AN EVALUATION OF NON-INFERIORITY IN ANTIBODY RESPONSE TO HUMAN PAPILLOMAVIRUS (HPV) 16 IN SUBJECTS VACCINATED WITH ACCEPTED

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1 CVI Accepts, published online ahead of print on 11 April 2007 Clin. Vaccine Immunol. doi: /cvi Copyright 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. AN EVALUATION OF NON-INFERIORITY IN ANTIBODY RESPONSE TO HUMAN PAPILLOMAVIRUS (HPV) 16 IN SUBJECTS VACCINATED WITH MONOVALENT (HPV 16) AND QUADRIVALENT (HPV 6, 11, 16, 18) L1 VIRUS LIKE PARTICLE VACCINES Suzanne M. Garland MD 1, Marc Steben MD 2*, Mauricio Hernandez-Avila MD 3, Laura A. Koutsky PhD 4, Cosette M. Wheeler PhD 5, Gonzalo Perez MD 6, Diane M. Harper MD, MPH, MS 7, Sepp Leodolter MD 8, Grace WK Tang MD 9, Daron G. Ferris MD 10, Mark T. Esser PhD 11, Scott C.Vuocolo PhD 11, Micki Nelson BS 11, Radha Railkar PhD 11, Carlos Sattler, MD 11, and Eliav Barr MD 11 on behalf of the 012 Study Investigators. 1 Microbiology and Infectious Diseases Department, Royal Women's Hospital, Carlton, Department of Obstetrics and Gynaecology, University of Melbourne, Victoria, Australia; 2 Direction Risques Biologiques, Environnementaux et Occupationnels, Institut National de Santé Publique du Québec, Montréal, Qc, Canada; 3 National Institute of Public Health, Cuernavaca, Morelos, Mexico; 4 Department of Epidemiology, University of Washington, Seattle WA, USA; 5 Departments of Molecular Genetics and Microbiology and Obstetrics and Gynecology, University of New Mexico, Albuquerque NM, USA; 6 National Research Center, Group Saludcoop, Bogotá, Colombia; 7 Norris Cotton Cancer Center, Departments of Obstetrics & Gynecology and Community & Family Medicine, Dartmouth Medical School, Hanover NH, USA; 8 Department of Gynecology and Obstetrics, Women's Health Clinic, Medical University of Vienna, Vienna, Austria; 9 Department of Obstetrics and Gynecology, University of Hong Kong, HKSAR; 10 Department of Family Medicine and Obstetrics and Gynecology, Medical College of Georgia, Augusta, GA, USA; 11 Merck Research Laboratories, West Point, PA USA Running head: Bridging of Anti-HPV 16 Response Source of support: Merck Research Laboratories, a division of Merck & Co *Correspondence: Marc Steben MD Direction risques biologiques, environnementaux et occupationnels Institut national de santé publique du Québec 190, boulevard Crémazie Est - Montréal (Québec) Canada H2P 1E2 Téléphone : (514) ext.3234 Télécopieur : (514) A listing of investigators can be found in the acknowledgements. 1

2 ABSTRACT Incorporation of multiple antigens into a single HPV vaccine may induce immune interference. To evaluate whether interference occurs when combining HPV16 VLPs in a multivalent vaccine, we conducted a study to evaluate anti-hpv16 responses among subjects receiving 3-dose regimens of either HPV16 vaccine or a quadrivalent HPV (types 6, 11, 16, 18) vaccine. (Trial registry #NCT ) 2

3 Natural history studies conducted over the past 20 years have demonstrated that Human Papillomavirus (HPV) infection is a necessary prerequisite for development of cervical cancer.[1] These studies have also demonstrated that among the 40 genital HPV types, only a subset of approximately 15 HPV types are oncogenic. Among these types, HPV16 is responsible for over 50% of cervical cancers, and an even higher proportion of HPV-related vulvar and vaginal cancers [2]. Of all HPV types with a tropism for genital tissues, HPV16 is the most likely to persist and result in abnormal Pap tests and cervical dysplastic lesions.[3] While combining several antigens into one vaccine is an efficient means to broaden the vaccine s coverage, such combination may result in immune interference, defined as the reduction in the immunogenicity of a vaccine antigen when it is administered as a component of a vaccine that includes multiple vaccine antigens. There are no physicochemical tests that can be used to accurately predict whether combining several immunogenic antigens into a single vaccine will result in interference. Thus, for the HPV vaccine candidates, the impact of combining HPV virus-like particles (VLPs) targeting oncogenic HPV types into a single vaccine on the immunogenicity of each VLP required a clinical evaluation. Here we present the results of a study that compared anti-hpv16 responses following administration of 3-dose regimens of either HPV 16 vaccine or a quadrivalent HPV (types 6, 11, 16, 18) vaccine. This report is representative of data from 3,882 subjects who were enrolled in Merck HPV vaccine protocol 012 between May 30th, 2002 and June 30th, Protocol 012 was part of a larger, randomized, double-blind (operating under in-house blinding 3

4 procedures), placebo-controlled, multicenter efficacy study in 16- to 23-year-old women of quadrivalent HPV (types 6, 11, 16, and 18) L1 VLP vaccine (GARDASIL, Merck & Co., Inc.) enrolling 5,455 subjects (protocol 013; FUTURE I).[4] Subjects participating in protocol 013 were enrolled in the context of 2 separate immunogenicity sub-studies: a concomitant hepatitis B vaccine (recombinant) administration sub-study (protocol 011, to be reported separately elsewhere) and a monovalent HPV16 vaccine immuno-bridging sub-study (protocol 012), results of which are presented herein (Figure 1). Protocol 012 randomized subjects in a 6:1:6 ratio to quadrivalent HPV vaccine, HPV16 vaccine, or placebo, respectively. Subjects must have reported 0 to 4 lifetime sex partners, no prior history of clinical HPV disease (CIN [cervical intraepithelial neoplasia], genital warts), and no allergy to vaccine components at day 1 to be enrolled. Subjects were enrolled regardless of day 1 HPV status or Pap test result. All eligible subjects received intramuscular (deltoid) injections of quadrivalent HPV vaccine, monovalent HPV 16 vaccine, or placebo at enrollment (day 1), month 2, and month 6. Subjects underwent cervicovaginal sampling for detection of HPV infection as well as serology testing. To improve precision, subjects were included in the primary immunogenicity analyses only if they did not have protocol violations that may have influenced vaccine-induced immune responses. The quadrivalent HPV vaccine and visually indistinguishable aluminumcontaining placebo have been previously described.[5] Allocation schedules were generated by computer with a blocking factor of 13. Subjects were randomized in a 6:1:6 ratio to quadrivalent HPV vaccine, HPV 16 vaccine, or placebo, respectively, at each 4

5 study site. An Interactive Voice Response System (IVRS ) was used to allocate subjects. Blood samples were obtained for anti-hpv serology testing at enrollment and month 7 for anti-hpv 6, anti-11, anti-16, and anti-18 assay (using competitive immunoassay developed by the sponsor using technology from the Luminex Corporation, Austin, TX, U.S.A.).[6] At each study visit all examinations and specimen collections took place prior to vaccination. Temperature, weight, and a serum or urine pregnancy test (β Human Chorionic Gonadotropin) were obtained prior to each injection. If the subject had a temperature of 100 F or 37.8 C (oral) within 24 hours prior to a vaccine dose, the injection was to be postponed. Comparisons of the immunogenicity of quadrivalent HPV vaccine and monovalent HPV 16 vaccine were carried out via competitive luminescence immunoassay (clia).[6] This assay was used to evaluate the serological response to the vaccine, to measure HPV infection induced antibody, and to exclude subjects with evidence of a current or past HPV infection from the primary analysis. For immunogenicity, the endpoints of interest were: (1) the anti-hpv 16 geometric mean titer (GMT) at week 4 post-dose 3; and (2) the percentage of subjects who seroconverted for HPV 16 by week 4 post-dose 3. As the HPV L1 VLP vaccines are intended to be prophylactic, the primary immunogenicity analysis was conducted in the per-protocol immunogenicity (PPI) population. This population included subjects who were-hpv 16 seronegative by clia at day 1 and PCR-negative to HPV 16 (swab and biopsy specimens) from enrollment through month 7. 5

6 The first primary hypothesis addressed the similarity (non-inferiority) in HPV 16 immune responses to the quadrivalent HPV (types 6,11,16,18) L1 VLP vaccine and the monovalent HPV 16 L1 VLP vaccine, as measured by anti-hpv 16 GMTs at week 4 post-dose 3. This hypothesis was addressed by an analysis of variance (ANOVA) model, which involved modeling the natural log of the post-dose 3 anti-hpv 16 levels of the subjects as a function of study country and vaccination group, both of which were fixed effects. The anti-log of: (1) the estimated treatment effect, and (2) its confidence interval were utilized to report the estimated GMT and 95% confidence interval for the GMT, respectively. This methodology tested a null hypothesis of a 0.5-fold difference or less in GMTs against the alternative hypothesis of >0.5 fold in the difference of GMTs. The second primary immunogenicity hypothesis addressed the similarity in anti-hpv 16 responses to the quadrivalent HPV vaccine and the monovalent HPV 16 vaccine, as measured by the percentage of subjects who seroconverted for HPV 16 by week 4 postdose 3 (seroconversion was defined as changing serostatus from seronegative to seropositive, where the anti-hpv 16 serum clia cutoff for determining serostatus was 20 mmu/ml; a subject with a clia titer at or above the serostatus cutoff was considered seropositive). This hypothesis was addressed by a one-sided test of non-inferiority conducted at the level. In order for the null hypothesis to be rejected, the lower bound of the 95% confidence interval for the difference in the percentage of subjects who seroconverted between the quadrivalent vaccine group and the monovalent HPV 16 vaccine group had to be greater than The test was based on the method of Miettinen and Nurminen [7]for testing the equivalence of 2 proportions which allows for 6

7 stratification by study size. Success of the study required both primary immunogenicity hypotheses to be successful and therefore no multiplicity adjustments were required. An audit conducted by Merck Research Laboratories concluded that there was a deviation from the Standard Operating Procedure (SOP) for testing a subset of serum samples from the protocol. Approximately 0.1% of day 1 serology results and 0.4% of post-vaccination serology results were determined to have been tested outside of the vaccination group corresponding to Standard Operating Procedure. All day 1 serum samples which were tested out of compliance with the actual clinical material received (i.e., HPV SOP) were reanalyzed. The remaining non-conformant test results were removed from the database. Of 3,882 subjects enrolled 3,875 received at least one dose of vaccine or placebo. A total of 95% of subjects in the quadrivalent HPV vaccine group and 96% of subjects in the HPV16 vaccine group received all 3 doses of vaccine. A total of 211 subjects discontinued study participation during the vaccination period. The percentage of subjects discontinuing treatment in each category was comparable among the 3 vaccination groups. Each subject serum sample was tested initially at a 1:4 dilution. As can be seen across the population stratified by percentage with certain GMTs, HPV 16 titers for HPV 16 monovalent vaccine and quadrivalent HPV vaccine were comparable (Figure 2). The observed GMT at month 7 was 2,310 mmu/ml (CI: 2,139.9, 2,493.9) for subjects receiving quadrivalent HPV vaccine, and 1,701 mmu/ml (CI: 1,461.7, 1,980.6) for subjects receiving monovalent HPV 16 vaccine (compared to a median of 28 mmu/ml for placebo subjects who were positive to HPV 16 at month 7 [n = 17]). If a result could 7

8 not be obtained in the quantifiable range, the sample was retested at dilutions of 1:40 and 1:400; and potentially at 1:4,000 if further dilution was required. Subjects who received placebo and were in the per-protocol analysis did not have detectable levels of anti-hpv 16 at month 7. The observed HPV 16 seroconversion rate for subjects receiving quadrivalent HPV vaccine was 99.8% (CI: ) compared to 100% (CI: ) for those subjects receiving HPV 16 monovalent vaccine. To address the primary analysis of immunogenicity, an analysis was conducted using a model that accounted for country of origin and vaccination group. In this analysis, anti-hpv 16 GMTs at week 4 post-dose 3 (as estimated using this model) and the percentage of subjects who seroconverted by week 4 post-dose 3 (as estimated using this model) were compared between the 2 vaccination groups (Table 1). The statistical criterion for non-inferiority with respect to GMT required that the lower bound of the 95% confidence interval for the fold difference in anti-hpv 16 GMTs (quadrivalent/monovalent) exclude a decrease of 2-fold or more. Using this statistical criterion, along with the statistical criterion of non-inferiority for seroconversion (Table 1) non-inferiority of the anti-hpv 16 GMT response in the quadrivalent HPV vaccine group, relative to the monovalent HPV 16 vaccine group, was established. Recent vaccines directed against Streptococcus pneumoniae have been engineered to include over 20 different capsular polysaccharide types. However, the combination of multiple antigens into a single vaccine presumes that the administration of this combination of antigens will not reduce the efficacy, safety and/or immunogenicity of the combination vaccine when compared to each individual antigen vaccine.[8] Caveats such as these are underscored by studies that have shown immune interference when effective 8

9 vaccines are combined into one product.[9] Another concern about combination vaccines is that naturally occurring immune response to closely related viruses may interfere with protection against the primary intended antigen. Vaccination against herpes simplex virus (HSV) type 2 provides a potential example of such unexpected findings. Despite antibody responses being generated against HSV-2 in both genders, the vaccine was not efficacious in women who were seropositive for HSV-1 and seronegative for HSV-2 at baseline. Further analysis showed that the vaccine was efficacious only in women who were seronegative for both HSV-1 and HSV-2 and that it was not efficacious in men regardless of their serological status.[10] The mechanisms by which preexisting antibodies against heterologous but related antigens interfere with vaccine induced immunity are not understood. Data in this report elaborate on the immunogenicity of the quadrivalent HPV vaccine by bridging data from the monovalent HPV 16 vaccine to the quadrivalent HPV vaccine. Immunization with quadrivalent HPV vaccine was found to induce immune response against HPV 16 (as measured by both anti-hpv 16 antibody titers at week 4 post-dose 3, and by the percentage of subjects who seroconverted for HPV 16 by week 4 post-dose 3) that were at least as good as those induced by the monovalent HPV 16 vaccine. This result helps to mitigate the theoretical concern that the inclusion of several antigens in an HPV vaccine will result in a reduction of the immune response to one or all of the component antigens. 9

10 ACKNOWLEDGEMENTS Investigators listed in alphabetical order according to country: Australia S. Garland; Austria S. Leodolter; Canada N. Ayotte, C. Bouchard, M. Boucher, L. Gilbert, J.P. Ouellet, M. Steben; Colombia J. Luna, G. Perez; Germany A.M. Funke, T. Grubert, F. Jaenicke, W. Lichtenegger; Hong Kong G. W. Tang; Italy G. Carosi, S. Greggi, L. Mariani, M. Moscarini, A. Perino; Mexico M. Hernandez-Avila; New Zealand S. Bagshaw, H. Roberts; Puerto Rico J. Romaguera; Russian Federation I. Manukhin, N. Mikhailova, N. Tsvetkova; Thialand P. Pitisuttithum; United Kingdom D. Jenkins, C. Lacey, A. Wade; United States M. Akin, R. Barnes, K. Beutner, D. Ferris, M. Gold, D. Harper, L. Koutsky, K. Kreutner, W. Ledger, S. G. McNeeley, Jr., W. Nebel, S. Sharma, S. Trupin, E. Wegner, C. M. Wheeler, D.M. Whitaker, M. Yardley. SOURCE OF SUPPORT Merck Research Laboratories, a division of Merck and Company, funded this study in its entirety. Dr Steben reports having received consulting or paid advisory board fees, and lecture fees from Digene, Merck Frosst, GlaxoSmithKline and Roche Molecular Diagnostic and grant support from Merck Frosst and GlaxoSmithKline. Dr Harper reports having received consulting or paid advisory board fees, lecture fees and grant support from Merck and Co., Inc. and GlaxoSmithKline. Dr Ferris reports having received consulting or paid advisory board fees and grant support from Merck and Co., Inc. and GlaxoSmithKline and lecture fees from Merck and Co., Inc. Dr Perez reports having received consulting or paid advisory board fees from Merck and Co., Inc. Dr Wheeler reports having received grant support from Merck and Co., Inc. and GlaxoSmithKline. Dr Garland reports having received paid advisory board fees from CSL (Commonwealth 10

11 Serum Laboratories), and GlaxoSmithKline, Victoria, Australia and grant support from CSL, GlaxoSmithKline, and lecture fees from Merck and Co., Inc. Dr Koutsky reports having received grant support from Merck and Co., Inc. Dr Hernandez-Avila reports having received consulting or paid advisory board fees from Merck and Co., Inc. Drs Railkar, Esser, Nelson, Vuocolo, Sattler, and Barr are employees of Merck Research Laboratories, a division of Merck & Co., Inc. and potentially own stock and/or hold stock options in the Company. No other potential conflict of interest relevant to this article was reported. 11

12 Reference [1] Walboomers JMM, Jacobs MV, Manos MM, Bosch FX, Kummer A, Shah KV, Snijders PJF. Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol. 189:12-9. [2] Cates W, Jr. Estimates of the incidence and prevalence of sexually transmitted diseases in the United States. American Social Health Association Panel. Sex Transm Dis. 26(4 Suppl):S2-S7. [3] Muñoz N, Kelsall G, Tellier P, Voyer H, Abrahamowicz M, Ferenczy A, Coutlée F, Franco EL. The natural history of human papillomavirus types associated with cervical cancer. N Engl J Med. 12: [4] Garland SM, Hernandez-Avila M, Wheeler.C.M., Perez G, Harper DM, Leodolter S, Tang GWK, Ferris DG, Steben M, Bryan JT, Taddeo FJ, Railkar R, Esser MT, Sings HL, Nelson M, Boslego J, Sattler C, Barr E, Koutsky LA. Efficacy and safety of a prophylactic quadrivalent human papillomavirus (types 6/11/16/18) L1 virus-like-particle vaccine against cervical, vulvar, and vaginal neoplasia and intraepithelial neoplasias and anogenital condylomata: a randomized controlled trial. N Engl J Med. submitted. [5] Villa LL, Costa RLR, Petta CA, Andrade RP, Ault KA, Giuliano AR, Wheeler CM, Koutsky LA, Malm C, Lehtinen M, Skjeldestad FE, Olsson S-E, Steinvall M, Brown DR, Kurman RJ, Ronnett BM, Stoler MH, Ferenczy A, Harper DM, Tamms GM, Yu J, Lupinacci L, Railkar R, Taddeo FJ, Jansen KU, Esser MT, Sings HL, Saah AJ, Barr E. Prophylactic quadrivalent human papillomavirus (types 6, 11, 16 and 18) L1 virus-like particle vaccine in young women: a randomised double-blind placebo-controlled multicentre phase II efficacy trial. Lancet Oncology. 6(5): [6] Dias D, Van Doren J, Schlottmann S, Kelly S, Puchalski D, Ruiz W, Boerckel P, Kessler J, Antonello JM, Green T, Brown M, Smith J, Chirmule N, Barr E., Jansen KU, Esser MT. Optimization and validation of a multiplexed Luminex assay to quantify antibodies to neutralizing epitopes on human papillomavirus 6, 11, 16, & 18. Clin Diagn Lab Immunol. 12(8): [7] Miettinen O, Nurminen M. Comparative analysis of two rates. Stat Med. 4: [8] Edwards KM, Decker MD. Combination vaccines consisting of acellular pertussis vaccines. Pediatr Infect Dis J. 16(4 Suppl):S [9] Trollfors B, Taranger J, Lagergard T, Sundh V. Reduced immunogenicity of diphtheria and tetanus toxoids when combined with pertussis toxoid. Pediatr Infect Dis J. 24(1):

13 [10] Stanberry LR, Spruance SL, Cunningham AL, Bernstein DI, Mindel A, Sacks S, Tyring S, Aoki FY, Slaoui M, Denis M, Vandepapeliere P, Dubin G. Glycoprotein-d-adjuvant vaccine to prevent genital herpes. N Engl J Med. 347(21):

14 Table 1. Analysis of Non-Inferiority As Measured by GMTs and Seroconversion in Subjects Who Received Quadrivalent HPV (Types 6,11,16,18) L1 VLP Vaccine and Subjects Who Received Monovalent HPV 16 L1 VLP Vaccine * Non-Inferiority by GMT Non-Inferiority by Seroconversion Quadrivalent HPV (Types 6,11,16,18) L1 VLP Vaccine (N=1783) Monovalent HPV 16 L1 VLP Vaccine (N=304) (Comparison Group A) (Comparison Group B) n Estimated GMT (mmu/ml) n Estimated GMT (mmu/ml) Estimated Fold Difference Group A / Group B (95% CI) p-value for Non- Inferiority (0.84, 1.35) <0.001 n Estimated Response (%) n Estimated Response (%) Estimated Percentage Point Difference Group A - Group B (95% CI) p-value for Non- Inferiority (-0.7, 2.0) <0.001 on September * The per-protocol immunogenicity population included all subjects who were not general protocol violators, received all 3 vaccinations within acceptable day ranges, were seronegative at Day 1 and PCR negative Day 1 through Month 7 for the relevant HPV type(s), and had a Month 7 serum sample collected within an acceptable day range. For the null hypothesis that GMTA/GMTB 0.5, a p-value <0.025 supported a conclusion that the anti-hpv 16 response in the Quadrivalent HPV 6,11,16,18 L1 VLP vaccine group was non-inferior to the response in the Monovalent HPV 16 L1 VLP vaccine group. Downloaded from 18, 2016 by Penn State Univ 14

15 For the null hypothesis that pa-pb -0.05, a p-value <0.025 supported a conclusion that the anti-hpv 16 seroconversion rate in the Quadrivalent HPV (Types 6,11,16,18) L1 VLP vaccine group was non-inferior to that in the Monovalent HPV 16 L1 VLP vaccine group. The estimated GMT, fold difference, associated confidence intervals, and p-values were based on a statistical analysis model adjusting for country. N = Number of subjects randomized to the respective vaccination group who received at least 1 injection. n = Number of subjects contributing to the analysis. Downloaded from September 18, 2016 by Penn State on Univ 15

16 Figure 1. Description of substudies 011 and 012 in the context of the larger efficacy study 013. Protocols 012 and 013 HPV 16 Bridging Study Protocol 013 Protocols 011 and 013 Quadrivalent HPV Efficacy Phase HPV+HBV Vaccination Study Day 0 Month 7 Month 48 Downloaded from September 18, 2016 by Penn State on Univ 16

17 Figure 2. Reverse Cumulative Distributions of Anti-HPV 16 clia Levels at Month 7 by HPV Vaccination Groups * % ,000 10, ,000 Titer, mmu/ml (log 10 scale) Quadrivalent (n=1,047, GMT=2,268) Monovalent (n= 181, GMT=1,794) * The per-protocol immunogenicity population included all subjects who were not general protocol violators, received all 3 vaccinations within acceptable day ranges, were seronegative at Day 1 and PCR negative Day 1 through Month 7 for the HPV 16, and had a Month 7 serum sample collected within an acceptable day range. The vertical line at 20 mmu/ml represents the seroconversion cutoff value for anti-hpv 16. n = Number of subjects contributing to the analysis. Downloaded from September 18, 2016 by Penn State on Univ 17