Abstract
Naturally induced serum antibodies against HPV may affect risks of subsequent incident genital infections by HPV 6, 11, 16, or 18 in men. In this study, we examined the hypothesis by following 4,123 healthy men every six months(median follow-up time 4.1 years). HPV antibodies were measured at baseline using a virus-like particle-based ELISA assay. Genital HPV genotypes were detected using Roche Linear Array. Incidence proportions and six-month persistence proportions were calculated at six-month intervals. Kaplan-Meier curves and Cox models were used to assess genotype-specific cumulative incidence and hazard ratios (HR), respectively. HPV 6, 11, 16, and 18 seroprevalence was 8.1%, 13.9%, 12.7%, and 10.8%, respectively. Significantly higher rates of incident infections were observed for HPV 16 among baseline seropositive men (adjusted HR 1.37, 95% CI 1.01–1.86), with similar but non-significant HRs for six-month persistent infections. Risk of persistent HPV 18 infection was significantly lower among seropositive men in the unadjusted model (HR 0.22, 95% CI 0.06–0.91), but not in the adjusted model (HR 0.19, 95% CI 0.03–1.37). Incident and six-month persistent infections for HPV 6 and 11 did not differ by baseline serostatus. Baseline serostatus among men was not associated with a reduction in subsequent incident genital HPV 6, 11, and 16 infections. However, protection against persistent HPV 18 infections was observed in unadjusted models. Our research suggests a need of further studies to examine the potentially protective effects of naturally induced HPV18 antibodies in men.
Keywords: human papillomavirus (HPV), serum antibodies, incident infection, persistent infections, HIM Study, anti-HPV antibodies
Introduction
Genital HPV prevalence among men exceeds 70% in some regions of the world (1), with HPV DNA detected in 29–82% of penile cancers (2, 3) and 80–100% of genital warts (4, 5). Furthermore, nearly 10,000 new cases of HPV-related oropharyngeal cancers among men are diagnosed in the U.S. each year (6). Although the antibodies produced following HPV vaccination among men provides protection against future ano-genital HPV infections and related diseases (7), it is unclear whether the antibodies produced after natural HPV infection are sufficient to protect against subsequent infection in men.
Among women, antibodies produced in response to natural HPV infection are markers of past infections and have been shown to provide partial immunity against subsequent infections and precancerous lesions (8–10); however, not all studies observed these protective effects (11–13). Differences in study findings may be due to the use of different antibody assays, serum antibody levels, and time since first exposure to HPV (14). Furthermore, the VLP based assay and reagents used in two prior studies (11, 12) were in early stages of investigation to assess the role of naturally acquired antibodies for immunity against subsequent HPV infections. A prospective study of HPV infection among men in Arizona did not show protective effects of circulating HPV antibodies (15). However, this study was limited by a short follow-up time, small sample size, and lack of a quantitative serum antibody assessment. An initial study of 2,187 participants in the multinational HPV Infection in Men (HIM) Study also did not show an association between serum antibodies and reduction in subsequent HPV 16 infections (14). However, this study was limited to only one HPV type with a median duration of two years follow-up. A recent study among HIV-negative and HIV-positive men also did not show protective effects against subsequent HPV infection for multiple HPV types, but the study was restricted to men who have sex with men (MSM) (16).
In the current study, we provide the first comprehensive evaluation of incident genital HPV 6, 11, 16, 18 (any duration infection and six-month persistent infections) by baseline antibody status among the entire HIM Study cohort (n=4,123) followed for a median 4.1 years.
Materials and Methods
Study Population
The HIM Study is an ongoing multinational study of the natural history of HPV among men in Tampa, Florida (U.S.), São Paulo (Brazil), and Cuernavaca (Mexico). Details of this study have been described previously (17). Briefly, healthy men were enrolled at each study site and followed for a median follow-up of 4.1 years, with an average interval of 6.9 months between visits. Men were eligible for the study if they: a) were 18–70 years of age; b) were residents of one of the study sites; c) had no previous diagnosis of penile or anal cancers; d) had never been diagnosed with genital or anal warts; e) had no symptoms of a sexually transmitted infection (STI) and were not receiving treatment for an STI; f) were not participating in an HPV vaccine study; g) had no history of HIV or AIDS; h) had no history of imprisonment, homelessness, or drug treatment during the past six months; and i) were willing to comply with 10 scheduled visits every six months for four years with no plans to relocate during that time. Extensive sexual history and health questionnaires were administered using computer-assisted self-interviewing (CASI) at baseline and at each follow-up visit. All eligible participants signed an informed consent, and approval was obtained from the human subjects committees of the University of South Florida (Tampa, FL), Ludwig Institute for Cancer Research (São Paulo, Brazil), Centro de Referencia e Treinamento em Doencas Sexualmente Transmissíveis e AIDS (São Paulo, Brazil), and Instituto Nacional de Salud Publica de Mexico (Cuernavaca, Mexico). After excluding subjects who reported never being sexually active (n=235) and subjects who received at least one dose of an HPV vaccine (n=27), a total of 4,123 HIM Study participants were eligible for inclusion in this study.
Baseline serum antibody testing
A 10 milliliter venous blood sample was collected at baseline to measure serum antibodies against four HPV genotypes (HPV 6, 11, 16, and 18). Serum anti-HPV antibody assessments for HPV genotypes (6, 11, 16, and 18) were carried out using a virus-like particle (VLP)-based enzyme-linked immunosorbent assay (ELISA) (18). Details of this assay have been described previously (19). Briefly, insect cells from recombinant baculoviruses expressing HPV L1 capsid proteins were used to produce HPV VLPs. Absorbance values in optical density (OD) were measured to assess seroreactivity. Seroreactivity from children (1–10 years old) was used as a negative control and to estimate the mean and standard deviation (SD) of the absorbance value for an antibody positive specimen. The cut-off point for seropositivity for each HPV genotype was selected as five SD above the mean absorbance value among the children. Positive and negative laboratory serum controls were used in each assay run for quality control purposes. Laboratory staffs were blinded to participants’ HPV DNA status.
External genital HPV DNA samples
Three prewetted Dacron swabs were used to collect genital cell specimens from the coronal sulcus/glans penis, penile shaft, and scrotum and were later combined to form a single sample (17, 20). All specimens were stored at −80°C until genotyping was conducted. The QIAamp DNA Blood Mini Kit (QIAGEN) was used to extract DNA from genital cell specimens. Roche Linear Array kits were used for PCR and HPV DNA genotyping to detect 37 different types of HPV (21). The presence of human β-globin was used to assess specimen adequacy with an overall β-globin positivity of >98%.
Statistical analysis
Demographic characteristics were compared between seronegative and seropositive men at baseline using Chi-square tests with Monte Carlo estimation of exact p-values. Only men who were DNA-negative for the respective HPV type at the baseline visit were included in analyses. Separate analyses were carried out for each of the four HPV genotypes (HPV 6, 11, 16 and 18) by duration of infection (any duration for incident infections and >6 months duration for persistent infection).
Incidence proportions (all incident HPV 6, 11, 16, and 18 infections regardless of duration) and six-month persistent incidence proportions were calculated at each six-month interval among subjects who had one or more follow-up visits after baseline. Follow-up times were calculated based on visit dates and participants who were late for their visit were included in the appropriate time interval for the analysis. To estimate incidence proportions, the first detection of HPV DNA at a follow-up visit was defined as an incident HPV infection (numerator). The number of subjects who tested negative for HPV DNA at the beginning of each study interval was defined as the at-risk population (denominator). For six-month persistent infections, a persistent infection was defined as HPV DNA detection at two or more consecutive visits. Persistent infections also included subjects with an intervening negative result (n= 20 for HPV 6, n=5 for HPV 11, n=22 for HPV 16 and n= 11 for HPV 18). Participants who were DNA- negative for the given HPV type (at risk of acquiring a new infection) at the beginning of each study interval were defined as at-risk subjects for six-month persistent infection.
Kaplan-Meier (KM) curves were constructed for incident and six-month persistent infections for each of the four HPV genotypes (6, 11, 16 and 18). Cumulative incidence for each genotype was compared between seropositive and seronegative subjects, with the log-rank test used to determine significant differences by serum antibody status. Cox proportional hazard models were used to calculate crude and adjusted hazard ratios (aHR) and 95% confidence intervals (CI). A list of candidate variables was created based on descriptive analyses, previous literature, and assessment of confounding by each variable. Variables were evaluated in both backward and forward stepwise models. Final models included variables based on best-fit approach based on the lowest Akaike information criterion (AIC) values. As sexual behavior prior to acquisition of incident infections strongly influences HPV acquisition, we included the following sexual behavior variables as time-varying covariates in final adjusted Cox models: lifetime number of female sexual partners, number of new female sexual partners in the past 6–12 months, frequency of sexual intercourse with female partners in the past 6–12 months, lifetime number of male sexual partners, and number of new male sexual partners in the past 6–12 months. Due to the differences in HPV seroprevalence by sexual orientation, stratified analyses were also conducted. To evaluate whether antibody levels were associated with subsequent detection of incident infections, we categorized serum antibody levels into the highest tertile and lowest two tertiles and compared these two groups to seronegative men (9, 16).
Results
Serum antibody and HPV genotyping data were available for 4,103 subjects at baseline. We excluded 250 (HPV 6), 55 (HPV 11), 311(HPV 16) and 90 (HPV 18) subjects who were DNA-positive at the baseline visit for the respective HPV type. Therefore, baseline seroprevalence analyses included 3,851 (HPV 6), 4,046 (HPV 11), 3,790 (HPV 16), and 4,011 (HPV 18) subjects who were DNA-negative for the specific HPV genotype. Among the excluded subjects with prevalent HPV 6, 11, 16 and 18 infections, baseline seroprevalence was 13.6%, 25.5%, 18.6%, and 12.2%, respectively. Among these men positive for a genital HPV infection at baseline, those who cleared their infections did not differ from those who reacquired infection with the same type with respect to baseline serum antibody status for HPV 6 (chi-square p-value= 0.626), HPV 11 (p=0.468), HPV 16 (p=0.457), and HPV 18 (p=0.448).
Overall, baseline HPV6, 11, 16, and 18 seroprevalence among eligible participants was 8.1%, 13.9%, 12.7%, and 10.8%, respectively. Significant differences were observed between seropositive and seronegative men for various covariates (Table 1). Seroprevalence increased with increasing age, with the highest seroprevalence for HPV 6 (9.4%), 11 (16.2%), and 16 (16.4%) observed among men ages 31–44, and the highest HPV 18 seroprevalence (17.9%) among men ages 45–70. When we compared seroprevalence across the three age categories, we found no differences between the two older age categories (31–44 and 45–70) for HPV 6, 11, and 16; however, HPV 18 seroprevalence was significantly higher among the oldest age group. Brazil had the highest seroprevalence for HPV 6 (10.8%), 16 (16.9%), and 18 (13.0%), while Mexico had the highest seroprevalence for HPV 11 (18.8%). Men who have sex with men (MSM) had the highest seroprevalence for all four genotypes (HPV 6: 23.2%, HPV 11: 27.6%, HPV 16: 28.6%, and HPV 18: 29.3%). Seroprevalence was highest among men reporting ≥10 lifetime male sexual partners (30.0%, 37.8%, 39.0%, and 40.4% for HPV 6, 11, 16, and 18 respectively).
Table 1.
Demographic characteristics of seronegative and HPV 6, 11, 16 and 18 seropositive HIM Study participants
| HPV 6 | HPV 11 | HPV 16 | HPV 18 | |||||
|---|---|---|---|---|---|---|---|---|
|
| ||||||||
| Characteristics | Seronegative | Seropositive | Seronegative | Seropositive | Seronegative | Seropositive | Seronegative | Seropositive |
| Country | ||||||||
| United States | 1216 (96.4) | 46 (3.6) | 1249 (94.0) | 80 (6.0) | 1086 (88.6) | 140 (11.4) | 1209 (93.2) | 88 (6.8) |
| Brazil | 1173 (89.2) | 142 (10.8) | 1155 (83.1) | 235 (16.9) | 1047 (81.7) | 234 (18.3) | 1199 (87.0) | 179 (13.0) |
| Mexico | 1149 (90.2) | 125 (9.8) | 1077 (81.2) | 250 (18.8) | 1176 (91.7) | 107 (8.3) | 1170 (87.6) | 166 (12.4) |
| p valuea | <0.0001 | <0.0001 | <0.0001 | <0.0001 | ||||
| Age | ||||||||
| 18–30 | 1723 (93.1) | 127 (6.9) | 1740 (88.2) | 232 (11.8) | 1655 (90.9) | 165 (9.1) | 1786 (91.8) | 159 (8.2) |
| 31–44 | 1354 (90.6) | 140 (9.4) | 1296 (83.8) | 250 (16.2) | 1225 (83.6) | 241 (16.4) | 1366 (88.3) | 181 (11.7) |
| 45–70 | 461 (90.9) | 46 (9.1) | 445 (84.3) | 83 (15.7) | 429 (85.1) | 75 (14.9) | 426 (82.1) | 93 (17.9) |
| p valuea | 0.0209 | <0.0001 | <0.0001 | <0.0001 | ||||
| Marital status | ||||||||
| Single | 1583 (92.6) | 127 (7.4) | 1606 (88.2) | 214 (11.8) | 1475 (87.9) | 203 (12.1) | 1612 (90.3) | 174 (9.7) |
| Married/Cohabitating | 1628 (91.3) | 156 (8.7) | 1555 (83.9) | 298 (16.1) | 1544 (87.6) | 218 (12.4) | 1648 (88.8) | 208 (11.2) |
| Divorced/Separated | 307 (91.6) | 28 (8.4) | 300 (85.7) | 50 (14.3) | 272 (82.4) | 58 (17.6) | 296 (85.3) | 51 (14.7) |
| p valuea | 0.3578 | 0.0016 | 0.0217 | 0.0209 | ||||
| Race | ||||||||
| Whites | 1590 (93.3) | 114 (6.7) | 1587 (87.7) | 223 (12.3) | 1419 (85.2) | 246 (14.8) | 1606 (90.5) | 169 (9.5) |
| African-Americans | 547 (91) | 54 (9.0) | 561 (88.6) | 72 (11.4) | 486 (83.5) | 96 (16.5) | 547 (87.4) | 79 (12.6) |
| Asian/Pacific Islander | 105 (98.1) | 2 (1.9) | 105 (93.8) | 7 (6.3) | 100 (92.6) | 8 (7.4) | 101 (93.5) | 7 (6.5) |
| Mixed race/Others b | 1236 (89.8) | 141 (10.2) | 1171 (81.9) | 258 (18.1) | 1254 (90.9) | 125 (9.1) | 1265 (87.9) | 174 (12.1) |
| p valuea | <0.0001 | <0.0001 | <0.0001 | 0.0213 | ||||
| Education | ||||||||
| ≤ 12 years | 1689 (89.9) | 189 (10.1) | 1653 (83.8) | 319 (16.2) | 1622 (86.9) | 244 (13.1) | 1750 (88.8) | 221 (11.2) |
| 13–15 years | 931 (95.0) | 49 (5.0) | 933 (91.3) | 89 (8.7) | 854 (89.9) | 96 (10.1) | 917 (90.9) | 92 (9.1) |
| ≥16 years | 895 (92.5) | 73 (7.5) | 875 (85.3) | 151 (14.7) | 813 (85.5) | 138 (14.5) | 888 (88.4) | 117 (11.6) |
| p valuea | <0.0001 | <0.0001 | 0.0113 | 0.1369 | ||||
| Alcohol use | ||||||||
| Non-drinkers | 834 (89.8) | 95 (10.2) | 822 (85.0) | 145 (15.0) | 803 (86.9) | 121 (13.1) | 846 (87.9) | 117 (12.1) |
| <0.5 drinks/day | 1173 (91.9) | 103 (8.1) | 1133 (85.1) | 199 (14.9) | 1110 (88.3) | 147 (11.7) | 1169 (88.3) | 155 (11.7) |
| 0.5–2 drinks/day | 817 (93.1) | 61 (6.9) | 813 (87.4) | 117 (12.6) | 760 (88.1) | 103 (11.9) | 832 (91.3) | 79 (8.7) |
| >2 drinks/day | 563 (92.9) | 43 (7.1) | 566 (87.3) | 82 (12.7) | 506 (85.6) | 85 (14.4) | 574 (89.4) | 68 (10.6) |
| p valuea | 0.0469 | 0.2322 | 0.3649 | 0.0649 | ||||
| Cigarette smoking | ||||||||
| Never | 2059 (92.9) | 158 (7.1) | 2020 (87) | 302 (13.0) | 1912 (87.5) | 272 (12.5) | 2072 (90.2) | 226 (9.8) |
| Former smoker | 644 (91) | 64 (9.0) | 636 (85) | 112 (15.0) | 612 (86.9) | 92 (13.1) | 646 (86.8) | 98 (13.2) |
| Current smoker | 811 (90.3) | 87 (9.7) | 802 (84.4) | 148 (15.6) | 760 (86.8) | 116 (13.2) | 832 (88.5) | 108 (11.5) |
| p valuea | 0.0244 | 0.1118 | 0.8047 | 0.0302 | ||||
| Circumcision | ||||||||
| No | 2152 (90.2) | 233 (9.8) | 2078 (83.2) | 421 (16.8) | 2045 (86.8) | 310 (13.2) | 2195 (88.0) | 299 (12.0) |
| Yes | 1386 (94.5) | 80 (5.5) | 1403 (90.7) | 144 (9.3) | 1264 (88.1) | 171 (11.9) | 1383 (91.2) | 134 (8.8) |
| p valuea | <0.0001 | <0.0001 | 0.2636 | 0.0016 | ||||
| Sexual orientation | ||||||||
| MSW | 2664 (93.5) | 186 (6.5) | 2640 (88) | 360 (12.0) | 2504 (89.3) | 300 (10.7) | 2704 (91) | 267 (9.0) |
| MSM | 86 (76.8) | 26 (23.2) | 84 (72.4) | 32 (27.6) | 80 (71.4) | 32 (28.6) | 82 (70.7) | 34 (29.3) |
| MSWM | 325 (83.8) | 63 (16.2) | 312 (75.5) | 101 (24.5) | 291 (76.6) | 89 (23.4) | 328 (80.2) | 81 (19.8) |
| p valuea | <0.0001 | <0.0001 | <0.0001 | <0.0001 | ||||
| Lifetime female sexual partners | ||||||||
| 0–1 partners | 764 (92.7) | 60 (7.3) | 487 (87.4) | 70 (12.6) | 483 (89.4) | 57 (10.6) | 518 (92.3) | 43 (7.7) |
| 2–9 partners | 1171 (92.7) | 92 (7.3) | 1393 (86.6) | 216 (13.4) | 1362 (89.4) | 162 (10.6) | 1449 (90.8) | 147 (9.2) |
| 10–49 partners | 1082 (92.6) | 87 (7.4) | 1080 (86.2) | 173 (13.8) | 987 (86.6) | 153 (13.4) | 1096 (88.5) | 142 (11.5) |
| ≥50 partners | 193 (88.9) | 24 (11.1) | 201 (87) | 30 (13.0) | 171 (80.7) | 41 (19.3) | 192 (85.3) | 33 (14.7) |
| p valuea | 0.2589 | 0.9058 | 0.0010 | 0.0041 | ||||
| New female partners in past 3–6 months | ||||||||
| None | 2020 (92.3) | 169 (7.7) | 1982 (86.6) | 306 (13.4) | 1898 (87.7) | 265 (12.3) | 2027 (89.5) | 238 (10.5) |
| 1 partner | 809 (93.1) | 60 (6.9) | 804 (86.7) | 123 (13.3) | 768 (89.6) | 89 (10.4) | 835 (90.6) | 87 (9.4) |
| ≥2 partners | 389 (93.3) | 28 (6.7) | 392 (88.5) | 51 (11.5) | 343 (85.3) | 59 (14.7) | 395 (91.0) | 39 (9.0) |
| p valuea | 0.6271 | 0.5583 | 0.0838 | 0.4886 | ||||
| Lifetime male sexual partners | ||||||||
| None | 2765 (93.6) | 188 (6.4) | 2745 (88.2) | 366 (11.8) | 2602 (89.6) | 302 (10.4) | 2808 (91.2) | 270 (8.8) |
| 1–9 partners | 306 (87.4) | 44 (12.6) | 299 (80.2) | 74 (19.8) | 282 (81.5) | 64 (18.5) | 317 (85.9) | 52 (14.1) |
| ≥10 partners | 105 (70.0) | 45 (30.0) | 97 (62.2) | 59 (37.8) | 89 (61) | 57 (39.0) | 93 (59.6) | 63 (40.4) |
| p valuea | <0.0001 | <0.0001 | <0.0001 | <0.0001 | ||||
| New male partners in past 3 months | ||||||||
| None | 3054 (92.7) | 239 (7.3) | 3015 (87) | 450 (13) | 2865 (88.6) | 370 (11.4) | 3100 (90.4) | 329 (9.6) |
| 1 partner | 57 (77) | 17 (23.0) | 59 (72) | 23 (28.0) | 51 (67.1) | 25 (32.9) | 60 (72.3) | 23 (27.7) |
| ≥2 partners | 64 (72.7) | 24 (27.3) | 66 (70.2) | 28 (29.8) | 57 (66.3) | 29 (33.7) | 56 (60.9) | 36 (39.1) |
| p valuea | <0.0001 | <0.0001 | <0.0001 | <0.0001 | ||||
Abbreviation: HPV, human papillomavirus; MSM, men who have sex with men; MSW, men who have sex with women; MSWM, men who have sex with women and men
p-values for categorical variables were calculated using the chi-square test with Monte Carlo estimation of p-values, α = 0.05.
Includes mixed race, American Indians, Alaska natives, Native Hawaiians or other races
For HPV 6, 11, 16, and 18, the total numbers of incident infections were 319 (10.3%), 111 (3.5%), 362 (12.4%), and 200 (6.2%) among seronegative men, and 30 (10.6%), 17 (3.3%), 70 (16.7%), and 22 (5.6%) among seropositive individuals, respectively. Cumulative incidence among seropositive individuals was higher after 18 months for HPV 16 (p-value= 0.007, Figure 1a). For HPV 11 and 18, incidence proportions were generally lower among seropositive individuals until the 13–18 month interval, followed by similar proportions (HPV 11 and 18) after 24 months. KM curves indicated a similar pattern with overall non-significant p-values (p = 0.76 for HPV 11 and p = 0.69 for HPV 18) for the association between serostatus and HPV acquisition. For HPV 6, cumulative incidences among seropositive and seronegative subjects was similar (p-value= 0.728).
Figure 1. Kaplan-Meier curves for acquisition of incident HPV 6, 11, 16, and 18 infections by serostatus among men in the HIM Study.
Serum status: Solid line = Seronegative subjects (0), Dashed line= Seropositive subjects (1)
P values were determined using the log-rank test and denote differences across the entire follow-up period, by serum status. Values < .05 are considered statistically significant
Median failure times were 47.6 months (HPV 6), 48.6 months (HPV 11), 47.9 months (HPV 16) and 48.5 months (HPV 18).
The total numbers of incident six-month persistent infections were 100 (3.5%), 37 (1.3%), 120 (4.5%), and 74 (2.5%) among seronegative men and 7 (2.8%), 2 (0.4%), 21 (5.5%), and 2 (0.6%) among seropositive individuals for HPV 6, 11, 16 and 18, respectively. Six month persistent HPV 16 infections did not differ significantly between seronegative and seropositive individuals (Figure 1b). The overall number of six-month persistent HPV 11 and 18 infections observed was low (two infections for each type over 4.1 year follow-up period). Six-month persistent infections were not observed prior to month 31 among HPV 11seropositive individuals and prior to month 19 for HPV18 seropositive individuals. Significantly lower incidence of six-month persistent HPV 18 infections was observed among seropositive individuals (p=0.02). Median duration of persistence was 28.8 months, 37.1 months, 34.8 months, and 46.2 months for HPV 6, 11, 16 and 18, respectively. Duration of persistent infection did not differ by seropositivity for HPV 6 (t test p value=0.586), HPV 11 (p=0.551), HPV 16 (p=0.769), and HPV 18 (p= 0.132).
Risk of an incident HPV 16 infection was significantly higher among seropositive compared to seronegative men (aHR 1.37, 95% CI 1.01, 1.86) (Table 2). A similar non-significant pattern was observed for risk of six-month persistent HPV 16 infections (aHR 1.26, 95% CI 0.79, 2.01). Serostatus was not associated with risk of HPV 6, 11, and 18 incident infections. However, risk of persistent HPV 18 infection was significantly lower among seropositive men in unadjusted analyses (crude HR 0.22, 95% CI 0.06–0.91), but failed to reach significance in the adjusted model (aHR 0.19, 95% CI 0.03, 1.37). In separate analyses adjusting for demographic and sexual behavior variables one at a time, similar results to those shown in Table 2 were observed.
Table 2.
Crude and adjusted hazard ratios (HR) according to serostatus for incident HPV 6, 11, 16 and 18 infections among HIM Study participants
| No. of men | No. of infections | Crude HR and 95% CI | Adjusted HR and 95% CI | |
|---|---|---|---|---|
|
| ||||
| Incident infection | ||||
|
| ||||
| HPV 6 | ||||
| Seronegative | 3105 | 319 | 1.00 | 1.00 |
| Seropositive | 283 | 30 | 1.08 (0.74, 1.56) | 0.91 (0.56, 1.48)a |
| HPV 11 | ||||
| Seronegative | 3132 | 111 | 1.00 | 1.00 |
| Seropositive | 513 | 17 | 0.92 (0.55, 1.54) | 0.80 (0.44, 1.45)b |
| HPV 16 | ||||
| Seronegative | 2912 | 362 | 1.00 | 1.00 |
| Seropositive | 420 | 70 | 1.40 (1.08, 1.81) | 1.37 (1.01, 1.86)c |
| HPV 18 | ||||
| Seronegative | 3202 | 200 | 1.00 | 1.00 |
| Seropositive | 391 | 22 | 0.92 (0.59, 1.43) | 0.90 (0.531, 1.57)d |
|
| ||||
| 6 month persistent infection | ||||
|
| ||||
| HPV 6 | ||||
| Seronegative | 3105 | 100 | 1.00 | 1.00 |
| Seropositive | 283 | 7 | 0.80 (0.37, 1.73) | 0.98 (0.39, 2.46)e |
| HPV 11 | ||||
| Seronegative | 3132 | 37 | 1.00 | 1.00 |
| Seropositive | 513 | 2 | 0.32 (0.08, 1.34) | 0.31 (0.04, 2.30)f |
| HPV 16 | ||||
| Seronegative | 2912 | 120 | 1.00 | 1.00 |
| Seropositive | 420 | 21 | 1.26 (0.79, 2.01) | 1.39 (0.80, 2.41)g |
| HPV 18 | ||||
| Seronegative | 3202 | 74 | 1.00 | 1.00 |
| Seropositive | 391 | 2 | 0.22 (0.06, 0.91) | 0.19(0.03 1.37)h |
Footnote: HPV- Human Papillomavirus, HR- Hazard ratios
Adjusted for marital status, alcohol use, lifetime male sexual partners (time varying), and new female partners in past 6–12 months (time varying).
Adjusted for marital status, race, lifetime female sexual partners (time varying), and new male partners in past 6–12 months (time varying).
Adjusted for age, marital status, lifetime female sexual partners (time varying), lifetime male sexual partners (time varying), and new female partners in past 6–12 months (time varying).
Adjusted for country, alcohol use, lifetime male sexual partners (time varying), and new female partners in past 6–12 months (time varying).
Adjusted for age, alcohol use, new female partners in past 6–12 months (time varying) and frequency of sexual intercourse with female partners in past 6–12 months.
Adjusted for age, alcohol use, new female partners in past 6–12 months (time varying) and new male partners in past 6–12 months (time varying)
Adjusted for age, country, lifetime male sexual partners (time varying) and new female partners in past 6–12 months (time varying)
Adjusted for marital status, alcohol use, lifetime male sexual partners (time varying), and new female partners in past 6–12 months (time varying).
Table 3 shows the risk of incident and six-month persistent HPV 16 and 18 infections by serostatus and sexual orientation. No significant associations were observed likely due to the relatively small sample sizes in each stratum. No evidence of effect modification was observed for HPV 18. However, serostatus was non-significantly associated with a decreased risk among MSM and increased risk among heterosexual men. Similarly, no significant associations were observed for HPV 6 and 11 (data not shown). In analyses by antibody levels, high levels of antibody were not significantly associated with reduced risk of subsequent genital HPV infection, with risk estimates similar to those presented for the whole cohort in Table 2 (data not shown). To address the issue of small sample size and to increase power, we conducted analyses by combining seropositive individuals for any of the four HPV types and compared them to seronegative individuals for all four HPV types. These analyses did not indicate significantly higher HR for subsequent HPV 16 incident infection, but for persistent HPV 18 infection a significant protective effect was observed in adjusted models (aHR 0.42, 95% CI 0.19, 0.93) (Table 4). Sensitivity analyses were conducted by 1) restricting the analysis to men who were negative to all 4 vaccine types at baseline and 2) restricting the analysis to men who were ages 18–50 years. The magnitude of the association for the various HPV types did not differ from what is presented in Table 2. However, as the sample size was reduced in these analyses, these associations were no longer statistically significant for risk of persistent HPV 18 infection. Furthermore, in a separate analyses adjusting for the same set of adjustment variables for each of the four HPV types and forcing the country variable into the model, results did not change from what is presented in Table 2.
Table 3.
Crude and adjusted hazard ratios (HR) stratified by sexual orientation for incident HPV 16 and 18 infections among HIM Study participants
| HPV 16 | HPV 18 | |||||||
|---|---|---|---|---|---|---|---|---|
|
| ||||||||
| Number at risk | Events | Crude HR | Adjusted HR | Number at risk | Events | Crude HR | Adjusted HR | |
| Incident infections MSW | ||||||||
| Seronegative | 2204 | 269 | 1.0 | 1.0 | 2419 | 138 | 1.0 | 1.0 |
| Seropositive | 261 | 42 | 1.37 (0.99, 1.90) | 1.31(0.90,1.93)a | 242 | 11 | 0.82 (0.45, 1.52) | 1.01 (0.51, 2.01)d |
| MSWM | ||||||||
| Seronegative | 268 | 47 | 1.0 | 1.0 | 305 | 27 | 1.0 | 1.0 |
| Seropositive | 82 | 14 | 1.06 (0.58, 1.93) | 0.88 (0.45, 1.73)b | 73 | 7 | 1.12 (0.49, 2.56) | 1.02 (0.41, 2.58)e |
| MSM | ||||||||
| Seronegative | 75 | 12 | 1.0 | 1.0 | 78 | 10 | 1.0 | 1.0 |
| Seropositive | 27 | 6 | 1.33 (0.50, 3.55) | 1.61 (0.58, 4.53)c | 31 | 2 | 0.44 (0.09, 2.03) | 0.98 (0.17, 5.67)c |
|
| ||||||||
| Six month persistent infections MSW | ||||||||
| Seronegative | 2204 | 93 | 1.0 | 1.0 | 2419 | 53 | 1.0 | 1.0 |
| Seropositive | 261 | 15 | 1.42 (0.82, 2.44) | 1.46 (0.79, 2.73)f | 242 | 1 | 0.19 (0.03, 1.41) | 0.25 (0.03,1.84)i |
| MSWM | ||||||||
| Seronegative | 268 | 13 | 1.0 | 1.0 | 305 | 9 | 1.0 | 1.0 |
| Seropositive | 82 | 3 | 0.83 (0.24, 2.93) | 0.85 (0.24, 3.00)g | 73 | 1 | 0.49 (0.06, 3.84) | 0.51 (0.06, 4.03)h |
| MSM | ||||||||
| Seronegative | 75 | 5 | 1.0 | 1.0 | 78 | 1 | 1.0 | 1.0 |
| Seropositive | 27 | 1 | 0.54 (0.06, 4.60) | 0.58 (0.07, 4.97)h | 31 | 0 | NE | NE |
HPV- Human Papillomavirus, HR- Hazard ratios, MSW- Men who have sex with women, MSWM- Men who have sex with women and men, MSM- Men who have sex with men
-Adjusted for race, alcohol use, lifetime female partners (time varying) and new female partners in past 6–12 months(time varying)
-Adjusted for marital status, alcohol use, new female partners in past 6–12 months(time varying) and new male partners in past 6–12 months(time varying)
Adjusted for new male partners in past 6–12 months(time varying)
Adjusted for marital status, alcohol use, new female partners in past 6–12 months(time varying) and frequency of sexual intercourses in past 6–12 months(time varying)
Adjusted for age, new male partners in past 6–12 months(time varying) and frequency of sexual intercourse with female partners in past 6–12 months(time varying)
Adjusted for age, country, and new female partners in past 6–12 months(time varying)
Adjusted for country
Adjusted for age
Adjusted for marital status, lifetime female partners(time varying) and new female partners in past 6–12 months(time varying)
Table 4.
Crude and adjusted hazard ratios (HR) according to seropositive for any four HPV types (HPV 6, 11, 16 and 18) compared to seronegative for all four types infections among men in the HIM study
| No. of men | No. of infections | Crude HR and 95% CI | Adjusted HR and 95% CI | |
|---|---|---|---|---|
|
| ||||
| Incident infection | ||||
|
| ||||
| HPV 6 | ||||
| Seronegativea | 2663 | 240 | 1.00 | 1.00 |
| Seropositiveb | 1082 | 109 | 1.12 (0.90, 1.41) | 1.08(0.81, 1.43)c |
| HPV 11 | ||||
| Seronegativea | 2858 | 88 | 1.00 | 1.00 |
| Seropositiveb | 1164 | 40 | 1.12 (0.77, 1.63) | 0.89 (0.55, 1.42)d |
| HPV 16 | ||||
| Seronegativea | 2609 | 307 | 1.00 | 1.00 |
| Seropositiveb | 1062 | 125 | 1.01 (0.82, 1.24) | 1.08 (1.84, 1.39)e |
| HPV 18 | ||||
| Seronegativea | 2803 | 157 | 1.00 | 1.00 |
| Seropositiveb | 1158 | 65 | 1.00 (0.75, 1.34) | 1.09 (0.77, 1.54)f |
|
| ||||
| 6 month persistent infection | ||||
|
| ||||
| HPV 6 | ||||
| Seronegativea | 2663 | 83 | 1.00 | 1.00 |
| Seropositiveb | 1082 | 24 | 0.72 (0.46, 1.13) | 0.82 (0.48, 1.40)g |
| HPV 11 | ||||
| Seronegativea | 2858 | 30 | 1.00 | 1.00 |
| Seropositiveb | 1164 | 9 | 0.73 (0.35, 1.54) | 0.49 (0.17, 1.44)h |
| HPV 16 | ||||
| Seronegativea | 2609 | 101 | 1.00 | 1.00 |
| Seropositiveb | 1062 | 40 | 0.98 (0.68, 1.41) | 1.27(0.83, 1.92)i |
| HPV 18 | ||||
| Seronegativea | 2803 | 64 | 1.00 | 1.00 |
| Seropositiveb | 1158 | 12 | 0.45 (0.24, 0.84) | 0.42 (0.19, 0.93)j |
Footnote: HPV- Human Papillomavirus, HR- Hazard ratios
Seropositive for any of the 4 HPV types (HPV 6, 11, 16 and 18)
Seronegative for all 4 HPV types (HPV 6, 11, 16 and 18)
Adjusted for marital status, alcohol use, lifetime male sexual partners (time varying), and new female partners in past 6–12 months (time varying).
Adjusted for country, lifetime male sexual partners (time varying), and new female partners in past 6–12 months (time varying).
Adjusted for country, lifetime female sexual partners (time varying), lifetime male sexual partners (time varying), and new female partners in past 6–12 months (time varying).
Adjusted for marital status, alcohol use, lifetime male sexual partners (time varying), and new female partners in past 6–12 months (time varying).
Adjusted for age, alcohol use and new female partners in past 6–12 months (time varying).
Adjusted for new female partners in past 6–12 months (time varying) and new male partners in past 6–12 months (time varying).
Adjusted for age, country and new female partners in past 6–12 months (time varying)
Adjusted for marital status, lifetime female sexual partners (time varying), and new female partners in past 6–12 months (time varying).
Discussion
This is the first study to report effect of baseline anti-HPV antibodies for subsequent HPV 6 and 11 infections among men, along with data for HPV 16 and 18. In this study, baseline seropositive status was not associated with a reduction in subsequent incident or six-month persistent HPV 6, 11, and 16 infections. In fact, HPV 16 seropositive men had a higher HPV 16 incident infection rate compared to seronegative men. A possible protective effect was observed for HPV 18 persistent infections among seropositive individuals, but the small number of infections requires a cautious interpretation of these results. In general, natural immunity after HPV infection was not associated with a reduced risk for subsequent HPV infections among men.
This is the first study to report statistically significant higher risk of incident genital HPV 16 infections and possible lower risk of persistent HPV 18 infections among seropositive men. In the few studies that have assessed the association of antibodies generated following natural HPV infection and protection against subsequent genital HPV infections in men, results have either been inconclusive or have not shown an association (14, 15, 16). The current study is an extended analysis of previous work from our group (14), in which we showed a non-significant increased risk for incident (aHR 1.22, 95% CI 0.83, 1.79) and persistent HPV 16 genital infections (aHR 1.05, 95% CI 0.51, 2.16).
A higher overall incidence of HPV 16 infections was observed in the current study. However, when examined over time, a similar or lower genital HPV 16 incidence was observed among seropositive and seronegative men up to month 18 of follow-up. Similar initial protective effects against HPV 16 and other HPV genotypes have been documented among women, followed by gradual waning of naturally induced IgG antibody levels over time (22–28). High-risk sexual behavior among seropositive men, compared to seronegative men, may have led to higher rates of incident infections among them. However, if this was the case, higher incidence among seropositive men should have been observed for other HPV types as well, and not just for HPV 16. Although we adjusted for sexual behavior variables as time-varying covariates, there is still a possibility that residual confounding occurred. There is also a possibility of not capturing precise sexual behavior related risk due to error in reporting sexual behavior in self-reported questionnaire. Higher risk of incident and persistent infections among HPV 16 seropositive individuals may also indicate possible reactivation of prior latent infection (29).
In this study, incident and persistent HPV 11 and 18 infections were generally lower among seropositive subjects. Lower incidence (especially for persistent HPV 18) may be suggestive of a transient protective effect, which gradually wanes after 18 months, as the KM analyses indicated. However, due to the small number of HPV 18 events, this association failed to reach statistical significance. Additional studies may be needed to understand the role of anti-HPV 18 antibodies among men.
Factors including differential immune response by gender, differences in assay techniques, and different durations of antibody levels have been suggested for the difference in antibody response observed among men and women (14, 23, 30, 31). Numerous studies have indicated a higher seroprevalence of both oncogenic and non-oncogenic HPV types among women compared with men (32–38). A lower level of antibody response following infection of keratinized epithelium among heterosexual men compared to mucosal epithelial infection in heterosexual women may be a possible explanation for this gender difference (14). Inclusion of non-neutralizing antibodies in our study may have led to overestimation of overall serum antibody levels and may have biased the results toward the null. Serum antibody cut-off levels may also affect the observed association. We therefore carried out analyses using the highest versus the lowest two tertiles of serum antibodies, and the results were not significantly different.
In our analysis, difference in seropositivity seen by anatomical genital location was not a marker for circumcision status. Due to overall large sample size and low seroprevalence among men, significant differences by seropositivity were observed for many covariates, including circumcision status, but circumcision status was not associated with HPV serostatus and risk of subsequent infection in further analyses. The highest seroprevalence was observed among men reporting ≥10 lifetime male sexual partners and having two or more new male sexual partners. These results are consistent with our previous report, which indicated that same sex anal intercourse among men is an independent risk factor for seroprevalence for HPV 6, 11, 16 and 18 (39).
To the best of our knowledge, our study is the first to assess the effect of serum antibody status for all four major vaccine type HPVs in a large, multicenter cohort of men with a long-term follow-up. Despite these strengths, some limitations may have influenced the results. Serum antibody levels were determined at a single baseline visit in our study. Men with prior exposure may have been misclassified as seronegative due to lower seroconversion rates, waning antibody response, and time-lag between exposure and antibody response. This misclassification is likely to be non-differential and may have attenuated findings toward the null. Furthermore, we did not have antibody information for participants at subsequent visits to assess changes in antibody status during the follow-up. Misclassification of HPV infection is also a possibility, although we utilized robust methods for DNA detection that are well-established (17). Furthermore, single baseline visit DNA negative subjects were assumed to be the at risk population and were considered eligible for the study. We ran a separate analysis using more stringent criteria of requiring two negative visits (baseline and first six months visit) to qualify for entry into the analyses. Use of this stringent criterion also did not alter study results. Information reported on the eligibility survey prior to study enrollment was self-reported and may have led to inclusion of older men with a history of genital warts; as such this is a potential limitation for our study.
We did not have information about infections that may have occurred prior to the study start. These infections may not have cleared but instead remained latent at low copy numbers (29). Latency for HPV infection has been described in animal models and recently, reactivation of latent infection has been demonstrated among immuno-compromised individuals (29). Assessment of previous infections is challenging, especially among men who have been sexually active for a long period of time. Furthermore, current HPV DNA detection methods may not be sensitive enough to detect low viral load of latent infection or reactivations that last for a short time period (14). Future studies are needed to explore the effect of latent HPV infections and their reactivation among healthy individuals.
This is the first study to provide results about the effect of anti-HPV antibodies on subsequent risk of HPV 6 and 11 infections, which are responsible for 90% of the genital warts (5). Genital warts associated with HPV 6 and 11 account for the majority of HPV-related diseases among men. This study indicates that men with naturally acquired HPV 6 and 11 antibodies remain susceptible to subsequent infections from these genotypes. Furthermore, our results confirm that seropositive men remain susceptible to subsequent HPV 16 and 18 infections as well. Only two previous studies have examined HPV 16 and 18 among men, which include a small study (n=285) of men in Tucson Arizona (15) and a study in Netherlands among men who have sex with men (MSM) (16). This is the first multinational study examining HVP 16 and 18 among a general population Our results have important public health implications supporting the need for HPV vaccination of both males and females. HPV infection is readily transmitted between sexual partners and male HPV infection significantly contributes to infection and subsequent disease in women (40). As natural antibodies do not protect men from subsequent HPV infection and vaccination coverage is low among men (13.9% for all three doses) and women (37.6% for all three doses) (41), it is important to promote vaccination and other preventive methods to target HPV associated diseases in both sexes.
In conclusion, among men participating in the HIM study, baseline seropositivity after natural infection with HPV 6, 11 and 16 was not associated with protection against subsequent type-specific genital infection, with a possible protective effect against persistent HPV 18 infections. For HPV 16, a significantly higher risk of incident infection was observed among seropositive men. The effect of anti-HPV antibodies may be influenced by duration of the antibody response, time period between initial and subsequent genital infection, and overall antibody levels. These results highlight the importance of HPV prevention measures, such as vaccination, which are proven to protect against subsequent HPV infections and related genital diseases.
Figure 2. Kaplan-Meier curves for acquisition of six-month persistent HPV 6, 11, 16, and 18 infections by serostatus among men in the HIM Study.
Serum status: Solid line = Seronegative subjects (0), Dashed line= Seropositive subjects (1)
P values were determined using the log-rank test and denote differences across the entire follow-up period, by serum status. Values < .05 are considered statistically significant
Median failure times were 47.8 months (HPV 6), 48.7 months (HPV 11), 48.0 months (HPV 16) and 48. 6 months (HPV 18).
Acknowledgments
Financial support: A.R. Giuliano, B.N Torres, D.J. Ingles, M. Abrahamsen, L.L. Villa, E. Lazcano-Ponce, J. Salmeron, and M. Quiterio received financial support from the NCI, NIH CA R01CA098803 (PI: AR Giuliano).
The authors thank the HIM Study teams in the U.S. (Huiyi Lin, Jane Messina, Christine Pierce Campbell, Bradley Sirak, Christine Gage, Kim Isaacs, Kayoko Kennedy, Andrea Bobanic, Shams Rahman, Matthew Schabath, Alan Nyitray, and Julie Rathwell), Brazil (Maria Luiza Baggio, Roberto Carvalho da Silva, Lenice Galan, Ricardo Cintra, Filomena Cernicchiaro, Graça Ribeiro, Rosária Otero, Roberta Bocalon, Juliana Antunes, Fernanda Silva, Rossana Terreri, and the CRT-DST/AIDS nursing team), and Mexico (Aurelio Cruz Valdez, René de Jesús Alvear Vásquez, Oscar Rojas Juárez, Rossana del Carmen González Sosa, Rosangel Ríos Vences, Martha Huerta Segura, Alicia Rodríguez Galván, Paula Román Rodríguez, Ana Laura Landa Vélez, Griselda Díaz García, Verónica Chávez Abarca, Gisela Flores Quevedo, María del Pilar Hernández Nevárez, Guillermina Sánchez Martínez, Adriana Ortiz Rojas, Carlos Omar Barrera Flores ).
Footnotes
Conflicts of interest: ARG and SLS receive research funding from Merck. ARG is a member of their Speaker’s Bureau. LLV and ARG are consultants of Merck for HPV vaccines. None of the other authors have conflicts of interest to report.
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