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Presented in part: 26th International Papillomavirus Conference, Oral Communications-Clinical Science 4 Section, Montreal, Canada, 6 July 2010.
Background.Although the prevalence of human papillomavirus (HPV) genital infection is similarly high in males and females, seroprevalence is lower in males. This study assessed rates and determinants of seroconversion after detection of genital HPV infection in young men.
Methods.We investigated HPV type-specific seroconversion in a cohort of heterosexual male university students who had an α9 HPV type (HPV-16, -31, -33, -35, -52, -58, or -67) detected in the genital tract (n = 156). HPV DNA and antibodies were detected and typed using liquid bead-based multiplex assays. We calculated seroconversion using Kaplan–Meier survival analysis. Cox proportional hazards models with generalized estimating equations were used to examine associations with seroconversion.
Results.Within 24 months of detecting genital HPV infection, type-specific seroconversion ranged from 4% for HPV–52 to 36% for HPV-31. HPV-16 seroconversion at 24 months was 13% (95% confidence interval [CI], 7%–25%). Among incident HPV infections, ever cigarette smoking and infection site(s) (shaft/scrotum and glans/urine vs shaft/scrotum or glans/urine only) were positively associated with type-specific seroconversion.
Conclusions.For each of the α9 HPV types, type-specific seroconversion within 24 months was observed in 36% or less of infected men. Seroconversion might be related to cigarette smoking and genital site(s) infected.
Human papillomavirus (HPV) is a common sexually transmitted infection in men , and high-risk HPV (hrHPV) is an etiologic agent for male anogenital cancers . Although these cancers are rare worldwide, hrHPV infection in heterosexual men is also important because it can be transmitted to women. In women, the most common clinical sequelae of hrHPV infections are cervical lesions, which can lead to cervical cancer, the second most common cancer in women worldwide .
The prevalence of genital HPV infection is equally high in both genders [1, 4], and the incidence is as high in males as it is in females, or higher [5, 6]. However, HPV seroprevalence is lower in males than in females, in both general [7, 8] and high-risk populations [9–13]. In one of the few studies of HPV seroconversion in women (a cohort study of female university students aged 18–20 years ), 94% of women with prevalent genital HPV-16 infection at enrollment seroconverted within 13 months, and 67% of women with incident HPV-16 infection seroconverted within 24 months of incident detection. A similar study of antibody response has not been conducted in men, with most serologic investigations limited by their cross-sectional design. In this longitudinal study, we examined HPV type-specific seroconversion among heterosexual male university students infected in the genital tract with one or more α9 HPV type (HPV-16, -31, -33, -35, -52, -58, or -67).
Participants were members of a male HPV cohort study . Male students recruited at the University of Washington in Seattle, Washington, between June 2003 and January 2009 (n = 452). Eligible men were 1) 18–21 years of age, 2) permanent residents of Washington state, 3) in general good health and immune competent (ie, had no history of HIV, ongoing chemotherapy for cancer or other chronic conditions), 4) able to provide informed consent, 5) reporting sexual activity with women, and 6) able to attend 3 years of follow-up. For this substudy on HPV seroconversion, inclusion was limited to men who were HPV DNA positive for at least 1 of 7 α9 HPV types (HPV-16, -31, -33, -35, -52, -58, or -67) in 1 of 4 genital specimens (penile shaft, glans, scrotum, or urine) and who reported a history of vaginal intercourse at enrollment. Of the 165 men that fit these criteria, we were able to test serum specimens for 156 men (95%) and among these men, serum specimens were available for 1084 visits (95%). Among the 156 men, 1 participant reported receiving the quadrivalent HPV vaccine at his 6th visit (31 months), at which time he was censored from follow-up.
Participants were seen approximately every 4 months. At each visit, the research nurse practitioner administered a medical- and sexual-history interview followed by a genital examination. The examination (described in detail elsewhere ) included collection of exfoliated cell specimens for HPV DNA genotyping from 3 separate sites: 1) penile shaft (including the outside of the foreskin in uncircumcised men), 2) glans (including the urethral meatus and the inside of the foreskin in uncircumcised men), and 3) scrotum. The exfoliated cells were placed in a site-specific vial that contained 1 mL of Specimen Transport Medium (STM, Qiagen). Approximately halfway through the study, the scrotum and shaft specimens were combined in the same vial. At each visit, urine (for detection of HPV DNA in the internal urethral meatus ) and blood (for antibodies) were also collected. Between visits, participants completed biweekly online diaries in which they recorded sexual behaviors for each day and each sex partner.
All participants provided informed consent. The study protocol was approved by the University of Washington Human Subjects Division.
The STM samples were digested with 20 μg/mL protease K at 37°C for 1 hour. For each sample, 400 μL were used to isolate DNA by QIAamp DNA blood minicolumn, following the manufacture’s protocol (Qiagen, cat. no. 51104). Each sample was digested with protease at 56°C for 10 min and then loaded onto 1 QIAamp column. The column was washed and DNA eluted in 50 μL heated Tris–ethylenediaminetetraacetic acid (EDTA) buffer (TE, 70°C). HPV detection was performed using dot blot hybridization after polymerase chain reaction amplification of genomic DNA isolated from samples. HPV-positive samples were subsequently genotyped for 37 α HPV types using a liquid bead microarray (LBMA) assay based on Luminex technology . Specimens that tested negative for both β-globin and HPV DNA were considered insufficient for HPV DNA testing.
Among participants who tested positive for α9 HPV DNA at least once, sera from all visits were tested for HPV type-specific antibodies. The expression and preparation of recombinant proteins and the multiplex antibody binding LBMA assays have been described in detail previously [17–19]. Assays were conducted on a BioPlex 200 instrument (BioRad Laboratories) that recorded the median fluorescence intensity (MFI) resulting from bead-bound streptavidin-phycoerythrin, which was an indirect measure of the amount of bound human immunoglobulin G. Laboratory personnel were blinded to the HPV DNA status of the men. The MFI values for each antigen (after subtracting the MFI for glutathione S-transferase–coated beads) were plotted as a histogram (data not shown) and a cut point selected such that the large peak that surrounded MFI = 0 was excluded. This resulted in cutoff values that ranged from 400 (HPV-16) to 1500 (HPV-35).
On each plate we tested the HPV-16 WHO international standard (10 units/mL; average MFI, 8009; 95% CI, 6574–9444)  (National Institute for Biological Standards and Controls) and a dilution series of immune serum ranging from 1:1600 to 1:204800 from quadrivalent HPV vaccine recipients. It was determined that the immune serum was 56.1 times (561 units/mL; 95% CI, 465–657 units/mL) more concentrated than the international standard and that using a cutoff point of MFI = 400 meant the assay was sufficiently sensitive to detect an anti–HPV-16 antibody concentration of ≥1.73 units/mL. In an informal examination of reliability, sera from men who were seropositive for HPV-16 or HPV-31 at some point during the study were retested (n = 246). Concordance between the first test and the retest for HPV-16 and HPV-31 was 92% and 74%, respectively and the Cohen’s kappa coefficients were 0.71 (substantial) and 0.44 (moderate).
International standards for HPV types other than HPV-16 were not available. However, it was possible to ensure that equivalent levels of fusion-proteins were bound to each bead set using a monoclonal antibody specific for the C-terminal epitope tag [21, 22]. The average variation between bead sets was 18.4% (coefficient of variation [CV]) and the difference was <40% between all bead sets. This antibody was used to calculate the variation between plates (CV = 8.7%, averaged over all antigens).
We estimated cumulative probabilities of seroconversion for each HPV type using Kaplan–Meier survival analysis. We calculated seroconversion estimates separately among men with prevalent or incident type-specific HPV infection, as well as among the combined group. We defined prevalent infection as a DNA positive genital result for a specific HPV type at enrollment, and incident infection as the first DNA positive genital result for a specific HPV type at any visit after a negative genital result for that HPV type at enrollment. For the survival analyses, time started at first HPV DNA detection and observations were censored at date of seroconversion, loss to follow-up, last study visit, report of HPV vaccination, or the end of the study. We resolved ties by assigning the seroconversion date as 1 day later than the DNA detection date. When seroconversion was detected prior to DNA detection, it was treated as a tie in the analyses of combined prevalent and incident infections, but excluded from analyses specific to incident infections.
We used marginal Cox proportional hazards models stratified according to HPV type within the model to estimate type-specific seroconversion among men with incident HPV infection. We accounted for correlated data using generalized estimating equations (GEE). We calculated univariate and multivariate hazard ratios (HR) and 95% CIs to assess the relationship between type-specific HPV seroconversion and demographic characteristics (age, ethnicity), health characteristics (circumcision status and cigarette smoking), sexual history (years since first vaginal intercourse and lifetime number of female sex partners), sexual behavior in the preceding 4 months (recent number of vaginal sex acts, presence of a new vaginal sex partner, and condom use frequency), and characteristics of the incident HPV infection (genital site(s) infected and number of visits DNA detected). The number of visits at which the incident HPV DNA type was detected was analyzed as a time-varying covariate. Lifetime number of vaginal sex partners at incident detection was the sum of the reported number of partners at enrollment and those reported in the online diary until incident detection. Recent number of vaginal sex acts and report of new partner were obtained from the online diary. Condom use was calculated as number of times a participant reported using a condom during vaginal sex (also recorded in the online diary) divided by the number of vaginal sex acts he reported. Characteristics were mainly assessed as categorical dummy variables, but when relevant and possible, categorical or continuous trends were also assessed. Characteristics that were marginally statistically significant in the univariate models (Wald P value < .10) were included in the multivariate model.
The male cohort participants who were infected with at least 1 α9 HPV type (HPV-16, -31, -33, -35, -52, -58, or -67) in the genital tract were similar to the parent cohort study participants, in that they had an average age of 19 years at enrollment, most were white, most were nonsmokers, and most were circumcised (Table 1, ). Their median follow-up time was 31.4 months (interquartile range, 15.9–36.3).
Among the 156 men, there were 241 genital α9 HPV infections, including 50 prevalent infections and 191 incident infections (Figure 1). In an analysis that combined prevalent and incident α9 HPV infections, type-specific seroconversion at 24 months varied by HPV type, ranging from 4.0% for HPV-52 to 36.0% for HPV-31 (Table 2). After a prevalent HPV infection, type-specific seroconversion at 24 months was 19.6% for HPV-16, 32.1% for HPV-31, 33.3% for HPV-33, 25.0% for HPV-67 (Figure 2A) and 0% for all other α9 HPV types. Type-specific seroconversion 24 months after incident HPV infection was 7.1% for HPV-16, 15.3% for HPV-31, 15.2% for HPV-67 (Figure 2B) and 0% for all other α9 HPV types.
Not included in the seroconversion estimates after incident detection was seropositivity detected prior to detection of incident HPV infection (n = 15, Figure 1). This occurred at least once for all 7 α9 HPV types. Excluding these seropositive results, and including concurrent detection of incident HPV infection and seropositivity (n = 3), the median time to type-specific seroconversion after incident genital HPV infection was 3.9 months (mean, 8.0 mo; standard deviation [SD], 9.3 mo). The median time to type-specific seroconversion after prevalent genital HPV infection (including concurrent prevalent HPV infection and seropositivity; n = 4) was 1.8 months (mean, 7.1 mo; SD, 9.4 mo). Among the prevalent and incident infections with at least 1 visit after antibody detection (n = 29), 62% were seropositive at a subsequent visit.
Type-specific seroconversion among incident genital HPV infections was more likely among men who reported ever smoking at enrollment and among men who had HPV detected in both the glans/urine and the shaft/scrotum sites (Table 3). Although information on ever smoking at time of incident HPV infection was only available for 60% of the infections, there was evidence of an association with seroconversion (univariate HR, 6.5; 95% CI, 0.7–56.0). There was also some evidence of associations with race, circumcision, and years since first vaginal intercourse, as well as sexual behavior in the preceding 4 months (number of new partners, number of vaginal sex acts, and condom use), though none of these associations was statistically significant. Age, lifetime number of female sex partners, and the number of visits at which the incident HPV type was detected did not appear to be associated with type-specific seroconversion.
In young heterosexual men with genital α9 HPV infection, rates of seroconversion were low at 24 months, ranging from 4% for HPV-52 to 36% for HPV-31. Generally, type-specific HPV seroconversion was higher, and time to seroconversion was shorter, after prevalent infection than after incident infection. Among men with incident genital HPV infection, report of ever smoking and genital site(s) infected were statistically significantly associated with seroconversion.
This is the first longitudinal study of HPV seroconversion in HPV infected men, and the low rates of seroconversion are consistent with the HPV-16 seroprevalence literature. In a study population representative of the general US population (2003-4 National Health and Nutrition Examination Survey (NHANES)), HPV-16 seroprevalence was 0.3% in males 20–24 years of age . In sexually transmitted disease (STD) clinic populations, male HPV-16 seroprevalence tends to be higher [9, 11, 13]. Among men recruited by multiple methods in 2 US cities (Tucson, Arizona, and Tampa, Florida), HPV-16 seroprevalence was approximately 5% in 18- to 24-year-olds . The seroprevalence observed in this study cannot be directly compared with most seroprevalence studies, because it is only among men with an observed genital α9 HPV infection. In 1 cross-sectional study of STD clinic patients in Demark and Greenland, HPV-16 seropositivity was observed among 20% of Danish men and 0% of Greenlandic men with prevalent genital HPV-16 infection .
There have been few investigations of type-specific HPV seroprevalence for types other than HPV-16, perhaps partially because of the assays used. Unlike the enzyme-linked immunoassay (ELISA) used in most seroprevalence studies thus far, LBMA is a multiplex assay that allows testing of multiple types. The level of concordance between the ELISA and LBMA assay is high . Using the LBMA assay on specimens from the general German population, seroprevalences for HPV-16, -33, -52, and -58 in males aged 15–24 years were all between 1% and 4%. HPV-31 and -67 were not included, and these estimates were not stratified by HPV infection status.
The gender difference observed in HPV-16 seroprevalence [7, 9–11, 13, 24] appears to extend to seroconversion among HPV-16 infected individuals. In this study of young men, HPV-16 seroconversion estimates 24 months after prevalent or incident genital HPV-16 infection (20% and 7%, respectively) were much lower than the published estimates of a female cohort of University of Washington students (94% and 67%, respectively) . It is possible that some of the difference between these studies is because of different serology assays (LBMA for males and ELISA for females). Using the LBMA assay in a more recent cohort of female university students (described in Winer et al ), cumulative HPV-16 seroconversion at 24 months among women with incident HPV-16 infections was 58% (authors’ unpublished data). It is not known why females might be more likely to seroconvert than males. Possible contributors to a lower seroconversion rate in men include a higher ratio of infections in more-keratinized (eg, penile shaft) versus less well-keratinized epithelium (eg, glans) [1, 5, 26, 27], lower HPV viral load , or lower likelihood of persistent infection [29–32]. It is also possible that because we are comparing across studies, the apparent difference between young males and females in seroconversion could be, in part, due to differences in seroconversion risk factors. However, regarding the nonanatomical factor associated with seroconversion in this study, females in the recent cohort of university students were only slightly more likely to be current smokers .
It should be noted that the consequences of seroconversion are not clear. Whereas the high levels of anti-HPV antibodies achieved following HPV vaccination prevent infection in both males and females [34, 35], the considerably lower anti-HPV titers achieved after natural infection appear to protect only partially against reinfection or redetection of the same HPV type . In this study, it is unknown whether the HPV infections detected after seroconversion were new infections of the same HPV type, reactivations, or prevalent infections that were missed at earlier visits.
A lower likelihood of seropersistence has been observed in males than in females , and in our study population, only 62% of seroconversions were followed by another seropositive visit. Lower seropersistence may make protection against subsequent type-specific infection less likely in men than in women. Interestingly, the median time to seroconversion following incident infection was shorter than that which has been observed in women [37, 38]. It is unlikely that this result is related to visit schedule (every 4 months vs every 6 months) because 1 of the studies of women  also used a 4-month visit schedule. Because of the small number of seroconversions observed in men, chance must be considered a possible explanation.
The association between ever cigarette smoking and seroconversion among men with incident HPV infection was strong and statistically significant when adjusted for genital site(s) of infection. An association between HPV seroprevalence in males and cigarette smoking has been found in some previous studies [23, 36], but not others [7, 8, 10]. The relationship between seroconversion and cigarette smoking seems inconsistent with the prevailing theory that cigarette smoking weakens the local immune system, which may leave a person more vulnerable to persistent HPV infection and, thus, HPV-related cancer [31, 39, 40]. Conversely, it is likely that persistent infection, at least initially, results in more virus production , thus more L1 antigen to induce an antibody response.
Seroprevalence studies among males have shown associations with sexual-history characteristics, though which characteristic(s) varies by study. For example, lifetime number of partners was associated with seroprevalence in some studies [7, 8, 11, 23] and not others [10, 13, 36], and the same is true of recent number of partners [13, 23]. Although these characteristics and other sexual history characteristics showed some evidence of an association with seroconversion after incident genital HPV infection in this study, they did not approach statistical significance. A sexual history characteristic that is consistently associated with seroprevalence in males is sex with males [7, 8, 36, 42]. We were not able to examine this characteristic in our heterosexual population.
In this study, we were able to examine infection characteristics in males, which have not been assessed in seroprevalence investigations. Seroconversion after incident genital infection with an α9 HPV type was associated with the genital site(s) where HPV was incidentally detected. A reason cited for lower seropositivity in males compared with that in females is the difference in the infected tissue type, with males less likely than females to be infected in minimally keratinized genital sites. However, in this study, none of the men who seroconverted were infected only in the less well-keratinized genital sites of the glans or urethral meatus. Men who were infected in the glans or urethral meatus as well as the more highly keratinized epithelium of the shaft or scrotum were most likely to seroconvert. This suggests that a higher viral load or infection of a larger surface area may be important predictors of seroconversion in men. Our study did not find a relationship between type-specific seroconversion and repeated detection of the same type of HPV DNA at 2 or more visits. In females, there is some evidence of an association between repeated detection of HPV DNA and seroconversion [38, 43], though not when examined as a time-varying covariate .
This is the first longitudinal study of seroconversion following α9 HPV infection in young men, but it was not without limitations. It was a relatively small study and, thus, larger studies are needed to verify our results. Also, given that male genital HPV infections tend to have low viral loads, it is possible that infections could have been missed, even though our DNA assay was sensitive and our specimen collection method was thorough. This could also lead to misclassification of HPV DNA detection as incident, as well as a bias toward a study population with higher viral loads. Additionally, this study did not measure HPV infection at nongenital sites, which, if present, could have resulted in seropositivity (eg, the oropharynx ). Although the LBMA assay has been used successfully in other investigations, standards for HPV types other than HPV-16 are not currently available. Lastly, the results of this study may not be generalizable to older men, men who have sex with men (MSM), or high-risk populations.
In summary, this study showed that detectable type-specific HPV seroconversion within 2 years of a genital type-specific α9 HPV infection was very low in young men. This suggests that seropositivity is an even less sensitive marker of prior HPV infection in men than it is in women. Among men with incident genital HPV infections, seroconversion was associated with cigarette smoking and genital site(s) infected. Further investigation into why α9 HPV infections appear to be poorly immunogenic in men is warranted.
Funding for this study was provided by the National Institutes of Health (R01 CA105181 to L. A. K. and R37 AI038382 to D. A. G.).
The authors would like to thank Michael Stern and Sandra O’Reilly for the excellent clinical care they provided to the participants in this study, as well as Shu-Kuang Lee for her skilled data management.