Department/Building 44 Rue de l'institut, 89 B-1330 Rixensart Belgium Telephone: or or or Fax: 1 The Biological Specimen Listing Form and the Biological Specimen Transfer Form are standard documents used in GSK Biologicals clinical trials. These documents are provided by GSK Biologicals monitor at study initiation. Final 07 February CARS Id : CLIN_200611_2714/ Version : 1.18,Admin. QC/ Modify Date : 07/02/2007 fc4511a37499ec c76c32be9dca75c110f 1d073efcfea318b54b165ad19806b01d1175fa

125 (HPV-028 ANC: 015) Appendix F Laboratory Assays Anti-HPV-16 and anti-hpv-18 ELISA (cervical samples) Antibody responses against HPV-16 and HPV-18 VLPs will be quantified by ELISA. These immunoassays are based on the direct test principle. Purified VLPs are coated onto a 96-well microtitration plate. After a washing and saturation step, serial dilutions of sera, control sera and standards are distributed and incubated in the coated wells to allow the specific antibody present in the sample to react with the corresponding antigen. Non-specific reactants are removed by washing and a peroxidase-conjugated anti-human polyclonal antibody is added to react with the specific antibody. Excess conjugate is removed by washing. Enzyme substrate and chromogen are added and the colour is allowed to develop. After adding the Stop Reagant, the resultant colour change is quantified and expressed in EL.U/mL. The intensity of the resultant yellow colour is directly proportional to the concentration of anti-vlp antibody present in the sample. Titres are calculated by reference to a standard serum using the 4-parameters equation for each serum dilution, and the final titre of a serum is the average of all titres falling in the proportional part of the reference curve. At the discretion of GSK, additional HPV-type antibodies including HPV-31, 45, 52, 33, 58 and 59 antibodies can also be tested. Total IgG by ELISA (serum and cervical samples) A commercially available assay or an assay developed by GSK will be used to determine the total IgG content in the cervical and serum samples. Final 07 February CARS Id : CLIN_200611_2714/ Version : 1.18,Admin. QC/ Modify Date : 07/02/2007 fc4511a37499ec c76c32be9dca75c110f 1d073efcfea318b54b165ad19806b01d1175fa

126 (HPV-028 ANC: 015) Cell Mediated Immune Response (subset 100 subjects) Cytokine flow cytometry assay The cell-mediated immune (CMI) response is assessed by GSK Biologicals, or a designated laboratory, on thawed peripheral blood mononuclear cells (PBMCs) by intracellular cytokine staining. This assay provides information on the frequency of CD4+ and CD8+ T cells responding to the antigen and producing secreted molecules involved in immunity; such as IFN-, IL-2, TNF-, and CD40-L. Briefly, the subjects PBMCs are stimulated for 2 hours in a pool of overlapping peptides covering the entire sequence of the vaccine antigen or the protein antigen. An intracellular block (Brefeldin A) is then added to inhibit cytokine secretion for a subsequent overnight stimulation. Cells are then harvested, stained for surface markers (CD4 and CD8) and fixed. Fixed cells are permeabilized and stained with anti-cytokinespecific antibodies, washed and analyzed by flow cytometry. Results will be expressed as frequency of cytokine(s)-positive CD4 or CD8 T cells within the CD4 or CD8 T cell sub-population. Extra analyses, not described in the protocol, related to the immune response to HPV vaccination might be performed if deemed necessary. B cell ELISPOT assay Memory B cells can be induced to differentiate into plasma cells in vitro following culture with CpG for five days. In vitro generated antigen-specific plasma cells can consequently be enumerated using the B cell ELISPOT assay. Briefly, in vitro generated plasma cells are incubated in culture plates coated with antigen. Antigen-specific plasma cells will form antibody/antigen spots, which can be detected by conventional immuno-enzymatic methods. VLPs or anti-human immunoglobulin will be used to coat culture plates to enumerate anti-hpv or total Ig-secreting plasma cells, respectively. Results will be expressed as the number of antigen-specific memory B cells per million of memory cells. Final 07 February CARS Id : CLIN_200611_2714/ Version : 1.18,Admin. QC/ Modify Date : 07/02/2007 fc4511a37499ec c76c32be9dca75c110f 1d073efcfea318b54b165ad19806b01d1175fa

127 (HPV-028 ANC: 015) Appendix G Vaccine Supplies, Packaging and Accountability No vaccine will be administered during this ancillary study. Adequate supplies will be provided either by GSK Biologicals or by Quest. 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, identification number for the subject {e.g. Subject number}, sampling timepoint {e.g., post vacc 3}, timing {e.g., study Month 12}. Other supplies provided by GSK Biologicals: In addition to study documentation and the sample labels, the investigator will receive the following supplies: tubes with screw caps for blood samples, racks and cardboard boxes for the tubes of blood, Merocel ophthalmic sponges and cryovials for collection of cervical samples. The investigator or pharmacist must sign a statement that he/she has received the clinical supplies for the study. It is NOT permitted to use any of the supplies provided by GSK Biologicals for purposes other than those specified in the protocol. The supplies receipt documents must then be returned to: Attention of Clinical Trial Supplies Unit GSK Biologicals Rixensart Fax : Final 07 February CARS Id : CLIN_200611_2714/ Version : 1.18,Admin. QC/ Modify Date : 07/02/2007 fc4511a37499ec c76c32be9dca75c110f 1d073efcfea318b54b165ad19806b01d1175fa

128 d8e2034af8cc182928ef2c9fdf2ace41 1d073efcfea318b54b165ad19806b01d1175fa

129 d8e2034af8cc182928ef2c9fdf2ace41 1d073efcfea318b54b165ad19806b01d1175fa

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132 Sample Case Report Form a49597d4c93feed7213c35c9422d4bf49e d1abed9cf986d2e478f2802d3ff6b050f783b

133 WORKBOOK Centre number Subject number Treatment number Protocol (HPV-028 ANC 015) Complementary immunology testing in a phase III, double-blind, randomized, controlled study to evaluate the safety, immunogenicity and efficacy of GlaxoSmithKline Biologicals HPV-16/18 L1 AS04 vaccine administered intramuscularly according to a three-dose schedule (0, 1, 6 month) in healthy adult female subjects aged 26 years and above enrolled in study (HPV-015) GlaxoSmithKline Biologicals Rue de l Institut 89 B 1330 Rixensart, Belgium Tel: CRF Template version 12.3 March 14, fc36fcc5dd88b61c d1abed9cf986d2e478f2802d3ff6b050f783b