Immunogenicity, efficacy and safety of quadrivalent HPV. vaccine in men: a systematic review & meta-analysis

Transcription

1 Immunogenicity, efficacy and safety of quadrivalent HPV vaccine in men: a systematic review & meta-analysis Keywords: Human papillomavirus, quadrivalent vaccine, men, systemic review, meta-analysis Word Count: Abstract: 298 Text without summary: 4940 Tables and figures: 4 tables, 6 figures

2 ABSTRACT Background: Human papillomavirus (HPV) infection is prevalent in males. The incidence of anal cancer is the highest among men having sex with men (MSM). High risk HPV infection (type 16, 18) has been casually linked to anal cancer while 90% cases of genital warts are accounted by infection of HPV-6 and HPV-11. In response, universal vaccination of quadrivalent HPV vaccine (qhpv) in adolescent boys has been proposed. Objectives: We conducted a systematic review and meta-analysis to summarize available data for the immunogenicity, efficacy, and safety of qhpv in males. Methods: Randomized controlled trials of qhpv reporting results on immunogenicity, efficacy, or safety in males were identified and assessed by a single reviewer from the Cochrane Central Register of Controlled Trials, MEDLINE, EMBASE, China Journal Net, WanFang Data, Google Scholar, Yahoo, and bibliographies of included studies before 31 st August Trials only reporting results of bivalent HPV vaccine, therapeutic vaccine, or female HPV vaccination were excluded. Summary estimates were synthesized by fixed effect models. Results: Nine unique trials enrolling totally 7,034 males were included. The pooled HPV6/11/16/18 seroconversion rates were 99.22% (95% CI %), 99.48% ( %), 99.37% ( %), and 99.10% ( %) respectively. The qhpv was efficacious against HPV6/11/16/18-related anal intraepithelial neoplasia in MSM and genital warts in both the intention-to-treat populations [Risk ratio (RR): 0.50 ( ) and 0.37 ( ) respectively] and per-protocol populations [RR: ( ) and ( ) respectively]. The risk of serious adverse events [RR: 1.96 ( )] did not differ significantly between vaccine and control groups. No serious adverse events or deaths were reported vaccine-related. Page 2 of 61

3 Conclusions: The qhpv is highly immunogenic, safe, well-tolerated, and efficacious against external genital and anogenital diseases associated with vaccine-hpv types among young males. Male vaccination of qhpv is recommended. Yet, the vaccine s long-term profile beyond 3 years post vaccination demands further investigations. Page 3 of 61

4 BACKGROUND Human papillomavirus (HPV) infection is common among males with prevalence ranged from 1.3% to as high as 72.9% 1,2. A recent meta-analysis even reported a summary prevalence estimate of 92.6% (95% CI %) in HIV-infected men having sex with men (MSM) 3. High risk HPV infection (type 16, 18) has been casually linked to anal cancer 4,5, accounting for over 90% cases of anal intraepithelial neoplasia (AIN) and 84.3% cases of anal cancer 6, and etiologically linked to penile cancer and oropharygneal cancer in men 7. In particular, HPV 16 has the highest degree of association with anal cancer as shown in 70% cases in population-based studies 8,9,10. Over the past two decades, the incidence of anal cancer has been rising for above 160% in the United States 11. Among MSM, the incidence was the highest, around 20 times greater than that in heterosexual males 10. HIV-infected MSM are at even greater risk 3,12,13. HPV infection can also lead to genital warts with around 90% caused by HPV 6 and HPV 11 14,15. The history of HPV-related genital warts, in turn, is a risk factor for anal cancer 16. Previous randomized control trials (RCTs) 17, 18, 19, 20, 21 and systematic reviews 22,23,24 of quadrivalent HPV vaccine (qhpv) in females has demonstrated seroconversion rates of near 100% 17 and stable Anti-HPV titres for each vaccine HPV types up to at least 5 years 18 for immunogenicity; 100% protection against adenocarcinoma in situ, external anogenital and vaginal lesions, and 99% against anogenital warts diseases for efficacy 19,20 ; and a generally safe and well tolerated profile 18,21,22,24 with other vaccines, such as Hepatitis B vaccine 18. Unfortunately, vaccine impact studies in Australia have already shown that MSM did not benefit from the herd immunity offered by the widespread uptake of qhpv in women younger than 27 years of age 25. Furthermore, the direct medical cost of treating HPV Page 4 of 61

5 infection was reported to be comparable to treating HIV infection (US$2.9 vs 3.0 billion) 26. To reduce HPV-related morbidity in MSM and the huge healthcare expenditure incurred, universal vaccination of adolescent boys before sexual debut has been suggested as one of the potential solutions 27. Meanwhile, results from RCTs investigating qhpv in males were recently published 28,29,30,31,32,33,34,35,36,37,38, which have not been reviewed systematically or conducted any meta-analysis. Thus, the present study aims to comprehensively summarize the published estimates of data for immunogenicity, efficacy, and safety of qhpv in the male populations using the techniques of systematic review and meta-analysis. METHODS Identification of Relevant Studies A systematic search on the Cochrane Central Register of Controlled Trials (2013), MEDLINE ( /7/2013), EMBASE ( /7/2013), China Journal Net ( /7/2013), WanFang Data ( /7/2013), Google Scholar, and Yahoo was carried out to identify articles of RCTs for qhpv published up to 31 st August, 2013 according to the protocol (available request via the authors ). The strategy for keywords search in the Cochrane Central Register of Controlled Trials, MEDLINE, and EMBASE were as following: Human Papillomavirus OR HPV AND Papilomavirus vaccine OR Gardasil OR Silgard and were complemented by the search of Medical Subject Headings (MeSH): Papillomavirus Vaccines for the Cochrane Central Register of Controlled Trials (2013) and MEDLINE, and Wart Virus Vaccine for EMBASE with all subheadings included. The detailed search terms and search steps for each electronic database were listed in Appendix A. Page 5 of 61

6 Reference lists of all included studies were reviewed to identify additional relevant publications. Inclusion and Exclusion Criteria The current review included RCTs of qhpv conducted among males and presented measurement of immunogenicity, efficacy or safety against HPV infection or diseases of interest [i.e. external genital lesions, AIN and anal cancer, penal intraepithelial neoplasia (PIN), penile/ perianal/ perineal cancer, condylomata acuminate (genital warts), and persistent HPV infection]. RCTs investigating both males and females with subgroup analyses or stratifications were included. Language of published trials was not limited in study selection. Trials reporting bivalent HPV vaccine, therapeutic vaccine, or female vaccination were excluded. Outcome Measures Immunogenicity, efficacy, and safety of qhpv were the three main outcomes in this review. Immunogenicity was referred as the ability to cause the production of antibodies or the degree to which a substance induces an immune response 39. Efficacy was referred as the protective or prophylactic efficacy which is the capacity to protect against the diseases caused by the virus contained in the vaccine 40. Safety was assessed by the incidence of adverse events (AEs) occurred among the vaccine recipients and the control group 41. The primary estimates for the current review was the HPV6/11/16/18 seroconversion rates, for measuring immunogenicity of qhpv, with the Anti-HPV6/11/16/18 serum Geometric Mean Titres (GMTs) as another estimate suggested by the World Health Organization (WHO) 42 and consistently reported in Page 6 of 61

7 most trials 28,29,34,35,36,37,38,43,44. Seven estimates were adopted for efficacy analyses: Risk ratios (RRs) of external genital lesions, AIN, anal cancer, PIN, penile/ perianal/ perineal cancer, genital warts, and persistent HPV infection respectively. Four estimates were used in safety assessment: RRs of injection-related AEs, systemic AEs, vaccine-related AEs, and serious AEs occurred within 14 days after vaccination. The outcomes and estimates used in this review have been adopted by the United States Food and Drug Administration (USFDA) for the evaluation of qhpv (Gardasil) regarding its licensure for men 45. Data Extraction Data were extracted by one single reviewer (CPL) under the supervision of Professor Lin-wei Tian. Detailed information on study design, inclusion and exclusion criteria, participant characteristics, vaccine and placebo administered, trial endpoints, efficacy populations, and methodological quality from all included studies were extracted. Statistical Analysis Effect sizes were summarized as HPV 6/11/16/18 seroconversion rates and Anti-HPV GMTs for immunogenicity; risk ratios (RRs) with 95% confidence interval, which was subsequently estimated as [1-RR] and expressed as percentage consistent with the WHO s suggestion, for efficacy 42 ; and incidence of AEs for safety. In efficacy analyses, the study populations were defined into 2 types, the per-protocol population (PPP) and the intention-to-treat population (ITT). Vaccine immunogenicity was primarily evaluated in the PPP while the safety assessment primarily analyzed subjects from the ITT cohort. Fixed effect model was applied to obtain pooled estimates of vaccine immunogenicity, efficacy, and safety. For observed moderate to Page 7 of 61

8 substantial heterogeneity, random effect model was used instead to pool estimates in the meta-analysis. Heterogeneity between studies was assessed by I 2 statistic. Sensitivity analysis according to methodological quality and study characteristics was planned a priori to explore possible sources of heterogeneity. Funnel plots were produced to assess potential publication bias. The present systematic review was performed according to the standards recommended by the Cochrane Collaboration 46. All statistical analyses were performed using the software RevMan (Version 5.2) 47. RESULTS Selection of Studies Of 4750 publications searched initially through databases, 150 were eligible for further selection after title-and-abstract screening. The detail study identification and selection was illustrated in the flow diagram in Figure 1. In total, 13 publications published in English language representing 9 unique RCTs were included in the current review (Appendix B) after the exclusion of 137 articles by applying the selection criteria stated in Figure 1. No relevant RCT published in Chinese language was found. Also, no additional relevant articles were identified after reviewing the bibliographies of obtained studies. Characteristics of Included Studies The characteristics of all RCTs included were summarized in Table 1. A total of 7,034 males were recruited. Among the included trials, 4 of them were multinational trials, including totally 6 geographical regions (Africa, Asia, Austria, Europe, Latin America, and North America). Seven of them included both male and female populations with subgroup analyses or stratifications. Eligible participants were males aged 8 to 26, including HIV-infected males and MSM. The prophylactic qhpv were Page 8 of 61

9 evaluated in 8 trials from Merck and 1 from Novartis Vaccines. The qhpv were administered in a three-dose regimen within a 6-month time frame in all trials. Five trials used placebo as control while 3 trials respectively administered MenACWY-CRM and Tdap, Menactra and Adacel, or Tdap concomitantly with qhpv in the experimental group and non-concomitantly in the control group. The remaining one was a single-arm open label trial. All 9 RCTs reported results on immunogenicity and safety. Participants were tested for seroconversion rates and serum Anti-HPV titres on day 1 of vaccine injection and month 7 (one month post-dose three). Subjects in Resinger et al (2007) 43 and Hillman et al (2012) 29 were also tested beyond month 7 (eighteen and/or thirty months post-dose three respectively). The immunogenicity results of all included trials were measured using a competitive Luminex immunoassay (clia) 48. For safety assessment, the majority of included RCTs observed participants for at least 30 minutes after each vaccination and used vaccine report cards for participants to report any adverse events 14 days following each vaccination. Only a single RCT with five publications 29,30,31,32,33 reported efficacy. Specimens for HPV testing by Polymerase Chain Reaction (PCR) assay and anogenital examinations were performed at a 6-month interval from day 1 of injection to month 36. The length of all included trials ranged from 7 to 36 months. Methodological quality of the included studies was generally acceptable (Table 1). Reviewed under the Cochrane Risk of Bias Tools (Appendix C), almost all trials demonstrated low risk in random sequence generation, incomplete outcome data, and selective outcome reporting. In contrast, 6 out of the 9 trials did not report on allocation concealment clearly; 3 of them were considered high risk in blinding of the Page 9 of 61

10 study participants and personnel due to open-label; 1 involved high risk in incomplete outcome data due to incorrect administration of vaccine, resulting in the removal of 92 participants from the analysis. There were no other significant sources of bias identified in the included trials. Outcomes I. Immunogenicity The seroconversion rates (Figure 2) and Anti-HPV GMTs for type 6/11/16/18 (Figure 3) at one month post-dose three were attained in males and HIV-infected males. The pooled seroconversion rates at one month post-dose three were similar in both populations, except for HPV18 which was notably higher in males than HIV-infected males (HPV6: 99.2% vs 99.7%; HPV11: 99.5% vs 99.6%; HPV16: 99.4% vs 99.6%; HPV18: 99.1% vs 96.6%). The pooled GMTs for Anti-HPV6/11/16/18 were higher in general males than in HIV-infected males (Anti-HPV6: 0.60MU/mL vs 0.43MU/mL; Anti-HPV11: 0.90MU/mL vs 0.67MU/mL; Anti-HPV16: 3.24MU/mL vs 1.42MU/mL; Anti-HPV18: 0.97MU/mL vs 0.20MU/mL). Across age, older males aged 16 to 26 in Hillman et al (2012) 29 showed a lower Anti-HPV GMTs than younger males aged 7 to 15 reported in other included RCTs (Anti-HPV6: MU/mL vs MU/mL ; Anti-HPV11: 0.624MU/mL vs MU/mL ; Anti-HPV16: MU/mL vs MU/mL ; Anti-HPV18: MU/mL vs MU/mL). A similar phenomenon was observed between older (aged over 18 years) and younger HIV-infected males (aged 12 to 17 years) in Figure 2. However, seroconversion rates were comparable and over 95% for all 4 vaccine-hpv types between the older and younger age groups for both Page 10 of 61

11 general males and HIV-infected males at one month post-dose three. In terms of immunogenicity beyond month 7, both Reisinger et al (2007) 43 and Hillman et al (2012) 29 presented type 6/11/16/18 seroconversion rates and Anti-HPV GMTs one year post-dose three (month 18) with Reisinger et al (2007) 43 yielding a higher immunogenicity result (For seroconversion rate, HPV6: 97.8% vs 90.9%; HPV11: 99.3% vs 95.6%; HPV16: 99.3% vs 99.1%; HPV18: 92.5% vs 62.3%; For Anti-HPV GMT, Anti-HPV6: 0.227MU/mL vs MU/mL; Anti-HPV11: 0.292MU/mL vs MU/mL; Anti-HPV16: 1.402MU/mL; Anti-HPV18: 0.233MU/mL vs MU/mL). Hillman et al (2012) 29 also reported immunogenicity results for seroconversion rates and Anti-HPV GMTs three years after the first dose (month 36). The seroconversion rates were reported at 88.9% ( %) for HPV6, 94.0% ( %) for HPV 11, 97.9% ( %) for HPV 16, and 57.0% ( %) for HPV 18. The Anti-HPV GMTs were reported at MU/mL ( MU/mL) for Anti-HPV6, MU/mL ( MU/mL) for Anti-HPV11, MU/mL ( MU/mL) for Anti-HPV16, and MU/mL ( MU/mL) for Anti-HPV 18. When qhpv was concomitantly administered with MenACWY-CRM and Tdap 38, Menactra and Adacel 36, or Tdap 37 respectively in different trials, it was observed that the estimated fold differences of concomitant group over non-concomitant group ranged from 0.72 to 1.06 (Table 2). By pre-defining the threshold of non-inferiority ratio at above ,37,38, the immunogenicity achieved after the administration of qhpv concomitantly with MenACWY-CRM and Tdap, Menactra and Adacel, or Tdap was found non-inferior to that administered non-concomitantly. Furthermore, seroconversion rates and Anti-HPV GMTs after Page 11 of 61

12 concomitant administration were similar or even higher than that after administration of qhpv alone in males (For seroconversion rates, HPV6: % vs %; HPV11: 100% vs %; HPV16: 100% vs %; HPV18: % vs %; For Anti-HPV GMTs, Anti-HPV6: MU/mL vs MU/mL ; Anti-HPV11: MU/mL vs MU/mL; Anti-HPV16: MU/mL vs MU/mL; Anti-HPV18: MU/mL vs MU/mL). II. Efficacy Only one included RCT enrolling 4065 participants (represented by five publications 29,30,31,32,33 ) investigated on qhpv efficacy in males. A statistically significant protection provided by qhpv against external genital lesions, AIN, anal cancer, condylomata acuminate (genital warts), and persistent infection was observed in males in both the ITT and PPP cohorts as summarized respectively in Table 3 and Table 4. 1) External genital lesions External genital lesions denotes a diagnosis of condyloma acuminatum, HPV, and PIN. In the ITT cohort, the protection of qhpv against HPV6/11/16/18 associated external genital lesions was even greater in MSM than that in heterosexual men and men in general [RR: 0.30 ( ) vs 0.37 ( ) vs 0.35 ( )]. The vaccine did not show a statistically significant protection against external genital lesions related to 10 additional non-vaccine HPV types (HPV 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59). In the PPP cohort, the efficacy of qhpv against HPV6/11/16/18 external genital Page 12 of 61

13 lesions in males, translated by the pooled RR, was comparatively higher than that in the ITT cohort [90.4% ( %) vs 65.5% ( %)]. However, the protective efficacy for PPP was not statistically significant in MSM. 2) AIN and anal cancer The qhpv demonstrated a statistically significant level of protection against HPV6/11/16/18 associated AIN and anal cancer in MSM in both the ITT [RR: 0.50 ( ); p= ] and PPP cohorts [RR: ( ); p= ]. The protective efficacy was slightly higher for high grade AIN (AIN 2 or 3) than low grade AIN (AIN 1) in the ITT [54.2% ( %) vs 49.6% ( %)] and PPP cohorts [74.9% ( %) vs 73.0% ( %)]. For ITT, the protection against AIN associated with 10 additional non-vaccine HPV types was not statistically significant. 3) PIN or penile/ perianal/ perineal cancer As shown in the ITT cohort, qhpv was not protective against all PIN lesions [RR: 1.20 ( )] but the result was not statistically significant (p= 0.77). For PPP, although there was no report of any PIN lesions in the vaccine group and efficacy against all PIN lesions reached 100%, the p-value was not estimable. In addition, no penile/ perianal/ perineal cancer associated with HPV 6/11/16/18 was reported in either vaccine or control group in both ITT and PPP cohorts. 4) Condylomata acuminate (Genital warts) Vaccination of qhpv produced a statistically significant protection against genital warts in the ITT [RR: 0.37 ( ); p< ] and PPP cohorts [RR: (0.021 to 0.345); p= ]. For MSM, the protection was also statistically Page 13 of 61

14 significant in ITT [RR: 0.42 ( ); p= 0.007] but that in PPP was not estimable, given that there was no report of genital warts in the vaccine group. 5) Persistent HPV infection Persistent infection was defined as the detection of the same HPV DNA (type 6, 11, 16, or 18) at two or more consecutive visits with an interval of at least 6 months between the visits 30,33. The protection against 6-month persistent infection was statistically significant in both ITT [RR: 0.54 ( ); p < )] and PPP cohorts [RR: ( ); p< ]. Converted from the estimates of the pooled RR, qhpv generated a higher efficacy against 6-month persistent infection in MSM than heterosexual men as demonstrated in ITT [59.4% ( %) vs 50.4% ( %)] and PPP [48.8% ( %) vs 43.6% ( %)]. III. Safety Reported by the included trials 28,30,35,43,44, the injection-site AEs (i.e. injection-site pain, swelling and redness) after administration of qhpv were common, ranging from % in vaccine groups and % in control groups. Systemic AEs were commonly described as headache, fever, pharyngeal pain, and upper respiratory tract infection. The incidence was similar between the vaccine groups and placebo groups ( % vs %) from the included trials. For HIV-infected males, the incidence of systemic AEs reported by Levin et al (2010) 35 was exceptionally low compared to that in general males, 2.08% for vaccine group and 3.33% for placebo group. In addition, there were no obvious effects of qhpv on plasma HIV viral load and CD4 cell counts since the viral load and CD4% after each injection did not differ significantly between vaccine group and placebo group 35. Reports of serious AEs included appendicitis, cellulitis, non-cardiac chest pain, Page 14 of 61

15 myocardial ischemia, hypersensitivity (peanut allergy), hypoglycaemia, acute supprative tonsillitis, chronic neutropenia, chickenpox-related seizure, severe fever (>39 C), muscular weakness, drug over dose, suicide, and injury resulting in death. Yet, none of them were determined to be vaccine-related by investigators of the included trials. As shown by the pooled RRs in Figure 4, only injection site AEs [1.11 ( ); p= 0.03] and vaccine-related AEs [1.08 ( ); p= 0.001], which included injection-site and systemic AEs determined to be related to the vaccine by trial investigators, showed statistically significant risk in vaccine groups compared with placebo groups. However, there was substantial heterogeneity among pooled studies for injection-site AEs (I 2 = 91%). Overall, the difference in risk of serious AEs between the vaccine groups and placebo groups was not statistically significant [RR: 1.96 ( ); p= 0.19]. There were also no vaccine-related serious AEs and death reported. Data from Moreira et al (2011) 31 further indicated that the occurrence of AEs did not increase with each successive injection [Report of at least one AE: 53.6% (after first dose); 42.4% (after second dose); 39.3% (after third dose)]. Also, only 0.2% (2/2020) of participants discontinued due to a vaccine-related AE and none of them discontinued after the second and third dose. As demonstrated in Figure 5, there was no statistically significant difference for injection-site AEs, systemic AEs, and vaccine-related AEs between concomitant and non-concomitant administration of qhpv with MenACWY-CRM and Tdap, Menactra and Adacel, or Tdap. Only serious AEs were found with a significantly higher risk in concomitant group than non-concomitant group [RR: 2.98 ( ); p= 0.008]. Yet, these serious AEs had a very low incidence of % and were deemed not related Page 15 of 61

16 to qhpv by trial investigators. In addition, the heterogeneity among pooled studies for serious AEs was significant (I 2 = 65%). Comparing concomitant administration of qhpv and administration of qhpv alone, the incidence of AEs were generally higher for concomitant administration (Injection-site AEs: % vs %; Systemic AEs: % vs %; Vaccine-related AEs: % vs %; Serious AEs: % vs %). There was no report of serious AEs and death determined to be vaccine-related in these trials 36,37,38. IV. Sensitivity Analysis It was noticeable that the heterogeneity among pooled studies was substantial for the estimate of Anti-HPV GMTs in all vaccine serotypes in general males and HIV-infected males, with I 2 > 60%. The heterogeneity among included trials was examined according to study characteristics, which included study population, inclusion and exclusion criteria, intervention and comparators, endpoints chosen, and participant baseline characteristics. As seen in Figure 6, the pooled Anti-HPV GMTs of Hillman et al (2012) 29 was significantly lower than those of other RCTs. When Hillman et al (2012) 29 was excluded, the subsequent heterogeneity decreased, with I 2 dropped by 3 to 17% compared to the original. This observed heterogeneity was likely due to the recruitment of older males aged 16 to 26 in Hillman et al (2012) 29, who induced lower Anti-HPV GMTs than younger males aged 7 to 15 reported in other RCTs. However, the resulted heterogeneity for Anti-HPV GMTs after the removal of Hillman et al (2012) 29 was still substantial (I 2 > 60%), suggesting other potential sources, such as ethnicity (subjects recruited from Africa, Asia, Austria, Europe, Page 16 of 61

17 Latin America, and North America among included trials), sexual maturity (sexually naïve vs number of lifetime sex partners 5), serostatus of the subjects at entry, the methodological quality (3 RCTs involved high risk in blinding and 1 was also high risk in incomplete outcome data), and also the different ways of qhpv administration (i.e. concomitant administration of qhpv vs administration of qhpv alone). V. Publication Bias Funnel plots were generated for the estimates of immunogenicity and safety respectively (Appendix D), but not efficacy because of only one included RCT. For immunogenicity, the assessment of funnel plots suggested that the included studies tend to report lower seroconversion rates and Anti-HPV GMTs. The asymmetry of funnel plots were more pronounced for Anti-HPV GMTs. For safety, the respective funnel plots for injection-site AEs, systemic AEs, vaccine-related AEs, and serious AEs were all asymmetric. However, the suggestion of publication bias from the asymmetric funnel plots was inconclusive given the few number of RCTs (less than 10) included for each estimate in this review. The limited number of included studies may already be an intrinsic factor contributing to the asymmetry 49. DISCUSSION The current systematic review was the first, according to the author s knowledge, in summarizing published estimates of qhpv in males, including HIV-infected males and MSM, through meta-analyses and demonstrating the vaccine s high immunogenicity, efficacy, and safety profile in these populations. Overall, qhpv was highly immunogenic in males with substantially higher HPV6/11/16/18 seroconversion rates (>95% at one month post dose three and Page 17 of 61

18 % at three years) after vaccination than that after natural HPV infection (a maximum of 56.7% eight months post infection and 37.0% within around one year) 50. Efficacy against HPV6/11/16/18 associated external genital lesions ( % in ITT and % in PPP cohorts), genital warts ( % in ITT and % in PPP cohorts), and persistent HPV 6/11/16/18 infection ( % in ITT and % in PPP cohorts) were the most impressive. The vaccine was also pronounced for protective efficacy against HPV6/11/16/18 associated AIN and anal cancer in MSM (50.3% in ITT and 77.5% in PPP cohorts) whose incidence for anal cancer was 20 times greater than that in heterosexual men 10. In immunogenicity analyses, it was observed that the Anti-HPV titres induced after vaccination was greater in general males than in HIV-infected males. However, it could not be concluded that the HIV-infection status has a direct effect on the lower antibody titres because the role of HIV status on the immune response to qhpv has yet to be determined 34,35. It was also presented that males generally exhibited the strongest immune response and maintained a higher immunity level for HPV16 [Pooled Anti-HPV 16 GMT: 3.24MU/mL ( MU/mL); Pooled HPV 16 seroconversion rate at three years: 97.9% ( %)]. Similar to the observation in females (aged 9 to 15 years vs aged 16 to 23 years) 51,52,53, younger adolescent males (aged 7 to 15 years) induced higher Anti-HPV titres after vaccination than older males (aged 16 to 26 years), suggesting an age-dependent response 29,43. However, this difference in immune response may not be relevant to protective efficacy of qhpv as high efficacy for all the vaccine-hpv types had already been demonstrated in males of 16 to 26 years of age 30. As we compared the immunogenicity in terms of seroconversion rates and Page 18 of 61

19 Anti-HPV GMTs achieved in males after vaccination with that in females and from natural infection, the strongest level of immune response induced was found in adolescent males aged 7 to 17 years 36,37,38,43,44, descending to HIV-infected males of similar age 35, then males and females aged 16 to 26 years 29,43, HIV-infected males or MSM aged above 18 years 29,34, and finally to those in natural infection 50. Yet, the clinical significance of the magnitude of Anti-HPV response was unknown since the minimum protective Anti-HPV level has not been determined 28,29,34,35,36,43,54. Additionally, concomitant administration of qhpv with other vaccines (i.e. MenACWY-CRM and Tdap/ Menactra and Adacel/ Tdap) did not show inferiority for immune response comparing to non-concomitant administration but demonstrated a similar immune response to that of injecting qhpv alone in advance 36,37,38. Furthermore, the immune response to other vaccine components (i.e. MenACWY-CRM and Tdap/ Menactra and Adacel/ Tdap) did not differ significantly between the concomitant group and non-concomitant group 36,37,38. The well-tolerated profile of qhpv with other vaccines supports the possibility of concomitant administration for more cost-effective immunization program in future. In efficacy analyses, the protective efficacy observed in PPP cohort was in general higher than in ITT cohort. Such discrepancies were likely attributable to differences in the populations evaluated and case counting method 30. The ITT cohort typically included male participants who had received 1 injection of vaccine or placebo, and had follow-up data regardless of HPV status at entry. In comparison, the PPP cohort had more restrictions and only included male subjects who received all three injections of vaccine or placebo, were DNA-negative and sero-negative for vaccine HPV types at enrolment, and remained DNA-negative throughout the study Page 19 of 61

20 without protocol violation. Moreover, case counting for ITT analyses began on day 1 of vaccination, rather than one month post-dose three in PPP analyses. The excessive cases in ITT cohorts were possibly the results of incomplete vaccine regimen and the inclusion of prevalent cases of HPV infection 30. The ITT cohort in the RCTs projects young men in the general population who may have exposure to infection of vaccine type HPV and have looser compliance with vaccination protocol. In contrast, the PPP cohort approximates pre-sexually active young male adolescents who are naïve to vaccine type HPV and with nearly perfect compliance. Since the young adolescents will soon become susceptible after initiation of sexual activities 55,56,57, the notably higher prophylactic efficacy against HPV infection and related diseases in the PPP cohort highlighted the public health benefits of early vaccination in males prior to their sexual debut. Our results could not find a statistically significant protection for qhpv against all PIN lesions in both ITT [RR= 1.20 ( ); p=0.77] and PPP [RR= 0 ( ); p= not estimable] cohorts 30. Moreover, no statistically significant cross-protection for 10 additional non-vaccine-hpv types (31, 33, 35, 39, 45, 51, 52, 56, 58 and 59) against external genital lesions and AIN were observed 32. Therefore, cross-protection of qhpv is not manifested in males from the current evidence. In safety assessment, the results indicated that the injection-related local and systemic AEs were generally mild, such as injection-site pain, swelling and redness, headache, and low-grade fever. The incidence of men reporting injection-site AEs after vaccination was lower than the incidence reported by a systematic review of qhpv for women (Vaccine group: % vs %; Control group: Page 20 of 61

21 % vs %) 22 and also lower than the incidence found in the post-licensure surveillance from the Vaccine Adverse Event Reporting System (VAERS) in females from 2006 to 2009 in the United States with 25 million doses distributed (Injection AEs or non-serious systemic AEs: 93% of 14,072 reports of AEs) 58. There was no evidence that the incidence of these local symptoms increased with each subsequent dose 31. The incidence of vaccine-related AEs was found to be similar or even lower in HIV-infected males reported in Levin et al (2010) 35 than that in general males reported in other studies. The possible explanations may be a different definition of systemic AEs adopted, which was not elaborated in the study, or the effect of HIV infection. However, the role of HIV status in the reactogenicity demands further exploration in future studies 34,35. Currently, it was only demonstrated that qhpv has no obvious effects on HIV viral load and CD4 cell counts 34,35. The risk of serious AEs after qhpv administration was not statistically significant. None of the serious AEs and deaths was considered attributable to qhpv by trial investigators. Likewise, the post-licensure VAERS surveillance supported the vaccine for continuing to be safe as only 7% out of 14,072 reports of AEs were identified as serious, half of the average number comparing to overall individual vaccines in the United States, and most of them, including the 26 confirmed cases of death, lacked evidence to suggest vaccine causality 58. Compared to non-concomitant administration of qhpv or administration of qhpv alone, our results demonstrated that administering qhpv concomitantly with MenACWY-CRM and Tdap/ Menactra and Adacel/ Tdap respectively showed a higher incidence of AEs. However, this finding could be biased towards an Page 21 of 61

22 over-estimation of AEs reporting in concomitant group since these participants were receiving more than one injection on the same day 37. In fact, the USFDA has approved the use of Gardasil in the prevention of AIN grades 1, 2, and 3 caused by the vaccine-hpv types; the prevention of anal cancer caused by HPV 16 and 18; and the prevention of genital warts caused by HPV 6 and 11 in males from 9 to 26 years of age 59. In addition, concomitant administration of qhpv with Menactra and Adacel has also been approved 59. However, lots of European countries and Asian countries, such as China, still have not granted the application of male HPV vaccination despite the high prevalence of HPV ( %) 60,61,62 and genital warts ( %) 63,64 reported in MSM. In view of the evidence presented here, the immunogenicity, efficacy, and safety of qhpv in males is warranted, given that our review is based on the summarized results of RCTs across four continents (Africa, Europe, Asia-Pacific, Latin America, and North America) and over 200 study sites. Future research could further investigate on the long-term immunogenicity and efficacy of qhpv in males. Continuous post-licensure surveillance would be useful to detect rare diseases or AEs that may be related to the vaccine. More studies for determining the role of HIV status on the immune response to qhpv would be crucial to explain the immunogenicity and reactogenicity of qhpv in HIV-infected males. Cost-effectiveness studies and acceptability studies in local setting hold the keys for future HPV vaccination campaign in men. Several limitations were identified in this review. Firstly, there were no published studies investigating the immunogenicity, efficacy, or safety of qhpv in males beyond three years post vaccination. Hence, discussion on the vaccine s long-term profile in men was not available in the current review. Secondly, the pooled Page 22 of 61

23 estimates presented were mainly from young males with limited sexual exposure to HPV, limiting the applicability to more mature and sexually active male populations. Thirdly, due to resources constraint, only one reviewer has conducted the search using the selection criteria and performed data extraction. No duplicated study selection was done by another independent reviewer. Lastly, the assessment of publication bias by funnel plots was inconclusive in this review due to the limited number of included studies (i.e. less than 10 for each estimate). In conclusion, our review has demonstrated that qhpv is highly immunogenic, safe, well-tolerated, and efficacious against external genital lesions, AIN, anal cancer, and genital warts up to 3 years post vaccination in young males, including HIV-infected males and MSM. Based on these summarized results, male HPV vaccination is recommended. However, the vaccine s long-term profile as well as the potential of male HPV immunization program as an effective public health measure against HPV-related diseases demands further investigations in future. Page 23 of 61

24 Table 1 Characteristics of included Randomized Controlled Trials Authors Study design Number of participants Number of study sites Countries included Year of study enrolment Funding source Li et al Hillman et al 30 Giuliano et al 31 Moreira et al Goldstone et al 33 Palefsky et al Wilkin et al Double-blinded 600 (male= 100; 1 1 (The USA) 07/ /2008 Merck Sharp & RCT female= 500) Dohme Corp. Double-blinded RCT 4065 men (heterosexual men= 3463; men having sex with men= 602) * 09/ /2008 Merck Sharp & Dohme Corp.; Qiagen; NIH/NCRR Single-arm, open- 112 HIV+ men 8 1 (The USA) 01/ /2008 Merck label trial 35 Levin et al Double-blinded RCT 126 HIV+ children - 1 (The USA) - - (boys= 55; girls=71) Reisinger et al Vesikari et al Phase III open-label RCT Open-label RCT children (boys= 394; girls= 648) 843 adolescents (male= 260; female= 583) 21 1 (The USA) 04/ /2006 Merck 17-06/ / Arguedas et al Reisinger et al Block et al Phase III open-label RCT Double blinded RCT RCT 1620 (male= 696; female=708) 1781 children (boys= 842; girls= 939) 1529 participants (boys= 510; girls= 506; women= 513) 1 1 (Costa Rica) 07/ /2008 Novartis Vaccines / /2004 Merck 61-12/ /2004 Merck Page 24 of 61

25 Table 1 Characteristics of included Randomized Controlled Trials (Continued) Authors Inclusion criteria Exclusion criteria Vaccine component Comparator Administration schedule Length of trial Li et al Chinese male aged 9-15 years; Chinese female aged 9-45 years; Number of lifetime sex partners 4 History of severe allergic reaction to any vaccine component; splenectomy; immune disorder HPV6, 11,16,18 L1 VLPs Placebo Day 1, month 2 and 6 7 months Hillman et al Giuliano et al Moreira et al Goldstone et al Palefsky et al HM aged 16 to 23 years; MSM aged 16 to 26 years; Number of lifetime sex partners 5 History of clinically detectable anogenital lesions; seropositive for HPV type 6/11 HPV6, 11,16,18 L1 VLPs Placebo Day 1, month 2 and 6 36 months Wilkin et al Men aged 18; HIV-1 infection; normal anal cytology result Abnormal cytology; PCR testing detected DNA for both HPV 16 and 18; prior receipt of HPV vaccines HPV6, 11,16,18 L1 VLPs Pre-vaccination (baseline) Day 1, month 2 and 6 7 months Levin et al HIV infected children aged 8-11 Immunosuppressive diseases; other vaccines 2-3 weeks before or after study vaccine HPV6, 11,16,18 L1 VLPs Placebo Day 1, month 2 and 6 7 months Reisinger et al 36 Boys and girls aged 10 to 17; Sexually naïve History of meningococcal / Tdap / HPV vaccination HPV6, 11,16,18 L1 VLPs and Menactra & Adacel (concomitantly) HPV6, 11,16,18 L1 VLPs and Menactra & Adacel (nonconcomitantly) QHPV: Day 1, month 2 and 6 Menactra & Adacel: Day 1 (concomitant group); Month 1 (non-concomitant group) 7 months Page 25 of 61

26 Table 1 Characteristics of included Randomized Controlled Trials (Continued) Authors Inclusion criteria Exclusion criteria Vaccine component Comparator Administration schedule Length of trial Vesikari et al Healthy boys and girls aged 11 to 17; Previous immunization against Tdap Sexually active throughout the study HPV6, 11,16,18 L1 VLPs and Tdap (concomitantly) HPV6, 11,16,18 L1 VLPs and Tdap (non-concomitantly) QHPV: Day 1, month 2 and 6 Tdap: Day 1 (concomitant group); Month 1 (nonconcomitant group) 7 months Arguedas et al Healthy persons aged years; sexually naïve Received meningococcal / HPV vaccine; taken other vaccines within 1 month of enrolment HPV6, 11,16,18 L1 VLPs, MenACWY- CRM, and Tdap (concomitantly) HPV6, 11,16,18 L1 VLPs, MenACWY- CRM, and Tdap (non-concomitantly) Concomitant group: QHPV: Day 1, month 2 and 6; MenACWY- CRM & Tdap: Day 1 Non-concomitant group: QHPV month 2, 4 and 8; MenACWY-CRM & Tdap: Day 1/month 1 8 months Reisinger et al 43 Healthy boys and girls aged 9-15 years; sexually naïve at enrollment and throughout the study Allergic to any vaccine component; received any blood product in the previous 6 months HPV6, 11,16,18 L1 VLPs Placebo Day 1, month 2 and 6 18 months Block et al Healthy boys and girls aged years, and females aged years Allergic to any vaccine component; received any blood product in the previous 6 months HPV6, 11,16,18 L1 VLPs HPV6, 11,16,18 L1 VLPs in girls aged years and women aged years Day 1, month 2 and 6 7 months Page 26 of 61

27 Table 1 Characteristics of included Randomized Controlled Trials (Continued) Authors Methodological quality Random sequence generation Allocation concealment Blinding Incomplete outcome data Selective outcome reporting Other sources of bias Li et al Low risk Unclear Unclear Low risk Low risk Low risk Hillman et al Low risk Low risk Low risk Unclear Low risk Low risk Giuliano et al Moreira et al Goldstone et al Palefsky et al Wilkin et al NA (single-arm) NA (single-arm) NA (single-arm) Low risk Low risk Low risk Levin et al Low risk Unclear Unclear Low risk Low risk Low risk 36 Reisinger et al Low risk Unclear High risk High risk Low risk Low risk Vesikari et al Low risk Unclear High risk Low risk Low risk Low risk Arguedas et al Low risk Unclear High risk Low risk Low risk Low risk 43 Reisinger et al Low risk Low risk Low risk Low risk Low risk Low risk Block et al Low risk Unclear Low risk Low risk Low risk Low risk * Five geographical regions were included: Africa, Asia-Pacific, Europe, Latin America, and North America. Four geographical regions were included: Northern America, Latin America, Asia-Pacific, and Europe. Five geographical regions were included: Asia, Australia, Europe, Latin America, and North America. VLPs: Vaccine-like Particles QHPV: Quadrivalent HPV vaccine Tdap: Tetanus, Diphtheria, Pertussis Vaccine High risk in blinding of the study participants and personnel due to the open-label in nature. High risk in incomplete outcome data due to incorrect administration of vaccine, resulting in the removal of 92 participants from the analysis. Page 27 of 61

28 Table 2 Anti-HPV GMTs in males after co-administration of qhpv with other vaccines Outcomes Concomitant group Nonconcomitant group Estimated fold difference Concomitant/ Nonconcomitant MU/mL (95%CI) (95% CI) P Value Studies Anti-HPV 6 GMT [0.926 to 1.211] [1.332 to 1.649] 0.72 [0.62 to 0.85] < Arguedas et al (2010)* [ to ] [ to ] 1.00 [0.84 to 1.20] <0.001 Reisinger et al (2010) [ to ] [ to ] 0.92 [0.77 to 1.11] <0.001 Vesikari et al (2010) [1.134 to 1.408] [1.576 to 1.838] 0.74 [0.65 to 0.85] < Arguedas et al (2010) Anti-HPV 11 GMT [ to ] [ to ] 1.06 [0.94 to 1.21] <0.001 Reisinger et al (2010) [ to ] [ to ] 0.92 [0.79 to 1.06] <0.001 Vesikari et al (2010) [4.711 to 5.945] [6.084 to 7.174] 0.80 [0.70 to 0.92] < Arguedas et al (2010) Anti-HPV 16 GMT [ to ] 7.37 [ to ] 0.98 [0.84 to 1.14] <0.001 Reisinger et al (2010) [ to ] [ to ] 0.90 [0.76 to 1.05] <0.001 Vesikari et al (2010) [0.798 to 1.032] [1.018 to 1.222] 0.81 [0.70 to 0.95] < Arguedas et al (2010) Anti-HPV 18 GMT [1.06 to ] [ to ] 0.89 [0.75 to 1.03] <0.001 Reisinger et al (2010) [ to ] [ to ] 0.84 [0.69 to 1.01] <0.001 Vesikari et al (2010) * Other vaccines concomitantly administered: MenACWY-CRM and Tdap. Other vaccine concomitantly administered: Menactra and Adacel. Other vaccine concomitantly administered: Tdap. GMT: Geometric mean titre Page 28 of 61

29 Table 3 Efficacy of qhpv in males in the Intention-to-Treat Population* Outcomes qhpv vaccine Control Risk ratio (95% CI) Efficacy % (95% CI) P Value Studies No. of cases/ No. of participants in each group External genital lesions Associated with HPV 6/11/16/18 27/ / [0.23 to 0.54] 65.5 (45.8 to 78.6) < Giuliano et al (2011) Associated with HPV 6/11/16/18 in HM 21/ / [0.22 to 0.60] 63.7 (39.3 to 79.1) < Giuliano et al (2011) Associated with HPV 6/11/16/18 in MSM 6/290 20/ [0.12 to 0.74] 70.2 (23.0 to 90.2) Giuliano et al (2011) Associated with any of 10 additional HPV 9/ / [0.197 to 50.3 (-16.5 to 80.3) Goldstone et al (2013) types 1.165] AIN and anal cancer Associated with HPV 6/11/16/18 in MSM 38/275 77/ [0.35 to 0.70] 50.3 (25.7 to 67.2) Palefsky et al (2011) Associated with any of 10 additional HPV types 38/275 44/ (-39.4 to 44.4) Goldstone et al (2013) AIN 1 in MSM 31/275 62/ [0.34 to 0.75] 49.6 (21.2 to 68.4) Palefsky et al (2011) AIN 2 or 3 in MSM 18/275 39/ [0.27 to 0.79] 54.2 (18.0 to 75.3) Palefsky et al (2011) Anal cancer in MSM 0/275 0/ Palefsky et al (2011) Page 29 of 61

30 Table 3 Efficacy of qhpv in males in the Intention-to-Treat Population* (Continued) PIN or penile/ perianal/ perineal cancer All PIN lesions 6/1943 5/ [0.37 to 3.91] ( to 69.7) 0.77 Giuliano et al (2011) PIN1 PIN 2 or 3 Penile/ perianal/ perineal cancer associated with HPV 6, 11, 16, or 18 3/1943 4/ [0.17 to 3.34] 25.6 ( to 89.1) 0.70 Giuliano et al (2011) 3/1943 2/ [0.25 to ( to 0.66 Giuliano et al (2011) 8.94] 82.9) 0/1943 0/ Giuliano et al (2011) Condylomata acuminate (Genital warts) All condylomata acuminate lesions All condylomata acuminate lesions in MSM Persistent infection Associated with HPV 6, 11, 16, or 18 Associated with HPV 6, 11, 16, or 18 in HM Associated with HPV 6, 11, 16, or 18 in MSM 27/ / [0.24 to 0.58] 13/275 31/ [0.23 to 0.79] 148/ / [0.45 to 0.65] 96/ / [0.41 to 0.65] 51/ / [0.34 to 0.60] 67.2 (47.3 to 80.3) < Giuliano et al (2011) 57.2 (15.9 to 79.5) Palefsky et al (2011) 47.8 (36.0 to 57.6) < Giuliano et al (2011) 50.4 (36.2 to 61.6) < Giuliano et al (2011) 59.4 (43.0 to 71.4) < Palefsky et al (2011) * Data shown are for subjects who had at least one follow-up visit after day1. Efficacy= 1- risk ratio External genital lesions denotes a diagnosis of condyloma acuminatum; HPV, human papillomavirus; and PIN, penile, perianal, or perineal intraepithelial neoplasia. Ten additional HPV types: HPV 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59. AIN: Anal Intraepithelial Neoplasia, severity graded from I to III with III being the most severe and as a potential precursor of anal cancer. PIN: Penal intraepithelial neoplasia, severity graded from I to III with III being the most severe and as a potential precursor of penile/ perianal/ perineal cancer. HM: Heterosexual men MSM: Men having sex with men Page 30 of 61

31 Table 4 Efficacy of qhpv in males in the Per-Protocol Population* Outcomes qhpv vaccine Control Risk ratio (95% CI) No. of cases/ No. of participants in each group Efficacy % (95% CI) P Value Studies External genital lesions Associated with HPV 6/11/16/18 3/ / (0.019 to Associated with HPV 6/11/16/18 in HM 2/ / (0.009 to Associated with HPV 6/11/16/18 in MSM 1/197 5/ (0.004 to 1.879) AIN and anal cancer 90.4 (69.2 to 98.1) Giuliano et al (2011) 92.4 (69.6 to 99.1) Giuliano et al (2011) 79.0 (-87.9 to 99.6) 0.44 Giuliano et al (2011) Associated with HPV 6/11/16/18 in MSM 5/194 24/ (0.067 to 77.5 (39.6 to 93.3) Palefsky et al (2011) 0.604) AIN 1 in MSM 4/194 16/ (0.066 to 73.0 (16.3 to 93.4) Palefsky et al (2011) 0.837) AIN 1 in HIV+ men 16/ Wilkin et al (2010) AIN 2 or 3 in MSM 3/194 13/ (0.046 to 74.9 (8.8 to 95.4) Palefsky et al (2011) 0.912) AIN 2 or 3 in HIV+ men 13/ Wilkin et al (2010) Anal cancer in MSM 0/194 0/ Palefsky et al (2011) Page 31 of 61

32 Table 4 Efficacy of qhpv in males in the Per-Protocol Population* (Continued) PIN or penile/ perianal/ perineal cancer All PIN lesions PIN1 PIN 2 or 3 Penile/ perianal/ perineal cancer associated with HPV 6, 11, 16, or 18 Condylomata acuminate (Genital warts) All condylomata acuminate lesions All condylomata acuminate lesions in MSM 0/1397 3/ (0 to 2.412) 100 ( to 100) - 0/1397 2/ (0 to 5.311) 100 ( to 100) - 0/1397 1/ (0 to ) 100 ( to 100) - 0/1397 0/ / / (0.021 to 89.4 (65.5 to 97.9) ) 0/194 6/208 0 (0 to 0.918) 100 (8.2 to 100) - Giuliano et al (2011) Giuliano et al (2011) Giuliano et al (2011) Giuliano et al (2011) Giuliano et al (2011) Palefsky et al (2011) Persistent infection Associated with HPV 6, 11, 16, or 18 15/ / (0.071 to 0.266) 85.6 (73.4 to 92.9) < Giuliano et al (2011) Associated with HPV 6, 11, 16, or 18 in HM 14/ / (0.085 to 0.289) 83.7 (71.1 to 91.5) < Giuliano et al (2011) Associated with HPV 6, 11, 16, or 18 in MSM 2/193 39/ ( to 0.196) 94.9 (80.4 to 99.4) < Palefsky et al (2011) * Data shown are for subjects who had received all three injections of vaccine or placebo, were DNA-negative and sero-negative for vaccine HPV types at enrolment, and remained DNA-negative throughout the study without protocol violation. Efficacy= 1- risk ratio External genital lesions denotes a diagnosis of condyloma acuminatum; HPV, human papillomavirus; and PIN, penile, perianal, or perineal intraepithelial neoplasia. AIN: Anal Intraepithelial Neoplasia, severity graded from I to III with III being the most severe and as a potential precursor of anal cancer. PIN: Penal intraepithelial neoplasia, severity graded from I to III with III being the most severe and as a potential precursor of penile/ perianal/ perineal cancer. HM: Heterosexual men MSM: Men having sex with men Page 32 of 61

33 Figure 1 Inclusion and exclusion of trials in study selection EMBASE* * MEDLINE The Cochrane Central China Journal Net Register of Controlled ( /8/2013) ( /8/2013) Trials (till 2013)* ( /8/2013) WanFang Data ( /8/2013) Google Scholar Yahoo articles 4600 records excluded through title and abstract screening 150 full-text articles assessed for eligibility 13 publications (9 unique RCTs) 137 records excluded by selection criteria 54 Reviews and commentary 14 Study population: females/ transplant patients 9 Therapeutic/ Bivalent vaccine 2 Vaccine study design: cohort studies 13 Vaccine cost-effective and impact studies 45 Duplications * Keywords and MESH search # Keywords search only First 10 pages of results Page 33 of 61

34 Figure 2 Seroconversion rates at one month post-dose three A. HPV 6 seroconversion rate In general males % In HIV-infected males % B. HPV 11 seroconversion rate In general males In HIV-infected males % % Page 34 of 61

35 Figure 2 Seroconversion rates at one month post-dose three (Continued) C. HPV 16 seroconversion rate In general males In HIV-infected males % % D. HPV 18 seroconversion rate In general males % In HIV-infected males % Page 35 of 61

36 Figure 3 Anti-HPV GMTs at one month post-dose three A. Anti-HPV 6 GMT In general males (MU/mL) In HIV-infected males (MU/mL) B. Anti-HPV 11 GMT In general males (MU/mL) In HIV-infected males (MU/mL) Page 36 of 61

37 Figure 3 Anti-HPV GMTs at one month post-dose three (Continued) C. Anti-HPV 16 GMT In general males (MU/mL) In HIV-infected males (MU/mL) D. Anti-HPV 18 GMT In general males (MU/mL) In HIV-infected males (MU/mL) Page 37 of 61

38 Figure 4 Adverse events of qhpv reported 14 days after each injection A. Injection-site adverse events B. Systemic adverse events C. Vaccine-related adverse events D. Serious adverse events Page 38 of 61

39 Figure 5 Adverse events of qhpv after concomitant and non-concomitant administration with other vaccines A. Injection-site adverse events B. Systemic adverse events C. Vaccine-related adverse events D. Serious adverse events Page 39 of 61

40 Figure 6 Sensitivity analysis for selected endpoints with significant heterogeneity A. Anti-HPV 6 GMT Included trials Excluded trials (MU/mL) B. Anti-HPV 11 GMT Included trials Excluded trials (MU/mL) Page 40 of 61

41 Figure 6 Sensitivity analysis for selected endpoints with significant heterogeneity (Continued) C. Anti-HPV 16 GMT Included trials Excluded trials (MU/mL) D. Anti-HPV 18 GMT Included trials Excluded trials (MU/mL) Page 41 of 61

42 Appendix A: The search steps in included electronic databases I. Cochrane Central Register of Controlled Trials Keywords search 1. HPV 2. Papillomavirus$ 3. 1 OR 2 4. Vaccin$ 5. 3 AND 4 6. Gardasil 7. Silgard 8. 5 OR 6 OR 7 MeSH search 9. Papillomavirus Vaccines (include all subheadings) Combining Keywords & MeSH search or Limit to Human II. MEDLINE Keywords search 1. HPV 2. Papillomavirus$ 3. 1 OR 2 4. Vaccin$ 5. 3 AND 4 6. Gardasil 7. Silgard 8. 5 OR 6 OR 7 9. Immunogenicity 10. Immune response 11. Immunity OR 10 OR AND Efficacy 15. Effect$ OR AND Safety 19. Adverse event$ Page 42 of 61

43 Appendix A: The search steps in included electronic databases (Continued) OR AND OR 17 OR Limit to Human MeSH search 24. Papillomavirus Vaccines (include all subheadings) 25. Limit to Human Combining Keywords & MeSH search or 25 III. EMBASE Keywords search 13. HPV 14. Papillomavirus$ OR Vaccin$ AND Gardasil 19. Silgard OR 6 OR Immunogenicity 22. Immune response 23. Immunity OR 10 OR AND Efficacy 16. Effect$ OR AND Safety 27. Adverse event$ OR AND OR 17 OR Limit to Human MeSH search 25. Wart virus vaccine (include all subheadings) Page 43 of 61

44 Appendix A: The search steps in included electronic databases (Continued) 26. Limit to Human Combining Keywords & MeSH search or 25 IV. China Journal Net Only keywords search available Input [KY= 人乳頭狀瘤病毒 or KY= 人乳頭瘤病毒 or KY= 乳頭狀瘤病毒 or KY= 乳頭瘤病毒 or KY=HPV) and KY= 疫苗 ] or (KY=Gardasil or KY=Silgard) V. Wanfang Data Only keywords search available Input [( 人乳頭狀瘤病毒 or 人乳頭瘤病毒 or 乳頭狀瘤病毒 or 乳頭瘤病毒 or HPV) and 疫苗 ] or (Gardasil or Silgard) VI. Google Scholar Only keywords search available Input Quadrivalent Human Papillomavirus Vaccine AND men VII. Yahoo Only keywords search available Input Quadrivalent Human Papillomavirus Vaccine AND men Page 44 of 61

45 Appendix B: List of Included Studies 1. Li R, Li Y, Radley D, Liu Y, Huang T, Sings HL, et al. Safety and immunogenicity of a vaccine targeting human papillomavirus types 6, 11, 16 and 18: A randomized, double-blind, placebo-controlled trial in Chinese males and females. Vaccine. 2012; 30: Hillman RJ, Giuliano AR, Palefsky JM, Goldstone SE, Moreira Jr. ED, Vardas E, et al. Immunogenicity of the quadrivalent human papillomavirus (type 6/11/16/18) vaccine in males 16 to 26 years old. Clinical and Vaccine Immunology. 2012; 19 (2): Giuliano AR, Palefsky JM, Goldstone SE, Moreira ED Jr., Penny ME, Aranda C, et al. Efficacy of quadrivalent HPV vaccine against HPV infection and disease in males. New England Journal of Medicine. 2011; 364(5): Moreira ED Jr., Palefsky JM, Giuliano AR, Goldstone SE, Aranda C, Jessen H, et al. Safety and reactogenicity of a quadrivalent human papillomavirus (types 6, 11, 16, 18) L1 viral-like-particle vaccine in older adolescents and young adults. Human Vaccines. 2011; 7(7): Goldstone SE, Jessen H, Palefsky JM, Giuliano AR, Moreira ED, Vardas E, et al. Quadrivalent HPV vaccine efficacy against disease related to vaccine and non-vaccine HPV types in males. Vaccine. 2013; 31(37): Palefsky JM, Giuliano AR, Goldstone SE, Moreira ED Jr., Aranda C, Jessen H, et al. HPV vaccine against anal HPV infection and anal intraepithelial neoplasia. New England Journal of Medicine. 2011; 365(17): Wilkin T, Lee JY, Lensing SY, Stier EA, Goldstone SE, Berry JM, et al. Safety and immunogenicity of the quadrivalent human papillomavirus vaccine in HIV-1- infected men. The Journal of infectious diseases. 2010; 202 (8): Levin MJ, Moscicki AB, Song LY, Fenton T, Meyer WA, Read JS, et al. Safety and immunogenicity of a quadrivalent human papillomavirus (types 6, 11, 16, and 18) vaccine in HIV-infected children 7 to 12 years old. Journal of Acquired Immune Deficiency Syndromes. 2010; 55(2): Reisinger KS, Block SL, Collins-Ogle M, Marchant C, Catlett M, Radley D. Safety, tolerability, and immunogenicity of gardasil given concomitantly with Menactra and Adacel. Pediatrics. 2010; 125: Vesikari T, Van Damme P, Lindblad N, Pfletschinger U, Radley D, Ryan D, et al. An open-label, randomized, multicenter study of the safety, tolerability, and immunogenicity of quadrivalent human papillomavirus (types 6/11/16/18) vaccine given concomitantly with diphtheria, tetanus, pertussis, and poliomyelitis vaccine in healthy adolescents 11 to 17 years of age. Pediatric Infectious Disease Journal. 2010; 29(4): Arguedas A, Soley C, Loaiza C, Rincon G, Guevara S, Perez A, et al. Safety and immunogenicity of one dose of MenACWY-CRM, an investigational quadrivalent meningococcal glycoconjugate vaccine, when administered to adolescents concomitantly or sequentially with Tdap and HPV vaccines. Vaccine. 2010; 28(18): Reisinger KS, Block SL, Lazcano-Ponce E, Samakoses R, Esser MT, Erick J, et al. Safety and persistent immunogenicity of a quadrivalent human papillomavirus types 6, 11, 16, 18 L1 virus-like particle vaccine in preadolescents and adolescents: A randomized controlled trial. Pediatric Infectious Disease Journal. 2007; 26(3): Block SL, Nolan T, Sattler C, Barr E, Giacoletti KE, Marchant CD, et al. Comparison of the immunogenicity and reactogenicity of a prophylactic quadrivalent human papillomavirus (types 6, 11, 16, and 18) L1 virus-like particle vaccine in male and female adolescents and young adult women. Pediatrics. 2006; 118(5): Page 45 of 61

46 APPENDIX C: The Cochrane Risk of Bias Tools for Randomized Controlled Trials Page 46 of 61

47 APPENDIX C: The Cochrane Risk of Bias Tools for Randomized Controlled Trials (Continued) Page 47 of 61

48 APPENDIX C: The Cochrane Risk of Bias Tools for Randomized Controlled Trials (Continued) Page 48 of 61

49 APPENDIX C: The Cochrane Risk of Bias Tools for Randomized Controlled Trials (Continued) Page 49 of 61

50 APPENDIX C: The Cochrane Risk of Bias Tools for Randomized Controlled Trials (Continued) Page 50 of 61