Human papillomavirus (HPV) 16 is the most commonly detected HPV type in the oral region1,2 and causes most HPV-positive head and neck squamous cell carcinomas.3 Human papillomavirus 16 infections, along with other HPV types, can lead to the production of type-specific antibodies to HPV proteins including to the L1 capsid. These naturally acquired IgG antibodies have been shown to reduce the risk of the subsequent acquisition of cervical HPV infection in some studies,4–8 but not others.9,10 Whether these antibodies impact the subsequent risk of oral HPV infection has not been explored.
Long-term persistent HPV16 infections are known to sometimes lead to the development of antibodies to HPV16’s E6 and E7 oncoproteins, usually late in carcinogenesis.11 E6 antibodies are strongly associated with HPV-positive oropharyngeal cancer12 and have been detected in some cases more than 10 years before their cancer diagnoses,13 but it is unknown if these antibodies are common among cancer-free individuals currently infected with oral HPV16.
Therefore, we examined the relationship between HPV16 L1, E6 and E7 seropositivity and oral HPV16 infection using a longitudinal cohort study of HIV-infected and at-risk HIV-uninfected individuals known to have a higher oral HPV16 prevalence.1
MATERIALS AND METHODS
These analyses included individuals from the Persistent Oral human Papillomavirus Study (POPS), a study nested within the Multicenter AIDS Cohort Study (MACS) of men who have sex with men (MSM) and the Women Interagency HIV Study (WIHS).1,14,15 There were 463 HIV-infected and 293 at risk HIV-uninfected participants who were tested for HPV16 L1 antibodies. These participants met the following criteria: enrolled in 2009 to 2010, not vaccinated with a prophylactic HPV vaccine by study baseline, and had 4 or more POPS follow-up visits. Banked serum was obtained from participant’s baseline POPS visit and tested for HPV L1 antibodies to HPV16 and to the other 2 most common oncogenic oral HPV types in the POPS: HPV33 and HPV45. The detected HPV L1 antibodies may have developed after an HPV infection at any number of anatomic (genital, anal, oral) regions.
Human papillomavirus 16 E6 and E7 antibody testing was performed on a subgroup of 273 participants without a history of any HPV-related cancer (cervical, anal, penile, or oropharyngeal). Participants with a detectable oral HPV16 infection at any POPS visit were included (n = 91), along with twice as many oral HPV16-negative controls (n = 182). These controls were a random sample selected after stratification by cohort and HIV status to match the distribution among oral HPV16-positive individuals. The MACS/WIHS executive committees and the institutional review boards from each site approved the study protocol, and participants provided written informed consent.
Antibody testing was performed on banked serum samples from participant’s POPS baseline visit by using virus-like particle-enzyme–linked immunosorbent assays (VLP-ELISAs) with HPV16, HPV33, and HPV45 capsids produced in insect cells from recombinant baculoviruses, following previously published methods.16,17 Seropositivty was defined as an optical density (OD) greater than 3 SDs above the mean OD of sera from 2-year-old children.17 For quality assurance, known positive controls were run on each ELISA plate throughout the testing period. When comparing all the samples with duplicates, the intra-assay coefficient of variations (CVs) were 5.8%, 7.4%, and 8.7% for HPV16, HPV33, and HPV45, respectively, whereas the interassay CVs (between different assays plates) were 16.1%, 13.3%, and 23.2% for HPV16, HPV33, and HPV45, respectively.
Banked baseline serum samples were also tested for antibodies to recombinantly expressed HPV16 E6 and E7 oncoproteins. Antibody testing was performed using ELISAs with a microtiter plate with HPV16 E6 and E7 GST-fusion proteins expressed in Escherichia coli according to the protocol of Sehr et al.18 For E6 and E7, the seropositivity cutpoint was defined as an OD greater than 3 SDs above the mean OD of sera of low-risk control cohort of 93 female US army recruits between the ages of 18 and 35 years after excluding positive outliers. Younger females were considered an adequate control population given their very low risk for HPV16 E6 and E7 seropositivity.13 We additionally considered a more stringent cutpoint defined as an OD greater than 5 SDs above the mean OD in the control cohort. When duplicates were compared, the intra-assay CV was 9.3% for E6 and 6.9% for E7.
Oral rinse samples were collected at up to 7 semiannual visits through a 30-second rinse and gargle with Scope mouthwash. DNA was isolated from these samples using a magnetic bead-based automated platform (QIAsymphony SP, Qiagen),19 and then tested for 37 HPV types using the Roche linear array with PGMY09/11 polymerase chain reaction primer pools and reverse line blot hybridization, as previously described.1,19
To compare baseline HPV16 L1 seroprevalence by each risk factor, we used χ2 tests for categorical variables and Mann-Whitney tests for continuous variables. We calculated prevalence ratios (PRs) and 95% confidence intervals (95% CIs) using Poisson regression with robust variance to analyze risk factors associated with baseline HPV16 L1 seroprevalence.
To evaluate the association of baseline L1 antibodies with subsequent risk of infection with the same HPV type, we excluded prevalent oral HPV infections and restricted outcomes to incidently detected infections. We calculated incidence rates and used unadjusted and adjusted Wei-Lin-Weissfeld modeling to evaluate the impact of seropositivity on oral HPV incidence. Seropositivity was also examined by antibody titer level, as titers were a priori categorized into tertiles to match the technique of a previous study.4
For the HPV16 E6/E7 antibody analysis, logistic regression was used to examine whether prevalent oral HPV16 infection was associated with E6 and/or E7 seropositivity at the same visit. Models were adjusted for variables that have been associated with prevalent or incident oral HPV infection.1,20 In different sensitivity analyses for HPV L1, we stratified by sex and HIV status and required 2 negative test results before classifying an infection as “incident.” We also examined the results when restricting to persistent infection (requiring 2 consecutive positive HPV test results) for both the HPV16 L1 and E6/E7 analyses. All statistical tests were 2 sided and considered significant using an α = 0.05 level. All analyses were performed by STATA-MP Version 12.0.
HPV L1 Seropositivity
Among the 756 eligible participants, there were 167 (22%) who were HPV16 L1 seropositive at baseline. Human papillomavirus 16 L1 seropositivity was similar by age and sex (P > 0.20, Table 1). However, never smoking cigarettes, increased number of recent oral sex partners, and HIV status were all associated with increased HPV16 L1 seroprevalence, even after adjustment for other risk factors (Table 1, all P < 0.05).
Baseline HPV16 L1 seroreactivity did not reduce the subsequent risk of oral HPV16 infection in either unadjusted (hazard ratio [HR], 1.4; 95% CI, 0.59–3.3) or adjusted analyses (adjusted HR [aHR], 1.1; 95% CI, 0.41–3.0; Table 2). Results were similar when restricted to HIV-infected (aHR, 1.0; 95% CI, 0.30–3.5) or HIV-uninfected (aHR, 1.4; 95% CI, 0.27–6.9) individuals, or among only individuals who reported having sex during the study. Results were also similar when requiring 2 consecutive negative test results before the first positive for an infection to be considered incident and when restricting to HPV16 persistent infection as an outcome (data not shown). Although we were underpowered to examine effect modification, we cannot exclude the possibility of an effect of seropositivity on subsequent oral HPV16 infection in certain subgroups such as females (aHR, 0.63; 95% CI, 0.13–3.1), particularly considering seropositive women had a higher HPV16 titer level than did seropositive MSM (OD = 0.51 vs 0.41, P = 0.02).
When stratifying the 167 HPV16 L1–seropositive individuals into antibody titer tertiles, the 55 individuals within the highest tertile had a nonsignificantly lower risk of oral HPV16 infection compared with the L1–seronegative group (aHR, 0.44; 95% CI, 0.05–3.7; Supplemental Table 1, http://links.lww.com/OLQ/A97). Results also seemed to differ among other HPV types, as HPV33 L1 seropositivity was associated with reduced risk of subsequent oral HPV33 infection (aHR, 0.11; 95% CI, 0.01–0.78; Table 2), whereas HPV45 L1 seropositivity was associated with a higher risk of subsequent oral HPV45 infection (aHR, 3.6; 95% CI, 1.1–11.8; Table 2).
HPV16 E6 and E7 Seropositivity
Among 195 HIV-infected and 69 at-risk HIV-uninfected participants evaluated, 7.6% (n = 20) were positive for antibodies to the HPV16 E6 oncoprotein, whereas 3.4% (n = 9) were positive for antibodies to the HPV16 E7 oncoprotein. E6 or E7 seropositivity was similar by sex (males vs. females: 8.2% vs. 8.5%, P = 0.94), but was doubled among HIV-infected compared with HIV-uninfected individuals, although the difference was not statistically significant (9.7% vs. 4.4%, P = 0.16).
The prevalences of both HPV16 E6 seroreactivity and HPV16 E7 seroreactivity were similar among individuals with an oral HPV16 infection detected during the POPS compared with those who never had an oral HPV16 infection (Table 3; E6: 10.2% vs. 6.3%, P = 0.25; E7: 4.6% vs. 2.8%, P = 0.47). After adjustment, the odds of E6 and E7 seroreactivity did not statistically differ when comparing those with and without an oral HPV16 DNA detected during POPS (Table 3; E6: adjusted OR, 2.0; 95% CI, 0.55–6.9; E7: adjusted OR, 2.0; 95% CI, 0.52–7.9). Results were also similar and nonsignificant when restricting to oral HPV16 infections detectable at baseline of this study (i.e., prevalent, P = 0.78) and when restricting to oral HPV16 infections persisting at least 6 months (P = 0.58).
When a more stringent seropositivity cutpoint was used (an OD that was 5 SDs above the mean OD in the control cohort), the number of E6 positive individuals declined from 20 to 6 individuals (prevalence, 2.3%), whereas the number of E7-positive individuals declined from 9 to 3 individuals (prevalence, 1.1%). However, there was still no association for either E6 or E7 seropositivity with HPV16 DNA (E6: OR, 1.2; 95% CI, 0.22–6.88; E7: OR, 1.2; 95% CI, 0.11–13.6).
This study found that HPV16 L1 seropositivity from natural infection did not reduce the subsequent risk of oral HPV16 infection. One potential explanation is that natural immunity may not protect against oral HPV16 infection. In addition, HPV16 E6/E7 seropositivity was not more common among individuals with concurrent oral HPV16 DNA suggesting that, like in cervical cancer, these E6/E7 antibodies may not be induced early in the carcinogenesis process and may not be suitable biomarkers for oral HPV16 infection in populations without HPV-related cancer.
Assessing the existence of naturally acquired (L1) immunity against oral HPV can be useful to evaluate the possible benefit of vaccinating sexually active individuals for HPV.21 Although we did not find evidence that HPV16 L1 seropositivity protects against subsequent oral HPV16 infection, it is unclear whether it may have a differential impact on infection risk at different anatomical sites. Several studies have suggested that HPV16 L1 seropositivity may partially protect against subsequent cervical HPV infection,4–6 but it is unclear if that protection is conferred against HPV at other anatomical sites as 2 other recent studies observed no protection for penile22,23 or anal HPV acquisition in men.23 However, there are several caveats to our finding that HPV16 L1 seropositivity may not impact risk of oral HPV16 infection that need to be considered as the results may differ by population, HPV type, titer level, or assay.
Although our results were similar by HIV status, results in this population may differ from other populations. The incident infections detected in this study may include many reactivated infections as well as some newly acquired infections, as HPV latency has been suggested, especially among immunosuppressed and older individuals.24,25 In addition, we cannot exclude the possibility that HPV16 L1 seropositivity has a different effect on oral HPV16 infection in women than in MSM. Although we had limited power in this study, particularly to examine potential effect modification; differences by sex should be further examined, as a genital HPV study suggested that HPV16 L1 antibodies had no protective effect in men.22
We also cannot preclude the possibility that high titers of HPV16 L1 antibodies may have a protective effect against oral HPV16 infection. A few cervical HPV studies have suggested that protection from naturally acquired antibodies may be stronger among those with higher antibody titers,4,6 and the protective ability of the considerably higher antibody titers induced by the HPV vaccine26 supports this notion. One recent study has suggested that the high titers from the L1-based HPV vaccine may protect against subsequent prevalent oral HPV16, but further examination is needed.27
Although HPV16 L1 seropositivity did not protect against oral HPV16 infection, results differed for the other HPV types examined. Indeed, HPV33 L1 seropositivity was associated with reduced oral HPV33 incidence, whereas HPV45 L1 seropositivity was paradoxically associated with increased HPV45 incidence in this study. Explanations from these incongruities include the following: potential limitations in the assays, residual or unmeasured confounding, or possible actual differences. A previous cervical HPV study also found differences in natural protection by HPV type (protection for HPV16, but no protection for other types such as HPV33 and HPV45).5 Similar to other serologic assays, the VLP-ELISAs used in this study are limited because there are no standard reference serum samples and the assay has been suggested to detect an antibody response for only 50% to 60% of women who previously had detectable cervical HPV DNA.16,28,29 The VLP-ELISA used in this study measured the total type-specific binding IgG antibodies which include both neutralizing and nonneutralizing antibodies. Further research is needed to determine if these results may differ across other serologic assays, particularly those that restrict to neutralizing antibodies.30
This study also observed that HPV16 E6/E7 seropositivity was not associated with concurrent oral HPV16 DNA. Although oral HPV16 DNA and HPV16 E6/E7 seropositivities have both been strongly associated with oropharyngeal cancer,12 this study suggests that the implications of oral HPV16 DNA and E6/E7 seropositivity may be less clear in populations without a diagnosed HPV-related cancer. Although it is unknown whether any participants had undetected oral premalignancies, the lack of an association between E6/E7 seropositivity and persistent oral HPV16 infection supports previous evidence from the cervical cancer field that these oncogenes are not normally expressed until late in the carcinogenesis process.11
Another recent study detected HPV16 E6 antibodies in some oropharyngeal cancer cases more than 10 years before their cancer diagnoses,13 conflicting with what has been seen for cervical cancer.11 Although E6 antibodies have been suggested to have a high specificity for HPV16-positive oropharyngeal cancer,13,31 our ELISA detected HPV16 E6 seroreactivity in 6.3% of our cancer-free participants who did not have an oral HPV16 infection, albeit our population is a higher-risk group. Although specificity may vary depending on the assay, the specificity of a potential HPV16 E6 serologic biomarker in high-risk groups such as HIV-infected individuals would need to be considered in any screening modality.
To our knowledge, this is the first longitudinal study to examine the relationship between HPV16 L1, E6, and E7 serostatus and oral HPV16 infection. This study, coupled with cervical HPV literature,4–6 raises the question of whether the relationship between natural HPV seropositivity and HPV infections at different anatomical sites may vary.
1. Beachler DC, Weber KM, Margolick JB, et al. Risk factors for oral HPV infection among a high prevalence population of HIV-positive and at-risk HIV-negative adults. Cancer Epidemiol Biomarkers Prev 2012; 21: 122–133.
2. Gillison ML, Broutian T, Pickard RK, et al. Prevalence of oral HPV infection in the united states, 2009–2010. JAMA 2012; 307: 693–703.
3. Kreimer AR, Clifford GM, Boyle P, et al. Human papillomavirus types in head and neck squamous cell carcinomas worldwide: A systematic review. Cancer Epidemiol Biomarkers Prev 2005; 14: 467–475.
4. Safaeian M, Porras C, Schiffman M, et al. Epidemiological study of anti-HPV16/18 seropositivity and subsequent risk of HPV16 and -18infections. J Natl Cancer Inst 2010; 102: 1653–62.
5. Wilson L, Pawlita M, Castle PE, et al. Seroprevalence of 8 oncogenic human papillomaviruses and acquired immunity against re-infection. J Infect Dis 2014; 210: 448–455.
6. Castellsague X, Naud P, Chow S, et al. Risk of newly detected infections and cervical abnormalities in women seropositive for naturally-acquired HPV-16/18 antibodies: Analysis of the control arm of PATRICIA. J Infect Dis 2014; 210: 517–534.
7. Malik ZA, Hailpern SM, Burk RD. Persistent antibodies to HPV virus-like particles following natural infection are protective against subsequent cervicovaginal infection with related and unrelated HPV. Viral Immunol 2009; 22: 445–449.
8. Wentzensen N, Rodriguez AC, Viscidi R, et al. A competitive serological assay shows naturally acquired immunity to human papillomavirus infections in the guanacaste natural history study. J Infect Dis 2011; 204: 94–102.
9. Viscidi RP, Snyder B, Cu-Uvin S, et al. Human papillomavirus capsid antibody response to natural infection and risk of subsequent HPV infection in HIV-positive and HIV-negative women. Cancer Epidemiol Biomarkers Prev 2005; 14: 283–288.
10. Palmroth J, Namujju P, Simen-Kapeu A, et al. Natural seroconversion to high-risk human papillomaviruses (hrHPVs) is not protective against related HPV genotypes. Scand J Infect Dis 2010; 42: 379–384.
11. Lehtinen M, Pawlita M, Zumbach K, et al. Evaluation of antibody response to human papillomavirus early proteins in women in whom cervical cancer developed 1 to 20 years later. Am J Obstet Gynecol 2003; 188: 49–55.
12. D’Souza G, Kreimer AR, Viscidi R, et al. Case-control study of human papillomavirus and oropharyngeal cancer. N Engl J Med 2007; 356: 1944–56.
13. Kreimer AR, Johansson M, Waterboer T, et al. Evaluation of human papillomavirus antibodies and risk of subsequent head and neck cancer. J Clin Oncol 2013; 31: 2708–2715.
14. Kaslow RA, Ostrow DG, Detels R, et al. The multicenter AIDS cohort study: Rationale, organization, and selected characteristics of the participants. Am J Epidemiol 1987; 126: 310–318.
15. Barkan SE, Melnick SL, Preston-Martin S, et al. The women’s interagency HIV study. WIHS Collaborative Study Group. Epidemiology 1998; 9: 117–125.
16. Viscidi RP, Ahdieh-Grant L, Clayman B, et al. Serum immunoglobulin G response to human papillomavirus type 16 virus–like particles in human immunodeficiency virus (HIV)–positive and risk-matched HIV-negative women. J Infect Dis 2003; 187: 194–205.
17. Wang SS, Schiffman M, Shields TS, et al. Seroprevalence of human papillomavirus-16, -18, -31, and -45 in a population-based cohort of 10000 women in Costa Rica. Br J Cancer 2003; 89: 1248–1254.
18. Sehr P, Zumbach K, Pawlita M. A generic capture ELISA for recombinant proteins fused to glutathione S
-transferase: Validation for HPV serology. J Immunol Methods 2001; 253: 153–162.
19. Broutian TR, He X, Gillison ML. Automated high throughput DNA isolation for detection of human papillomavirus in oral rinse samples. J Clin Virol 2011; 50: 270–275.
20. Beachler DC, Sugar EA, Margolick JB, et al. Risk factors for oral HPV infection acquisition and clearance among HIV-infected and HIV-uninfected adults. Am J Epidemiol 2014.
21. Franceschi S, Baussano I. Naturally-acquired immunity against HPV: Why it matters in the HPV vaccine era. J Infect Dis 2014; 210: 507–509.
22. Lu B, Viscidi RP, Wu Y, et al. Prevalent serum antibody is not a marker of immune protection against acquisition of oncogenic HPV16 in men. Cancer Res 2012; 72: 676–685.
23. Mooij SH, Landen O, van der Klis FR, et al. No evidence for a protective effect of naturally induced HPV antibodies on subsequent anogenital HPV infection in HIV-negative and HIV-infected MSM. J Infect 2014; 69: 375–86.
24. Beachler DC, D’Souza G, Sugar EA, et al. Natural history of anal versus oral HPV infection in HIV-infected men and women. J Infect Dis 2013; 208: 330–9.
25. Gravitt PE. Evidence and impact of human papillomavirus latency. Open Virol J 2012; 6: 198–203.
26. Safaeian M, Porras C, Pan Y, et al. Durable antibody responses following one dose of the bivalent human papillomavirus L1 virus–like particle vaccine in the Costa Rica vaccine trial. Cancer Prev Res (Phila) 2013; 6: 1242–1250.
27. Herrero R, Quint W, Hildesheim A, et al. Reduced prevalence of oral human papillomavirus (HPV) 4 years after bivalent HPV vaccination in a randomized clinical trial in costa rica. PLoS One 2013; 8: e68329.
28. Wilson LE, Pawlita M, Castle PE, et al. Natural immune responses against eight oncogenic human papillomaviruses in the ASCUS-LSIL triage study. Int J Cancer 2013; 132: 2172–2181.
29. Kreimer AR, Alberg AJ, Viscidi R, et al. Gender differences in sexual biomarkers and behaviors associated with human papillomavirus-16, -18, and -33 seroprevalence. Sex Transm Dis 2004; 31: 247–256.
30. Lin SW, Ghosh A, Porras C, et al. HPV16 seropositivity and subsequent HPV16 infection risk in a naturally infected population: Comparison of serological assays. PLoS One 2013; 8: e53067.
31. Kreimer AR, Johansson M, Hildesheim A, et al. Reply to P.E. castle. J Clin Oncol 2013; 32: 361–362.