Human Papillomavirus Seroprevalence and Association with Anal HPV Infection and Squamous Intraepithelial Lesions in Australian Gay and Bisexual Men

More than two decades of study of the natural history of human papillomavirus (HPV) in the cervix has shown that persistent high-risk HPV (HRHPV) infection is the precursor to precancerous high-grade squamous intraepithelial lesions (HSIL). If left untreated, some HSIL will eventually progress to invasive cancer, at a rate of around 1% per year (1). It is generally accepted that a broadly similar natural history applies to HPV infection at all sites in the lower anogenital tract (2). However, in gay and bisexual men (GBM), the prevalence of anal HPV infection is substantially higher than that of HPV infection of the cervix in women. In fact, even though GBM experience very high rates of anal cancer, the prevalence of anal HRHPV and associated HSIL is so high in GBM that it is clear that many, if not most infections must clear or never progress to cancer (3). Thus, it is evident that persistence, regression, and progression of anal HPV-related disease are not fully analogous to cervical HPV–related disease.

A better understanding of the immune response to anal HPV is likely to improve our understanding of the natural history of HPV at this site. Type-specific serum antibodies against the late protein (L1) of the viral capsid and the early HPV oncoproteins (E6 and E7) may be produced after infection. Infection-induced antibodies against HPV L1 are considered markers of cumulative exposure to the virus, representing past and present infection. Antibodies targeting E6 and E7 proteins are potential markers for HPV-associated cancer (4, 5). Seropositivity for HPV16 E6 antibodies has been shown to be a highly sensitive and specific marker HPV-driven oropharyngeal and anal cancer but is less common in cervical cancer (6–8). As yet, there have been no studies published reporting the association of HPV16 E6 and E7 seropositivity with anal HSIL.

We aimed to characterize the relationship between the serologic response to HRHPV L1, E6, and E7 and prevalent anal HPV infections and anal HSIL in a community-based cohort of Australian GBM.

The Study of the Prevention of Anal Cancer (SPANC) is a prospective cohort study exploring the epidemiology of anal HPV infection and related cytologic and histologic anal abnormalities in a community-recruited cohort of GBM (9). It is an ongoing study based in Sydney, Australia, involving HIV-negative and HIV-positive GBM ages 35 years and older. Participants were predominantly men recruited from gay community social events and organizations, referrals from other participants, or from previous community-based studies conducted by the Kirby Institute. Participants attend six visits over a period of three years, including a baseline enrollment visit, four follow-up clinical visits, and a final visit to discuss all study results. At each visit, an anal swab is collected for cytology screening and for HPV DNA genotyping using the Roche Linear array (Roche Molecular Systems), to determine the presence of 37 HPV genotypes. Serum samples are collected annually. A high resolution anoscopy (HRA) is performed and any potential HPV-related abnormalities are biopsied. The baseline data collection commenced in September 2010 and the study was fully recruited by August 2015. Follow-up of participants will continue to early 2018.

This article reports the serologic results from the baseline serum samples from SPANC participants who provided informed consent to storage and testing of their sera. Serum samples were analyzed at the German Cancer Research Center (DFKZ), Heidelberg, Germany, for L1, E6, and E7 antibodies to 10 mucosal HPV types: HPV6, 11, 16, 18, 31, 33, 35, 45, 52, and 58. Samples were analyzed using multiplex serology, an antibody detection method based on glutathione S-transferase (GST) capture ELISA, in combination with fluorescent bead technology (10). Briefly, full-length viral proteins are expressed in Escherichia coli bacteria in fusion with an N-terminal GST domain. Glutathione cross-linked to casein is coupled to fluorescence-labeled polystyrene beads (SeroMap, Luminex Corp.) and GST fusion proteins are affinity purified on the beads. Each fusion protein is bound to a spectrally distinct bead set, and fusion protein–loaded bead sets are mixed. Sera are incubated with the mixed bead sets at a final dilution of 1:100, and bound antibodies are detected with biotinylated goat anti-human IgG secondary antibody and streptavidin-R-phycoerythrin. A Luminex analyser (Luminex Corp) is used to identify the internal color of the individual beads and to quantify their reporter fluorescence [expressed as median fluorescence intensity (MFI) of at least 100 beads per set per serum]. Glutathione-casein–coupled bead sets are loaded with their respective antigens in one batch. Autofluorescence of each bead set and background reactions resulting from binding of secondary reagents to the antigen-loaded beads are determined in one well per plate without human serum. Mean bead background values are subtracted from raw MFI values and antigen-specific reactivity is determined by subtraction of the MFI of GST alone from the MFI of the specific antigen. MFI values were dichotomized as antibody positive or negative, using previously established seropositivity cutoffs that were calculated independently for each HPV antigen (10, 11). In this report, HRHPV includes HPV16, 18, 31, 33, 35, 45, 52, and 58.

Type-specific HPV seroprevalence was calculated and the exact binomial method was used to calculate 95% confidence intervals (CIs). ORs with corresponding 95% CIs were calculated for seroprevalent HPV type–specific associations with age group and HIV status. Among HIV-positive participants, the association between reported current and nadir CD4 T-cell count and seropositivity was analyzed. Univariate and multivariate logistic regressions were performed to determine the association between type-specific anal HPV DNA detection, anal low-grade squamous intraepithelial lesions (LSIL), HSIL, and HPV L1 capsid seropositivity at baseline. Composite “normal,” LSIL, and HSIL endpoints were used, comprising cytologic and/or histologic LSIL and HSIL diagnoses (12). Composite normal included normal cytology, normal histology, and/or biopsies not taken due to normal HRA appearances. Composite LSIL included histologic LSIL and cytologic LSIL and atypical squamous cells of undetermined significance (ASC-US). Composite HSIL included histologic HSIL and cytologic HSIL and atypical squamous cells—cannot exclude HSIL (ASC-H; ref. 12). HSIL detection was analyzed by pooled HRHPV serostatus (for the 8 included HRHPV types) overall and then stratified by anal HRHPV DNA detection. The association between HPV E6 and E7 seropositivity and anal HSIL was analyzed by logistic regression. Data analyses were performed using STATA version 14 (Stata Corporation).

Ethics approval for the study was granted by the St. Vincent's Hospital (Sydney, Australia), Australia Human Research Ethics Committee (file number 09/203). Written informed consent was obtained from all individuals before any study-specific procedures were performed. Separate, optional written informed consent was obtained for collection and storage of serum for future testing, including for HPV serology and data linkage studies.

A total of 617 men were enrolled in SPANC, with a median age of 49 years (range 35–79 years). Nearly all men (588, 95.3%) identified as gay or homosexual and 17 (2.9%) identified as bisexual. More than one-third (220, 35.7%) were HIV-positive. The great majority of these men (206, 93.6%) were currently receiving antiretroviral therapy, reported a viral load of less than 50 copies/mL (197, 89.5%) and had a CD4 T-cell count of more than 350 cells/μL (194, 88.0%). Of the 606 men with adequate anal samples, 525 (86.6%) had at least one anal HPV type detected by DNA genotyping. Among these, a single HPV type was detected in 101 participants (16.7%), while the rest had multiple HPV types detected. The prevalence of any anal HPV was 94.5% (207) in HIV-positive men and 82.2% (318) in HIV-negative men (P < 0.001). HPV16 was the most common HPV type (178, 29.4%), followed by HPV6 (109, 18.0%) and HPV45 (84, 13.9%; ref. 13).

Of the 617 men enrolled, 603 consented to storage and additional testing of collected specimens and had adequate serum samples for antibody analyses. Fifteen participants reported receiving prior HPV vaccination, four in the past year and 11 more than a year ago. Vaccinated participants had mean HPV16 L1 MFIs of 3,205 (vaccine in the past year) and 1,540 (more than a year ago), compared with a mean MFI of 569 for participants who reported never receiving HPV vaccination. These 15 participants were excluded from further analyses on L1 because their immune response was likely to be vaccine-induced rather than infection-induced.

Of the remaining 588, 436 (74.2%) were seropositive for at least one of the 10 HPV types analyzed. The seroprevalence for each HPV type is presented in Table 1. Almost half of the participants had L1 antibodies to HPV6 (48.5%) and over a third had L1 antibodies to HPV11 (36.4%) and HPV16 (34.5%). There was no significant association between increasing age and seropositivity. In fact, for every HPV type and for HPV overall, seroprevalence was numerically lower in men aged 65 and above than in younger men, but did not reach statistical significance for any HPV type. HIV-positive men were more likely to be L1 seropositive for each specific type, and this was significant for six of the 10 types (HPV16, P < 0.001; HPV18, P = 0.005; HPV31, P = 0.005; HPV35, P = 0.012; HPV45, P = 0.001 and HPV52, P = 0.017). HIV-positive men reporting a nadir CD4 T-cell count of less than 200 cells/μL were more likely to be HPV16 (P = 0.044) and HPV18 seropositive (P = 0.028). HIV-positive men with a higher current CD4 T-cell count were more likely to be seropositive for HPV45 (P = 0.042). This association was not seen with other HPV types.

The relationship between HPV L1 serology and DNA detection is shown in Table 2. Approximately 50% of participants were both L1 seronegative and DNA negative for each of HPV6 and HPV 16. For all other HPV types, more than 60% were L1 seronegative and anal HPV DNA negative (Table 2). In general, for each HPV type, more men who were L1 seropositive were also DNA positive. The exception was HPV45, for which 13% of the seropositive and 14% of the seronegative were DNA positive. For HPV16, anal HPV16 DNA was detected in 86 (22.8%) of the seronegative and 85 (42.7%) of the seropositive.

The relationships between HPV DNA and serologic positivity with anal HPV-associated disease are presented in Table 3. The four categories examined were: double negatives, serology positive/DNA negative, serology negative/DNA positive, and double positives. For most HPV types, HSIL detection increased across these four categories. Compared with men who were HPV16 seronegative/DNA negative, participants who were HPV16 seronegative/DNA positive were 4 fold more likely (OR = 4.26; 95% CI, 2.23–8.12) to have HSIL detected, whereas participants who were HPV16 seropositive/DNA positive were almost 13-fold more likely (OR = 12.93; 95% CI, 4.99–33.48) to have HSIL detected (Table 3).

Men who were L1 seropositive for at least one HRHPV type were significantly more likely to have HSIL detected (P = 0.032) than men who were seronegative to all. Among men with detectable HRHPV DNA, there was a nonsignificant trend toward more HSIL being detected among seropositive than seronegative participants (P = 0.088). There was no difference in HSIL detection by serostatus in participants with no detectable HRHPV DNA (P = 0.349; Table 4).

The association of E6 and E7 antibodies with HSIL is presented in Table 5. The association of HPV16E6 seropositivity with composite HSIL approached statistical significance (OR = 2.97; 95% CI, 0.92–9.60; P = 0.068). Of the 14 men who had composite HSIL and tested HPV16 E6 positive, three participants were also positive for E7 (14). One participant was HPV33 E6 and E7 double seropositive. He had possible LSIL on cytology and normal histology. No participant was HPV18, 31, 35, 45, 52, or 58 E6 and E7 double seropositive.

In this cohort of GBM, three quarters of men had L1 antibodies to at least one HPV type, and HPV6, HPV11, and HPV16 L1 antibodies were most commonly detected. HIV-positive men were more likely to be HPV L1 seropositive, and a nadir CD4 T-cell count of less than 200 cells/μL was predictive of HPV16 and HPV18 L1 seropositivity. Type-specific anal DNA was more prevalent in men who were L1 seropositive for the same HPV type. For most HRHPV types, HSIL detection was highest among participants who were both HPV serology and DNA positive. The presence or absence of HPV L1 antibodies was not significantly associated with HSIL among participants who were anal HPV DNA negative. There was a borderline significant increased risk of HSIL in those who were HPV16 E6 seropositive.

The high rates of HPV L1 seroprevalence we have found are comparable with findings from other serologic studies involving GBM. HPV L1 seroprevalence ranged from 31% to 86% of participants in these studies (15–21). In a Dutch population-based cross-sectional survey, men who reported sexual attraction to men had an HPV16 L1 seroprevalence of 31.4% compared with 16.8% among heterosexual men (22). Similarly, in U.S. cross-sectional national surveys, HPV16 L1 seroprevalence was 37.7% among men who reported ever having had sex with a man (23), and was 30% among 169 Austrian HIV-positive men, 59 of whom were homosexual (24).

Inconsistent relationships of HPV L1 antibody status with age have been described. Increasing HPV seroprevalence up to age 35 years was described in the multinational HIM study (18, 25). In the Amsterdam-based H2M study, seroprevalence increased across the three age groups of less than 35, 35–44, and 45 and above. The HYPER study, which enrolled 200 GBM aged between 16 and 20 years in Melbourne, Australia found very low seroprevalence (3.0% for HPV18 to 12.5% for HPV6) for the quadrivalent vaccine HPV types (26). In a San Francisco study of HIV-positive MSM, no association with age was found. Other studies group men into age group inclusive of 45–70 years or older than 45 years (15, 25) or include separate groups of less than 10 men older than 60 (17) and thus cannot identify whether a decline occurs at ages older than 60 years. In SPANC, HPV seroprevalence was nonsignificantly lower in men ages 65 years and older.

HPV L1 seroprevalence in most studies of GBM is more common among HIV-positive men. In a GBM population recruited from a sexual health clinic in the Amsterdam-based H2M study, HIV-positive men were more likely than HIV-negative men to be HPV16 seropositive (65% vs. 31%; OR = 4.05; 95% CI, 2.84–5.77; ref. 27). This is a larger difference than seen in SPANC (44% vs. 30%) despite both studies reporting higher numbers of lifetime and recent sexual partners among HIV-positive participants (9, 27).

HPV16 L1 seroprevalence was associated with concurrent detection of HPV16 DNA in the anal canal. Although L1 seropositivity is believed to reflect lifetime history of HPV infection, in our study, type-specific seropositivity was associated with current infection and with current disease (HSIL). This suggests that HPV seropositivity may predict persistent infection, but this requires confirmation in longitudinal data. These results correlate with findings on HPV infection in the vagina in women. The HELIUS study from the Netherland reported that in adjusted analyses, vaginal HPV DNA detection was significantly associated with type-specific HPV seropositivity (28).

The SPANC study is the first study to report on the relationship between HPV seropositivity and HPV-associated noninvasive anal disease. We found that HPV16 L1 seroprevalence was a strong predictor of concurrent HSIL. Compared with men who were HPV16 seronegative/DNA negative, participants who were HPV16 seronegative/DNA positive were 4-fold more likely to have HSIL detected, whereas participants who were HPV16 seropositive/DNA positive were 13-fold more likely to have HSIL detected. Thus, HPV16 seropositivity added incrementally to the likelihood of HSIL detection in participants who were anal HPV16 DNA positive. In addition, men who were seropositive for antibodies against at least one HRHPV type were significantly more likely to have HSIL detected (P = 0.018) than HRHPV seronegative men.

Previous studies have clearly demonstrated that HPV16 E6 seropositivity is associated with anal cancer. A nested case–control study within the European Prospective Investigation into Cancer and Nutrition (EPIC) found that 7 of 24 (29%) people who subsequently developed anal cancer and 0.6% of controls (4 of 718) were HPV16 E6 seropositive, translating to a 75-fold increase in cancer risk (7). In a case–control study nested in the Swiss HIV Cohort antibodies against HPV16 L1 (OR = 4.5) and E6 (OR = ∞; 95% CI, 4.6–∞) were strongly associated with anal cancer. In that study, antibodies against HPV16 E6 were 100% specific, in that the prevalence was zero in people without anal cancer, but were a relatively insensitive marker for anal cancer (29). HPV16 E6 seropositivity has been shown to be rare in cancer-free populations (30). A pooled analysis of almost 5,000 controls from cancer studies found that only 32 people (0.7%) were HPV16 E6 seropositive (31). The prevalence of HPV16 E6 antibodies in SPANC participants, presumed to be cancer-free on enrollment, was 2.3%, slightly higher than in the other cancer-free populations described above. However, there are few data on HPV16 E6 seropositivity in populations of homosexual men (32). The association between HPV16 E6 seropositivity and HSIL that we found was of borderline significance, with a 3-fold increase in the likelihood of anal HSIL (OR, 2.97; 95% CI, 0.92–9.60). There have been no previous studies of the association between HPV E6 antibodies and anal HSIL. Although the seroprevalence of HPV16 E6 is low (4% in participants with HSIL), the association should be explored further, to determine whether HPV16 E6 serology has a role as a biomarker in an anal cancer screening program.

The SPANC study is a large prospective study and one of the few studies globally to perform anal cytology, HRA screening, and HPV genotyping at the same visit on all participants. Serologic results were available for the vast majority of participants. Recruitment for the study was mostly from community-based settings, with broad inclusion criteria and thus we believe that results are likely to be generalizable to the GBM population in Australia.

There were several limitations in this research. Differences in serologic methods and variations between cut-off definitions for positivity between laboratories limit direct comparisons with other studies. The results presented are cross sectional and thus we cannot determine whether HPV anal infections or anal HSIL are acute or persistent or whether the serologic response is related to past or current infection with a specific HPV type. This will also be examined in subsequent analyses of SPANC longitudinal data. In addition, multiple HPV types were often present and it was not possible to conclusively attribute a HPV type based on a cytology specimen to an HSIL lesion.

In summary, HPV L1 seropositivity was common in this cohort of GBM, particularly among HIV-positive men. Concurrent detection of type-specific anal DNA was twice as common in men who were L1 seropositive for the same type and HSIL detection was highest among participants who were both HPV serology and DNA positive. These results suggest that HPV L1 seropositivity, in conjunction with anal HPV DNA, predicts concurrent HSIL and could be evaluated as part of an algorithm for current HSIL risk. The apparent association between HPV16 E6 antibodies and prevalent HSIL is a finding with potential clinical significance that needs further exploration.

A. Cornall has received speakers bureau honoraria from Seqirus (formerly bioCSL). S.M. Garland reports receiving commercial research grants from Merck, Glaxo Smith Kline, CSL, and the Commonwealth Department of Health; has received speakers bureau honoraria from MSD and SPMSD; and is a consultant/advisory board member for Merck. A.E. Grulich reports receiving a commercial research grant from Gilead as well as other commercial research support from Viiv and Seqirus; has received speakers bureau honoraria from Merck; and is a consultant/advisory board member for Viiv. No potential conflicts of interest were disclosed by the other authors.

The views expressed in this publication do not necessarily represent the position of the Australian Government.

Conception and design: F. Jin, R.J. Hillman, C.K. Fairley, A.E. Grulich

Development of methodology: T. Waterboer, F. Jin, R.J. Hillman, S. Tabrizi, A.E. Grulich

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): I.M. Poynten, T. Waterboer, D.J. Templeton, R.J. Hillman, C. Law, A. Cornall, S. Tabrizi, J.M. Roberts

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): I.M. Poynten, T. Waterboer, F. Jin, D.J. Templeton, S. Tabrizi, J.M. Roberts, S.M. Garland, A.E. Grulich

Writing, review, and/or revision of the manuscript: I.M. Poynten, T. Waterboer, F. Jin, D.J. Templeton, R.J. Hillman, A. Cornall, S. Tabrizi, J.M. Roberts, S.M. Garland, C.K. Fairley, A.E. Grulich

Study supervision: A.E. Grulich

This research is supported by a National Health and Medical Research Council Program New Investigator Grant (to I.M. Poynten). The SPANC study is funded by a National Health and Medical Research Council Program Grant (Sexually Transmitted Infections: Causes, Consequences and Interventions Grant #568971; to A.E. Grulich and C.K. Fairley) and a Cancer Council New South Wales Strategic Research Partnership Program Grant (Preventing Morbidity and Mortality from Anal Cancer Grant #13-11; I.M. Poynten, F. Jin, R.J. Hillman, D.J. Templeton, S. Tabrizi, S.M. Garland, AF, C.K. Fairley). Cytologic testing materials were provided by Hologic (Australia) Pty Ltd. The Kirby Institute is affiliated with the Faculty of Medicine, University of New South Wales and funded by the Australian Government of Health and Ageing.

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