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Serum Antibodies to Human Papillomavirus (HPV) Type 6, 11, 16 and 18 and Their Role in the Natural History of HPV Infection in Men

Dissertation
Author: Beibei Lu
Abstract:
Our understanding of humoral immune response to human papillomavirus (HPV) infection has been mainly derived from studies in women. Very little is known about humoral immune response to HPV in men. There is also a growing interest in understanding the burden of HPV exposure in the subgroups of the male population, including men who have sex with women (MSW), men who have sex with men (MSM) and men who have sex with both men and women (MSMW). This dissertation was undertaken to understand and characterize humoral immune response, measured by detectable serum antibody IgG, to HPV 6, 11, 16 and 18 infection, to estimates seroprevalence of HPV 6, 11, 16 and 18, to determine the associations of socio-demographic and sexual behavioral factors with seroprevalence of individual HPV types, and to evaluate the role of serum antibodies in the subsequent acquisition of infection with the same HPV type, genetically related and un-related HPV types. Three studies that compose of this dissertation were conducted within the framework of two longitudinal studies of HPV infection in men: a single-site natural history study of male residents of Tucson, Arizona (the 1st study: N=285); and a multi-national natural history study of healthy men residing in São Paulo, Brazil, Cuernavaca, Mexico, and Tampa, Florida (the 2 nd study: N=1477; the 3rd study: N=2187). Men were recruited using similar eligibility criteria in both natural history studies and followed every 6 months for a maximum of 18 months in the single-site study and 48 months in the multi-national study. HPV DNA status was assessed using the PGMY09/11 L1 consensus primer system and the Linear Array HPV Genotyping Protocol. Testing of serum antibodies to HPV 6, 11, 16 and 18 was performed with virus-like particle-based ELISA assays. Data from our studies indicate that exposure to HPV 6, 11, 16 and 18, the four HPV types targeted in the currently license HPV vaccines, is common. Of 285 male residents of Tucson, Arizona, 28.8% of them were seropositive to HPV 16 and/or 18 at study entry. Similarly, approximately one third of 1477 participants of the multi-national male HPV natural history study were seropositive to at least one vaccine HPV type, with the percentage of 21.8% in U.S. site, 33.4% in Mexico site, and 49.1% in Brazil site. It is also noted that seroprevalence of individual vaccine HPV types is greatly elevated among men of different sexual practices. Seroprevalence of HPV 6, 11, 16 and/or 18 was twice as high among MSM and MSMW compared to MSW. Likewise, seroprevalence of individual HPV types was two fold or higher among MSW and MSMW. Our findings suggest that the predominant predictors of seropositivity to HPV 6, 11, 16 and 18 are age and same-sex sexual behaviors. Seroprevalence increased with age among young-to-middle-aged men with significant upward age trends observed for HPV 11, 16 and 18. MSM, compared to MSW, more likely to be seropositive to HPV 16 or 18. Similarly, men who practiced same-sex anal sex, compared to those who did not, were significantly more likely to be seropositive to HPV 6, 11, 16 and 18, respectively. Among 276 men free of HPV 16 at enrollment in Tucson, We did not detect statistically significant associations between the baseline serum antibodies to HPV 16 and/or 18 and subsequent risk of infection with homogeneous HPV types or related-HPV types. Of 2187 men residing in three countries who tested HPV 16 negative at enrollment, the risk of subsequent HPV 16 infection was not associated with enrollment HPV 16 serum antibodies status. Our data provide important estimates of population exposure to vaccine HPV types for future studies modeling potential vaccine impact and vaccine cost effectiveness in men. Our findings also support strategic vaccination of males as an effective preventive measure for HPV-related diseases and cancers in men and their sex partners, men and women alike.

TABLE OF CONTENTS

LIST OF TABLES.............................................................................................................iii

LIST OF FIGURES...........................................................................................................iv

ABSTRACT.......................................................................................................................v

CHAPTER 1: INTRODUCTION........................................................................................1

CHAPTER 2: THE FIRST MANUSCRIPT: EPIDEMIOLOGIC FACTORS ASSOCIATED WITH SEROPOSITIVITY TO HUMAN PAPILLOMAVIRUS TYPE 16 AND 18 VIRUS-LIKE PARTICLES (VLPS) AND RISK OF SUBSEQUENT INFECTION IN MEN....................................................................4 Abstract.................................................................................................................5 Introduction............................................................................................................6 Methods.................................................................................................................6 Study Population.......................................................................................6 HPV DNA Testing......................................................................................7 HPV Serum Antibody Testing....................................................................7 Statistical Analysis.....................................................................................7 Results..................................................................................................................8 Discussion...........................................................................................................10

CHAPTER 3: THE SECOND MANUSCRIPT: HUMAN PAPILLOMAVIRUS (HPV) 6, 11, 16 AND 18 SEROPREVALENCE IS ASSOCIATED WITH AGE AND SEXUAL PRACTICE: RESULTS FROM THE MULTI-NATIONAL HPV INFECTION IN MEN STUDY (HIM STUDY)...............................................18 Abstract...............................................................................................................19 Introduction..........................................................................................................20 Methods...............................................................................................................21 Study Population......................................................................................21 Study Protocol.........................................................................................21 HPV Serum Antibody Testing..................................................................22 HPV DNA Sampling and Testing.............................................................22 Statistical Analysis...................................................................................22 Results................................................................................................................23 Discussion...........................................................................................................26

CHAPTER 4: THE THIRD MANUSCRIPT: A PROSPECTIVE STUDY OF GENITAL HUMAN PAPILLOMAVIRUS (HPV) 16 INFECTION IN ASSOCIATION WITH BASELINE SERUM ANTIBODIES TO HPV 16...............43 Introduction..........................................................................................................44 Methods...............................................................................................................45

i

ii Study Population......................................................................................45 HPV Serum Antibody Testing..................................................................46 HPV DNA Sampling and Testing.............................................................46 Statistical Analysis...................................................................................46 Results................................................................................................................48 Discussion...........................................................................................................50

CHAPTER 5: CONCLUSIONS AND RECOMMENDATIONS.........................................65

REFERENCES CITED....................................................................................................68

APPENDIX A: LITERATURE REVIEW...........................................................................76

iii

LIST OF TABLES

TABLE 2.1: Factors Associated with HPV-16/18 Seropositivity among 285 Men in Tucson, Arizona........................................................................................... 14 TABLE 2.2: Risk of Subsequent Infection with Type- and Group-Specific HPV by Baseline Serum Antibody Status.................................................................17 TABLE 3.1: Participant characteristics and serum antibody titers for HPV 6, 11, 16 and 18 among seropositive men.................................................................31 TABLE 3.2: Factors associated with seroprevalence of HPV 6, 11, 16 and 18 among 1477 men in univariate analyses .................................................................34 TABLE 3.3: Factors associated with seroprevalence of HPV 6, 11, 16 and 18 among 1477 men in multivariable analyses............................................................40 TABLE 4.1: Participant characteristics in the current cohort and in the full study cohort of the HIM Stu dy...............................................................................55 TABLE 4.2: Select participant characteristics between seronegative and seropositive men in Tampa, Cuernavaca and São Paulo ................................................57 TABLE 4.3: Incidence proportion of HPV 16 infection and 6-month persistent infection by study visit and baseline serostatus...........................................60 TABLE 4.4: Association of incident H PV 16 infection and 6-month persistent HPV 16 infection with baseline serostatus (seronegative vs. seropositive) among 2187 men in Tampa, Cuernavaca and São Paulo.......................................61 TABLE 4.5: Association of incident HPV 16 infection and 6-month persistent HPV 16 infection with baseline serostatus (seronegative vs. seropositive) for MSW and MSM in Tampa, Cu ernavaca and São Paulo.............................62 TABLE 4.6: Association of incident H PV 16 infection and 6-month persistent HPV 16 infection with baseline serum antibody titer (continuous) among 2187 men in Tampa, Cue rnavaca and São Paulo...............................................63 TABLE 4.7: Association of incident H PV 16 infection and 6-month persistent HPV 16 infection with baseline serostatus (seronegative vs. seropositive) in the restricted analysis........................................................................................64

iv v

LIST OF FIGURES

FIGURE 3.1.A: Seroprevalences of HPV 6, 11, 16 and 18 Overall and by Sexual Practice ..................................................................................................42

FIGURE 3.1.B: Seroprevalences of HPV 6, 11, 16 and 18 by Age.................................42

FIGURE 3.1.C: Seroprevalences of HPV 6, 11, 16 and 18 by Country of Residence....42

FIGURE 3.1.D: Seroprevalences of HPV 6, 11, 16 and 18 by Lifetime Number of Female Sex Partners among Me n Who Had Sex with Women (MSW).42

FIGURE 3.1.E: Seroprevalences of HPV 6, 11, 16 and 18 by Lifetime Number of Male Anal Sex Pa rtners.........................................................................42

v

ABSTR ACT

Our understanding of hu moral immune response to human papillomavirus (HPV) infection has been mainly derived from studies in women. Very little is known about humoral immune response to HPV in men. There is also a growing interest in understanding the burden of HPV exposure in the subgroups of the male population, including men who have sex with women (MSW), men who have sex with men (MSM) and men who have sex with both men and women (MSMW). This dissertation was undertaken to understand and characterize humoral immune response, measured by detectable serum antibody IgG, to HPV 6, 11, 16 and 18 infection, to estimates seroprevalence of HPV 6, 11, 16 and 18, to determine the associations of socio- demographic and sexual behavioral factors with seroprevalence of individual HPV types, and to evaluate the role of serum antibodies in the subsequent acquisition of infection with the same HPV type, genetically related and un-related HPV types. Three studies that compose of this dissertation were conducted within the framework of two longitudinal studies of HPV infection in men: a single-site natural history study of male residents of Tucson, Arizona (the 1 st study: N=285); and a multi- national natural history study of healthy men residing in São Paulo, Brazil, Cuernavaca, Mexico, and Tampa, Florida (the 2 nd study: N=1477; the 3 rd study: N=2187). Men were recruited using similar eligibility criteria in both natural history studies and followed every 6 months for a maximum of 18 months in the single-site study and 48 months in the multi-national study. HPV DNA status was assessed using the PGMY09/11 L1 consensus primer system and the Linear Array HPV Genotyping Protocol. Testing of

vi serum antibodies to HPV 6, 11, 16 and 18 was performed with virus-like particle-based ELISA assays. Data from our studies indicate that exposure to HPV 6, 11, 16 and 18, the four HPV types targeted in the currently license HPV vaccines, is common. Of 285 male residents of Tucson, Arizona, 28.8% of them were seropositive to HPV 16 and/or 18 at study entry. Similarly, approximately one third of 1477 participants of the multi-national male HPV natural history study were seropositive to at least one vaccine HPV type, with the percentage of 21.8% in U.S. site, 33.4% in Mexico site, and 49.1% in Brazil site. It is also noted that seroprevalence of individual vaccine HPV types is greatly elevated among men of different sexual practices. Seroprevalence of HPV 6, 11, 16 and/or 18 was twice as high among MSM and MSMW compared to MSW. Likewise, seroprevalence of individual HPV types was two fold or higher among MSW and MSMW. Our findings suggest that the predominant predictors of seropositivity to HPV 6, 11, 16 and 18 are age and same-sex sexual behaviors. Seroprevalence increased with age among young-to-middle-aged men with significant upward age trends observed for HPV 11, 16 and 18. MSM, compared to MSW, more likely to be seropositive to HPV 16 or 18. Similarly, men who practiced same-sex anal sex, compared to those who did not, were significantly more likely to be seropositive to HPV 6, 11, 16 and 18, respectively. Among 276 men free of HPV 16 at enrollment in Tucson, We did not detect statistically significant associations between the baseline serum antibodies to HPV 16 and/or 18 and subsequent risk of infection with homogeneous HPV types or related-HPV types. Of 2187 men residing in three countries who tested HPV 16 negative at enrollment, the risk of subsequent HPV 16 infection was not associated with enrollment HPV 16 serum antibodies status. Our data provide important estimates of population exposure to vaccine HPV types for future studies modeling potential vaccine impact and vaccine cost effectiveness

vii in men. Our findings also support strategic vaccination of males as an effective preventive measure for HPV-related diseases and cancers in men and their sex partners, men and women alike.

- 1 -

CHAPTER 1:

INTRODUCTION Human papillomavirus (HPV) infection is one of the most common sexually transmitted infections (STIs) in the United States [1]. HPV infection in men contributes significantly to infection and subsequent cervical disease in women [2-5]. Studies of HPV infection in heterosexual couples have shown that males’ sexual behavior and HPV infection status are significantly associated with their female partners’ risk of precancerous lesion and cervical cancer [2-8]. High prevalence and high incidence of HPV infection in men are also associated with lesions [9-11] and cancers of anal canal and penis [12-16]. It has been reported in recent studies [17-19] that 25.8-72.9% of HIV- negative adult men test positive for genital HPV. Anal and oral HPV, though less common, is present in 8.0-16.6% [20-22], and 2.9-7.6% [23] of HIV-negative adult men, respectively. There is also a growing interest in understanding the burden of HPV infection among men with different sexual practices including men who have sex with women (MSW), men who have sex with men (MSM) and men who have sex with both men and women (MSMW). Compared to MSW, MSM and MSMW are several times more likely to be infected with HPV at different anatomic sites, and hence may be at increased risk for HPV-associated diseases and cancers [24-28]. HPV serology reflects cumulative exposures to type-specific HPV over time and across anatomic sites. It provides a useful means for estimating cumulative HPV exposures in a population and can provide insights into the natural history of HPV

- 2 - infection. With the recent licensure of the quadrivalent HPV vaccine for use in males, information on seroprevalence of vaccine-type HPV (HPV 6, 11, 16 and 18) in the general male population is needed to provide guidance for strategic planning of vaccination. Our understanding of humoral immune response to HPV infection has been mainly derived from serological studies in women with a focus on serum antibodies to HPV 16. In contrast, little is known about humoral immune response to HPV in men. This dissertation was undertaken to understand and characterize humoral immune response, measured by detectable serum antibody IgG, to HPV 6, 11, 16 and 18 infection, to determine the associations of socio-demographic and sexual behavioral factors with seroprevalence of individual HPV types, and to evaluate the role of serum antibodies in the prevention of subsequent infection with the same HPV type, genetically related and un-related HPV types. Three studies were conducted to achieve these objectives. The first is a study of serum antibodies to HPV 16 and/or 18, its relationship with potential risk factors and subsequent development of HPV 16 and/or 18 infections among a cohort of U.S. men; in the second study, investigation of serum antibodies and associated risk factors was expanded to four HPV types targeted in current HPV vaccines (HPV 6, 11, 16 and 18) among a large multi-national male cohort; and the third study further examined if risk of acquiring HPV 16 infection over a three-year follow-up period is modified by the enrollment HPV 16 serum antibody status in the same cohort of men. The sources of study population are two natural history studies of HPV infection in men: (1) a single-site, prospective study of male HPV infection conducted between September 2003 and December 2005 and funded by the Arizona Disease Control Research Commission, consisting of male residents aged 18-44 from Tucson, Arizona; (2) an ongoing multi-national prospective study of HPV Infection in Men (HIM Study)

- 3 - beginning in June 2005, comprising men aged 18-70 residing in Tampa, United States, São Paulo, Brazil, and Cuernavaca, Mexico.

- 4 -

CHAPTER 2:

THE FIRST MANUSCRIPT EPIDEMIOLOGIC FACTORS ASSOCIATED WITH SEROPOSITIVITY TO HUMAN PAPILLOMAVIRUS TYPE 16 AND 18 VIRUS-LIKE PARTICLES (VLPS) AND RISK OF SUBSEQUENT INFECTION IN MEN Beibei Lu 1, 2 , Michael E. Hagensee 3 , Ji-Hyun Lee 1, 2 , Yougui Wu 2 , Heather G. Stockwell 2 , Carrie M. Nielson 4 , Martha Abrahamsen 1 , Mary Papenfuss 1 , Robin B. Harris 5 , Anna R. Giuliano 1, 2 Affiliations: 1 H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida; 2 Department of Epidemiology & Biostatistics, College of Public Health, University of South Florida, Tampa, Florida; 3 Department of Internal Medicine, School of Medicine, Louisiana State University Health Sciences Center; 4 Oregon Health & Science University, Portland, Oregon; and 5 Arizona Cancer Center and Mel and Enid Zuckerman College of Public Health, University of Arizona, Tucson, Arizona

Running Title: Determinants of Male HPV Seropositivity and Risk of New Infection Key Words: HPV, seropositivity, epidemiologic factors, risk of infection Word Count: Abstract Word Count (limited to 250): 202 Text (without Abstract): 2206 Tables and Figures: 2 tables

- 5 - ABSTRACT

Our understanding of humoral response to HPV infection has been mainly derived from studies in women. The role of serum antibodies in the natural history of HPV in men has yet to be investigated. Data from 285 male participants of a natural history study were used to determine epidemiologic factors associated with HPV 16/18 seropositivity and explore the role of HPV 16 and 18 serum antibodies in subsequent HPV infections. Serum antibodies were detected by use of HPV 16 and 18 virus-like particles ELISA. Logistic regression and generalized estimating equation was employed for evaluation of risk factors. The risk of subsequent HPV infection by baseline antibody status was assessed by Incidence Rate Ratio and its confidence intervals. Men aged 36- 44 compared to men aged 18-25 were four times more likely to be seropositive to HPV 16/18. In addition, being divorced, separated or widowed, being former smoker and having sex with men was positively and independently associated with HPV 16/18 seropositivity. Our findings on the protective role of HPV 16 or 18 serum antibodies in subsequent infection were inconclusive. Large prospective studies are warranted to adequately address questions on the role of natural immunity in the natural history of HPV infections in men.

- 6 - INTRODUCTION Type-specific Human Papillomavirus (HPV) serology can provide insights into the natural history of HPV infection and associated HPV diseases. Our understanding of HPV serology has been mainly derived from studies in women with a focus on serum antibodies to HPV 16. A limited number of studies have evaluated the prevalence of HPV 16 serum antibodies and its determinants, and fewer have evaluated the prevalence of serum antibodies to other HPV types. Among 21 published studies that investigated serum antibodies to HPV 16 L1 VLPs and factors associated with HPV 16 seropositivity, only nine enrolled men [29-37]. Only two published studies to date have examined the potential protective role of serum antibodies in subsequent infection in women [38, 39] and none have been conducted among men. To address this information gap, we investigated epidemiologic factors associated with HPV 16/18 seropositivity and explored the role of HPV 16 and 18 serum antibodies in subsequent HPV infections among 285 U.S. men. METHODS Study Population. A prospective cohort was established for the HPV Infection in Men study in Tucson, Arizona between September 2003 and December 2005. Details of this cohort have been reported previously [18, 40]. In brief, 337 men (89.4% of eligible men screened) aged 18-44 years were enrolled. Men were residents of southern Arizona who reported no prior diagnosis of penile or anal cancers, or genital warts; and no current diagnosis or treatment of sexually transmitted infections (STIs) including genital warts, genital Herpes, Chlamydia, gonorrhea, syphilis, non-gonococcal urethritis, hepatitis B virus, hepatitis C virus and Human Immunodeficiency Virus (HIV) infection. All participants were consented prior to enrollment. Men were followed at 6-month intervals for approximately 18 months. At each study visit, participants completed a self- administered risk factor questionnaire, and had penile and scrotal cell samples and a

- 7 - venous blood sample collected. The current analysis included 285 men who completed at least 2 study visits, had adequate samples for HPV DNA detection and serum antibody testing, and had available questionnaire information from each visit. HPV DNA Testing. Cellular materials collected at each visit were tested for presence of HPV DNA using the PGMY09/11 L1 consensus primer system. HPV genotyping was conducted using the reverse line blot method [41] to detect 37 genital HPV types, regardless of the PCR result (Roche Molecular Diagnostics). Only samples that tested β-globin positive were deemed adequate and included in this analysis. HPV Serum Antibody Testing. HPV VLPs were prepared using BSC-1 cells (monkey kidney cell line) infected with the recombinant vaccinia virus expressing the L1 gene of HPV 16 and 18. IgG antibodies to HPV 16 and 18 VLPs were measured with enzyme-linked immunoassay (ELISA) as described previously [42]. Monoclonal antibodies used for Capture ELISA were H16.V5-HPV-16 and H18.J4-HPV-18 kindly provided by Dr. N. Christensen at Pennsylvania State Medical Center, Hershey, PA. The cutpoints were determined using a serum bank from children less than 10 years old with no prior history of warts, and calculated as the average reactivity plus two standard deviations [42]. Statistical Analysis. To determine factors associated with HPV 16/18 serum seropositivity, we employed logistic regression models with HPV 16/18 serum antibody status as the dependent variable. All participants with available serum antibody measurements were included in the analysis regardless of their baseline serum antibody status. Repeated measurements taken throughout the study period were incorporated to capture changes in serum antibody status. The Generalized Estimating Equation (GEE) [43] was applied to account for the correlation between repeated observations of the same individuals. An unstructured working correlation was assumed. We examined the likelihood of seropositivity in relation to sociodemographic characteristics, such as age,

- 8 - race, marital status, and education; and lifestyle and behavioral factors obtained at enrollment, including cigarette smoking, average alcohol use, circumcision, age at first sexual intercourse, sexual practice, and lifetime number of sex partners. We also included risk factors that changed over time and were repeatedly surveyed at each study visit, including the number of recent sex partners, self-reported STI status for self and partner since last visit, recent condom use and HPV 16/18 DNA status. Factors demonstrating a p-value of 0.10 or smaller in univariable models were included in the multivariable models and further evaluated by score chi-squared statistics. Odds Ratio (OR) and 95% confidence intervals (CIs) were calculated. The risk of subsequent infection with type-specific or group-specific HPV was evaluated among men who were DNA negative to corresponding HPV type(s) at study entry, and measured by Incidence Rate Ratio (IRR) and its 95% CIs calculated using the method proposed by Rothman and Greenland [44]. RESULTS Two hundred and eighty-five men were followed for approximately 18 months. Of the 285 men, 153 (53.7%) completed four study visits including the enrollment visit, 87 (30.5%) completed three visits, and 45 (15.8%) only completed two visits. The median duration of follow-up was 15.5 months (range, 3.7–24.7 months) and the median follow- up interval was 5.3 months. The mean age of the cohort was 29.8 years (SD: 8.1). Overall, HPV 16/18 seroprevalence was 28.8% at study entry, and 26.4, 30.0 and 29.8% at the 6-, 12- and 18-month visit, respectively. At enrollment, 14.8% of men were seropositive to HPV 16, followed by 14.4, 18.9 and 24.8% of men at each follow-up visit. The seroprevalence for HPV 18 was 21.1, 18.8, 21.6 and 19.9% throughout the study period. In crude analyses, HPV 16/18 serum antibody status was significantly associated with age, marital status, smoking status, sexual practice and the number of lifetime

- 9 - sexual partners (Table 2.1). Characteristics that remained statistically significantly associated with serum antibody status in the final adjusted model included age, marital status, cigarette smoking and sexual practice. Compared to men aged 18-25 years, men aged 36-44 years were more likely to be seropositive to HPV 16/18 (OR, 4.2, 95% CI: 2.2-8.1). Divorced, separated or widowed men compared to single men were twice as likely to be seropositive to HPV 16/18 (OR, 2.3, 95% CI: 1.02-5.3). Smokers were more likely to be seropositive compared with never smokers (former smoker: OR, 2.0, 95% CI: 1.03-3.9; current smoker: OR, 1.5, 95% CI: 0.8-2.8), although the latter did not reach statistical significance. A higher likelihood of seropositivity for men having sex with men (MSM) compared to men having sex with women only (MSW) was observed (OR, 2.6, 95% CI: 1.05-6.7). We examined the potential protection that HPV 16 and 18 serum antibodies confer against subsequent infection by assessing the risk of infection with homologous HPV types over the study period among men who had no detectable HPV DNA of interest at the baseline by their baseline serum antibody status (Table 2.2). The risk of subsequent infection with HPV 16 among 276 men who had no detectable HPV 16 infection at study entry did not differ significantly by their baseline HPV 16 serum antibody status (IRR, 1.1, 95% CI: 0.3-4.0). Similarly, no statistically significant difference in the risk of subsequent infection with HPV 18 was detected between HPV 18 seropositive and seronegative men at the baseline (IRR, 1.8, 95% CI: 0.2-20.2). We also explored the potential cross-protection provided by HPV 16 or 18 antibodies (Table 2.2). Seropositivity to HPV 16 appeared to be protective against new infections with HPV 16-related types (HPV 31, 33, 35, 52, 58 and 67) with an IRR of 0.6 (95% CI: 0.1-2.6), although the IRR did not achieve statistical significance. Similarly, seropositivity to HPV 18 yielded a statistically insignificant IRR of 0.6 (95% CI: 0.2-2.0) for infection with HPV 18-related types (HPV 39, 45, 59, 68, and 70). No significant

- 10 - reduction in the risk of subsequent infection with other HPV types was observed for HPV 16 or 18 seropositive men as compared with seronegative men. DISCUSSION This is one of few studies to examine factors associated with HPV serum antibody status in men and the first study to explore the protective role of serum antibodies against future infections in men. In this study the predominant predictor of HPV 16/18 seropositivity was age. Men aged 36-44 years were four times more likely to be seropositive than men aged 18-25 years. In previous studies, age has been positively associated with seroprevalence in both men and women [33, 35, 45-48]. In two studies that included participants across a broad age range an inverse U-shaped association with age was reported, with seroprevalence peaking at age 35-49 and declining with increasing age afterwards [45, 46]. The observed age effect in the current study is likely to be a result of cumulative lifetime sexual exposure to HPV infection. In the current study, smoking status was significantly associated with HPV antibody status in men. Smokers, compared to never smokers, were more likely to be HPV 16/18 seropositive. Recent studies of HPV seroprevalence and smoking in men have yielded mixed results [31, 34, 48]. No significant association with smoking status or tobacco use was found in the studies of Kreimer et al. [31] or Stone et al. [34], while a significant association with current smoking was reported by Dunne et al. among male residents of two U.S. cities (OR, 1.9, 95% CI: 1.1-3.2) [48]. Our finding is in agreement with the suggested immune suppressive effect of tobacco smoking which may facilitate persistence of viral infection and result in a higher likelihood of seroconversion for smokers [49]. An important observation from the current study was that MSM and MSMW (men who had sex with both men and women) were more likely to be seropositive. Similar findings have been reported in other studies. Stone et al. demonstrated that MSM were

- 11 - 6 times more likely to be seropositive to HPV 16 [34]. Kreimer et al. reported a significant association (OR, 2.9, 95% CI: 1.2-7.1) between seropositivity to HPV 16/18/33 VLPs and same-sex oral sexual intercourse among male participants of an oral HPV study [31]. History of having same-sex anal or oral sexual intercourse may serve as a surrogate marker for increased sexual exposure to the virus via multiple transmission routes including oral-penile, oral-anal and penile-anal transmission. With the absence of information on oral and anal HPV infection among this cohort of men, we could only hypothesize that an increased risk of oral or anal HPV infection in addition to genital infection may have contributed to increased seroconversion observed among MSM and MSMW. This hypothesis is also supported by findings from several recent studies. D’Souza et al. reported a higher prevalence of oral HPV infection with genital HPV types among MSM and MSMW compared to heterosexual men [26]. A high prevalence of anal HPV infection was reported among HIV-positive MSM from HIV/AIDS clinics in Montreal [50] as well as HIV-negative MSM in a community-based study [51]. Further studies of serum antibody development following incident HPV infection at different anatomic sites are needed to test this hypothesis. Results of the current study were inconclusive regarding whether HPV 16 or 18 serum antibodies were effective in protecting against subsequent infection with the homologous or phylogenetically related HPV types. A protective role of serum antibodies to HPV 16 was shown by Ho and colleagues in a prospective study of 247 female college students [38]. Women with a high titer level of IgG antibodies (OD>400-800) to HPV 16 in 2 or more consecutive visits had a lower risk for subsequent infection with HPV 16 (RR=0.49; p=0.037) and with HPV 16-related types (p=0.010) [38]. In contrast, no protective effect was reported by Viscidi et al. in the natural history study of 7,046 Guanacaste women for whom the risk of subsequent infection was measured at the follow-up visit scheduled 5-7 years after the baseline serology. It is likely that the

- 12 - inconclusive results on protective effect of serum antibodies in this study compared with female studies are in part attributable to potential gender difference in viral shedding and antibody response as observed in several serology studies that enroll both men and women [31, 33-37], and further compounded by the relative small sample size in the this study. Therefore, caution should be taken in interpreting the inconclusive finding of this study in men. The current study has a number of limitations. First, we only enrolled men aged 18-44 years. The narrow age range prohibits an evaluation of age effect in older men. Another limitation of the current study is that we were not able to evaluate the risk of subsequent infection based on a quantitative measure of antibody titer. The percent of HPV-infected men who seroconvert remains unknown, as is the longevity of serum antibodies in men. Serum antibodies elicited by distant, past infection may wane over time like serum antibodies to other viral infections such as hepatitis B virus, leading to potential exposure misclassification. Additional limitations include the relatively small sample size and limited duration of follow-up which may have lowered statistical power needed to detect a statistically significant difference in the risk of type-specific or group- specific infections given the low incidence for HPV types of interest. In summary, the current study identified age, marital status, smoking status and sexual practice as factors significantly and independently associated with HPV 16/18 serum antibody status. We did not detect statistically significant associations between the baseline serum antibody status and subsequent risk of infection. Large prospective studies that employ quantitative assessment of HPV antibody titers are needed to adequately address fundamental questions regarding the role of natural immunity in the natural history of HPV infections in men.

- 13 - Table 2.1 Factors associated with HPV-16/18 seropositivity in 285 men in Tucson, Arizona

Baseline Seroprevalence Crude Adjusted §

Characteristics No. Subjects (% Seropositive) OR 95% CI OR 95% CI Age 18-25 113 (13.3) 1.0 1.0 26-35 86 (19.8) 1.3 (0.7-2.4) 0.6 (0.3-1.2) 36-44 86 (58.1) 5.6 (3.4-9.3) * 4.2 (2.2-8.1) * Race White 237 (29.1) 1.0 -- Non-white 38 (26.3) 0.7 (0.3-1.3) -- Marital Status Single/Never married 182 (23.6) 1.0 1.0 Married/Cohabiting 69 (34.8) 1.6 (0.99-2.7) 1.5 (0.8-2.6) Divorced/Separated/Widow 34 (44.1) 3.2 (1.7-6.1) * 2.3 (1.02-5.3) * Education High school graduate or less 69 (26.1) 1.0 -- Some college/vocational school 98 (24.5) 1.0 (0.6-1.8) -- College graduate/graduate school 118 (33.9) 1.2 (0.7-2.1) -- Cigarette smoking Never 95 (21.1) 1.0 1.0 Former 54 (33.3) 2.2 (1.2-4.1) * 2.0 (1.03-3.9) * Current 67 (35.8) 1.8 (0.99-3.2) 1.5 (0.8-2.8) Alcohol use (drinks per month) 0-13 66 (33.3) 1.0 -- 14-52 103 (29.1) 1.0 (0.7-1.3) -- ≥ 53 56 (21.4) 0.8 (0.5-1.1) -- Circumcision (clinical assessment) No 35 (31.4) 1.0 --

- 14 - Baseline Seroprevalence Crude Adjusted §

Characteristics No. Subjects (% Seropositive) OR 95% CI OR 95% CI Yes 250 (28.4) 1.1 (0.6-2.0) -- Age at first sexual intercourse <18 160 (33.1) 1.4 (0.9-2.2) -- ≥18 113 (23.9) 1.0 -- Sexual practice Sexual intercourse with women 242 (26.4) 1.0 1.0 Sexual intercourse with men 13 (46.2) 2.4 (0.9-6.2) 2.6 (1.05-6.7) * Sexual intercourse with both 13 (53.8) 2.9 (1.01-8.4) * 2.1 (0.7-5.8) Lifetime no. of sex partners (either sex) 0-4 82 (19.5) 1.0 1.0 5-16 130 (23.1) 1.1 (0.6-1.8) 0.6 (0.3-1.3) ≥17 68 (51.5) 2.3 (1.3-4.1) * 1.0 (0.4-2.1) No. of new sex partners in the past 3 months None 160 (27.5) 1.0 -- One or more 94 (29.8) 1.0 (0.7-1.3) -- Diagnosed with other STI since last visit ** No 204 (26.5) 1.0 -- Yes 75 (36.0) 1.5 (0.9-2.4) -- Had partner(s) with other STI since last visit ** No 103 (18.4) 1.0 -- Yes 124 (34.7) 1.1 (0.9-1.5) -- Condom use in the past 3 months Never 86 (30.2) 1.0 -- Sometimes 41 (34.1) 1.0 (0.7-1.5) -- Frequently 40 (25.0) 0.8 (0.5-1.1) -- Always 45 (20.0) 1.1 (0.7-1.8) -- HPV 16/18 infection(s)

Full document contains 95 pages
Abstract: Our understanding of humoral immune response to human papillomavirus (HPV) infection has been mainly derived from studies in women. Very little is known about humoral immune response to HPV in men. There is also a growing interest in understanding the burden of HPV exposure in the subgroups of the male population, including men who have sex with women (MSW), men who have sex with men (MSM) and men who have sex with both men and women (MSMW). This dissertation was undertaken to understand and characterize humoral immune response, measured by detectable serum antibody IgG, to HPV 6, 11, 16 and 18 infection, to estimates seroprevalence of HPV 6, 11, 16 and 18, to determine the associations of socio-demographic and sexual behavioral factors with seroprevalence of individual HPV types, and to evaluate the role of serum antibodies in the subsequent acquisition of infection with the same HPV type, genetically related and un-related HPV types. Three studies that compose of this dissertation were conducted within the framework of two longitudinal studies of HPV infection in men: a single-site natural history study of male residents of Tucson, Arizona (the 1st study: N=285); and a multi-national natural history study of healthy men residing in São Paulo, Brazil, Cuernavaca, Mexico, and Tampa, Florida (the 2 nd study: N=1477; the 3rd study: N=2187). Men were recruited using similar eligibility criteria in both natural history studies and followed every 6 months for a maximum of 18 months in the single-site study and 48 months in the multi-national study. HPV DNA status was assessed using the PGMY09/11 L1 consensus primer system and the Linear Array HPV Genotyping Protocol. Testing of serum antibodies to HPV 6, 11, 16 and 18 was performed with virus-like particle-based ELISA assays. Data from our studies indicate that exposure to HPV 6, 11, 16 and 18, the four HPV types targeted in the currently license HPV vaccines, is common. Of 285 male residents of Tucson, Arizona, 28.8% of them were seropositive to HPV 16 and/or 18 at study entry. Similarly, approximately one third of 1477 participants of the multi-national male HPV natural history study were seropositive to at least one vaccine HPV type, with the percentage of 21.8% in U.S. site, 33.4% in Mexico site, and 49.1% in Brazil site. It is also noted that seroprevalence of individual vaccine HPV types is greatly elevated among men of different sexual practices. Seroprevalence of HPV 6, 11, 16 and/or 18 was twice as high among MSM and MSMW compared to MSW. Likewise, seroprevalence of individual HPV types was two fold or higher among MSW and MSMW. Our findings suggest that the predominant predictors of seropositivity to HPV 6, 11, 16 and 18 are age and same-sex sexual behaviors. Seroprevalence increased with age among young-to-middle-aged men with significant upward age trends observed for HPV 11, 16 and 18. MSM, compared to MSW, more likely to be seropositive to HPV 16 or 18. Similarly, men who practiced same-sex anal sex, compared to those who did not, were significantly more likely to be seropositive to HPV 6, 11, 16 and 18, respectively. Among 276 men free of HPV 16 at enrollment in Tucson, We did not detect statistically significant associations between the baseline serum antibodies to HPV 16 and/or 18 and subsequent risk of infection with homogeneous HPV types or related-HPV types. Of 2187 men residing in three countries who tested HPV 16 negative at enrollment, the risk of subsequent HPV 16 infection was not associated with enrollment HPV 16 serum antibodies status. Our data provide important estimates of population exposure to vaccine HPV types for future studies modeling potential vaccine impact and vaccine cost effectiveness in men. Our findings also support strategic vaccination of males as an effective preventive measure for HPV-related diseases and cancers in men and their sex partners, men and women alike.