Highlights
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Lower HPV16/18 prevalence among women recipients of two or three HPV vaccine doses.
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One HPV vaccine dose may be less efficient in preventing oral HPV infection.
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No difference was observed between one dose vaccinated and unvaccinated women.
Keywords: Papillomavirus, HPV vaccine, Upper respiratory tract, Head and neck cancers, Cervical cancer, India, Oral cavity, HPV genotyping
Abstract
Background
Human papillomavirus (HPV) is one of the most common sexually transmitted infections worldwide. Although the efficacy of the HPV vaccine in preventing the development of cervical pre-malignant lesions has been well demonstrated, the efficacy of the HPV vaccine in preventing HPV infection in the upper respiratory tract has been poorly studied.
Methods
In the context of the IARC cohort study of two versus three doses of HPV vaccine in India, we compared the HPV type prevalence in the oral cavity of women vaccinated with three doses, two doses, or a single dose of quadrivalent HPV vaccine with that of unvaccinated women. A total of 997 oral samples, from 818 vaccinated women and 179 unvaccinated women, were collected at three study sites. All the participants were sexually active at the time of sample collection.
Results
The age-standardized proportion (ASP) of HPV16/18 infections was 2.0 % (95 % CI, 1.0–3.0 %) in vaccinated women and 4.2 % (95 % CI, 1.2–7.2 %) in unvaccinated women. HPV16 was detected in 3.5 % of single-dose recipients, 1.2 % of two-dose recipients (days 1 and 180), and 1.5 % of three-dose recipients (days 1, 60, and 180), whereas 3.3 % of the unvaccinated women tested positive for HPV16. The same trend was observed for HPV18.
Discussion
Our findings agree with those of previous studies on the efficacy of HPV vaccination in reducing oral HPV infections and provide indications that a single vaccine dose may be less efficient than two or three doses in preventing oral HPV infection.
Introduction
The Papillomaviridae family includes more than 200 human papillomavirus (HPV) types that infect cutaneous and/or mucosal epithelia of a wide variety of anatomical sites, including the skin and the anogenital and upper respiratory tracts (reviewed in [1]). Mucosal HPV infections are one of the most common sexually transmitted infections in females and males [2]. They are subdivided into low-risk (LR) and high-risk (HR) HPV types, according to their ability to induce benign or malignant lesions, respectively. The mucosal HR HPV types have been associated with the development of several types of anogenital cancers (e.g. cervical cancer and anal cancer) as well as with a subset of head and neck squamous cell carcinoma (HNSCC). The International Agency for Research on Cancer (IARC) Monographs program has classified the mucosal HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59 as carcinogenic (Group 1), HPV68 as probably carcinogenic (Group 2A), and HPV types 26, 53, 66, 67, 70, 73, and 82 as possibly carcinogenic (Group 2B) to humans. In contrast, the LR HPV types (e.g., HPV6 and HPV11) are the causative agents of genital warts, such as condyloma acuminatum [3].
Epidemiological studies have reported that approximately 70 % of cervical cancers worldwide are associated with HPV16 and HPV18. In HNSCCs, HR HPV types are involved mainly in the development of oropharyngeal cancers (OPC), whereas their role in other sites of the head and neck, such as the oral cavity or the larynx, is marginal [4]. In contrast to the situation for cervical cancer, HPV16 appears to be the main causative agent for OPC in different geographical regions, ranging from approximately 60 % to 96 % of OPC [5], [6], [7]. In recent decades, while a decline in rates of head and neck cancers has been observed as a result of anti-tobacco campaigns, the proportion of HR HPV DNA-positive HNSCC has been increasing rapidly, mainly in the most developed countries, as seen in the United States (71.0 %) [8] and eastern Denmark (62 %) [7]. Castellsagué et al. (2016) [4] conducted a comprehensive assessment of HPV-related biomarkers (i.e., HPV DNA, HPV E6*I mRNA, and p16INK4a) in multiple countries excluding North America; they found that South America had the highest HPV-attributable fraction of OPC (53.6 %), while in other geographical areas the HPV-attributable fractions were 50.0 % (Central, Northern, and Eastern Europe), 22.4 % (Eastern Asia), 19.7 % (Central America), 19.4 % (Western Europe), and 9.4 % (Southern Europe). In central India, a reported 9.4 % of OPC was related to HR HPV infections [9].
The existing prophylactic HPV vaccines approved by the US Food and Drug Administration (i.e., Cervarix, Gardasil, and Gardasil-9) have demonstrated their effectiveness in preventing anogenital cancers [10] and genital warts [11]. High coverage of adolescent girls with the vaccine in combination with effective cervical cancer screening programs could lead to the elimination of cervical cancer globally [12]. Many well-organized vaccination programs have been implemented worldwide [13], [14], [15], [16], [17], in which the efficacy of the HPV vaccine in preventing the development of cervical pre-malignant and malignant lesions has been well demonstrated. In contrast, very limited epidemiological data are available on the efficacy of the HPV vaccine in preventing HPV infection in the upper respiratory tract [18].
Here, in the context of the IARC randomized clinical trial of two versus three doses of HPV vaccine in India [19], [20], which was converted into a cohort study after the suspension of HPV vaccination in trials by the Indian Government, we compare the HPV type prevalence in the oral cavity of women previously vaccinated with different doses of quadrivalent HPV vaccine with that of unvaccinated women.
Materials and methods
Study participants
For this substudy, 818 vaccinated and 179 unvaccinated married female participants from the IARC cohort study were recruited. A detailed description of this study has been published previously [21]. This substudy on HPV vaccination and HPV-related oral infections was set up using deep oral gargle specimens collected from participants enrolled from June 2015 to October 2016.
Vaccinated and unvaccinated married women, matched by current age and site, who were participants in the IARC cohort study of two versus three doses of HPV vaccine in India, irrespective of the number of vaccine doses received, were invited to participate in this substudy. All participants provided informed consent. The original study was approved by the IARC Ethics Committee and the ethics committees of all participating institutions. The substudy was approved by ethics committees from the participating study sites and registered with ISRCTN, ISRCTN98283094, and ClinicalTrials.gov, NCT00923702.
Collection of oral cells
In each of the collection centers (i.e., Ambilikkai, Barshi, and Pune), deep oral gargle specimens were collected from participants eligible for collection of cervical cells for HPV genotyping (i.e., 18 months after marriage or 6 months after delivery, and at least 10 months after a previous collection of cervical cells). Briefly, 15 ml of sterile 0.9 % sodium chloride solution was used to swirl in the entire buccal cavity for 15 s and then deep oral gargled for 15 s. The participants were asked to spit into a 50 ml Falcon tube containing 7.5 ml of ethanol 90 % (ratio 2:1). After centrifugation at 3,000 g for 10 min at 4 °C, 14 ml of the supernatant was carefully removed without disturbing the pellet. The oral cell pellets were re-suspended with repeated pipetting in the remaining volume (1 ml) and transferred to a barcoded safe-lock 1.5 ml Eppendorf tube and stored at 4 °C. Samples were transferred to the Rajiv Gandhi Centre for Biotechnology (RGCB) in Trivandrum for laboratory analysis.
DNA extraction
DNA extraction was performed at RGCB using the Qiagen BioRobot EZ1 Advanced XL instrument with the EZ1 DNA tissue kit according to the manufacturer’s instructions (Qiagen, Hilden, Germany). Briefly, oral cell pellets were incubated in proteinase K and buffer G2 (Qiagen, Hilden, Germany) at 56 °C for 3 h before proceeding with DNA extraction according to the manufacturer’s instructions.
HPV DNA genotyping
HPV DNA was measured in all samples at RGCB using the E7-MPG assay [22] for the detection of 19 possible or probable HR HPV types (16, 18, 26, 31, 33, 35, 45, 51, 52, 53, 56, 58, 59, 66, 68a, 68b, 70, 73, and 82) and two LR HPV types (HPV6 and HPV11). The assay combines two different techniques: multiplex PCR using HPV type-specific primers for amplification of viral DNA, and a Luminex analyzer (Luminex Corp., Austin, TX) to identify the internal bead color and quantify the fluorescence, expressed as median fluorescence intensity (MFI). Two primers for amplification of the beta-globin gene were also included to control for the quality of the template DNA.
Statistical analysis
The characteristics of participants, stratified by the number of vaccine doses received, were presented as proportions. The observed and age-standardized prevalence of the type-specific oral HPV infections were shown as proportions. Age standardization was done to remove the influence of age on the HPV prevalence estimates and make them comparable across vaccine dose groups. The age standardization was done using the direct standardization method obtained from the formulae of the improved estimate by Dobson et al. (1991) [23] and using the 2011 Indian female population as the standard population. To assess the effect of being vaccinated on the HPV type-specific prevalence, odds ratios (ORs) and their 95 % confidence intervals (CIs) were estimated using exact logistic regression and adjusted for age at oral sample collection.
Results
The characteristics of women enrolled in the IARC cohort study of two versus three doses of HPV vaccine in India and selected for this substudy are shown in Table 1. A total of 997 oral samples, from 818 vaccinated and 179 unvaccinated women, were collected from Ambilikkai (n = 328, 32.9 %), Barshi (n = 270, 27.1 %), and Pune (n = 399, 40.0 %). The number of participants in the different dose groups was as follows: three doses (days 1, 60, and 180), 323 (32.4 %); two doses (days 1 and 180), 190 (19.0 %); two doses (days 1 and 60), 101 (10.1 %); a single dose, 204 (20.5 %); and unvaccinated, 179 (18.0 %) (Table 1).
Table 1.
Characteristics of women for oral HPV genotyping by the vaccine dose received.
| Vaccine dose received |
||||||
|---|---|---|---|---|---|---|
| 3 doses (Days 1, 60 and ≥ 180) |
2 doses (Days 1 and ≥ 180) |
2 doses (Days 1 and 60) |
A single dose |
Vaccinated women |
Unvaccinated women |
|
| n (%) | n (%) | n (%) | n (%) | n (%) | n (%) | |
| Women assessed | 323 | 190 | 101 | 204 | 818 | 179 |
| Birth cohort | ||||||
| <1995 | 190 (58.8) | 129 (67.9) | 54 (53.5) | 140 (68.6) | 513 (62.7) | 150 (83.8) |
| 1995+ | 133 (41.2) | 61 (32.1) | 47 (46.5) | 64 (31.4) | 305 (37.3) | 29 (16.2) |
| Religion | ||||||
| Hindu | 317 (98.1) | 183 (96.3) | 91 (90.1) | 195 (95.6) | 786 (96.1) | 159 (88.8) |
| Others | 6 (1.9) | 7 (3.7) | 10 (9.9) | 8 (3.9) | 31 (3.8) | 18 (10.1) |
| Education | ||||||
| Nil, primary or middle | 56 (17.3) | 25 (13.2) | 12 (11.9) | 51 (25.0) | 144 (17.6) | 50 (27.9) |
| High or college | 267 (82.7) | 165 (86.8) | 89 (88.1) | 152 (74.5) | 673 (82.3) | 129 (72.1) |
| Monthly household income (rupees)* | ||||||
| <15,000 | 226 (74.1) | 131 (70.4) | 63 (67.7) | 140 (71.4) | 560 (71.8) | 106 (59.9) |
| 15,000–29,999 | 42 (13.8) | 33 (17.7) | 23 (24.7) | 34 (17.3) | 132 (16.9) | 46 (26.0) |
| 30,000+ | 37 (12.1) | 22 (11.8) | 7 (7.5) | 22 (11.2) | 88 (11.3) | 25 (14.1) |
| Total no. of pregnancies | ||||||
| None | 96 (29.7) | 31 (16.3) | 14 (13.9) | 25 (12.3) | 166 (20.3) | 9 (5.0) |
| 1 | 141 (43.7) | 98 (51.6) | 60 (59.4) | 89 (43.6) | 388 (47.4) | 47 (26.3) |
| 2+ | 86 (26.6) | 61 (32.1) | 27 (26.7) | 90 (44.1) | 264 (32.3) | 123 (68.7) |
| Mean (SD) | 1.0 (0.8) | 1.2 (0.8) | 1.2 (0.8) | 1.4 (0.8) | 1.8 (0.8) | 1.2 (0.8) |
| (Range) | (0–4) | (0–3) | (0–4) | (0–4) | (0–5) | (0–4) |
| Age at oral sample collection (years) | ||||||
| 18–20 | 129 (39.9) | 59 (31.1) | 49 (48.5) | 56 (27.5) | 293 (35.8) | 13 (7.3) |
| 21–25 | 194 (60.1) | 131 (68.9) | 52 (51.5) | 148 (72.5) | 525 (64.2) | 166 (92.7) |
| Mean (SD) | 20.9 (1.9) | 21.4 (1.8) | 20.7 (1.9) | 21.6 (1.8) | 21.2 (1.9) | 22.3 (1.2) |
| (Range) | (18–25) | (18–25) | (18–24) | (18–25) | (18–25) | (19–25) |
| Interval between marriage and oral sample collection (years) | ||||||
| <2 | 78 (26.9) | 41 (21.7) | 34 (33.7) | 25 (12.3) | 178 (22.7) | 0 (0.0) |
| 2–<3 | 61 (21.0) | 59 (31.2) | 37 (36.6) | 33 (16.3) | 190 (24.3) | 8 (4.5) |
| 3–<4 | 71 (24.5) | 37 (19.6) | 10 (9.9) | 36 (17.7) | 154 (19.7) | 26 (14.5) |
| 4-<5 | 58 (20.0) | 33 (17.5) | 12 (11.9) | 63 (31.0) | 166 (21.2) | 61 (34.1) |
| 5+ | 22 (7.6) | 19 (10.1) | 8 (7.9) | 46 (22.7) | 95 (12.1) | 84 (46.9) |
| Mean (SD) | 3.2 (1.3) | 3.2 (1.3) | 2.7 (1.2) | 3.9 (1.4) | 3.2 (1.4) | 5.0 (1.4) |
| (Range) | (0.8–6.3) | (0.8–6.3) | (1.2–5.4) | (1.2–6.5) | (0.0–6.5) | (2.3–10.5) |
50 rupees ≈ 1 US dollar; SD: standard deviation.
The mean age of the participants at the time of oral sample collection was similar across the dose groups (three doses: 20.9 ± 1.9 years; two doses (days 1 and 180): 21.4 ± 1.8 years; two doses (days 1 and 60): 20.7 ± 1.9 years; a single dose: 21.6 ± 1.8 years; unvaccinated: 22.3 ± 1.2 years. The age range of the participants was 18–25 years for the vaccinated group and 19–25 years for the unvaccinated group. The mean time lag between marriage and oral sample collection was 3.2 ± 1.4 years for the vaccinated group and 5.0 ± 1.4 years for the unvaccinated group. Of note, 33.3 % of the vaccinated women and 81.0 % of the unvaccinated women provided an oral sample ≥ 4 years after marriage (Table 1).
Oral samples from 818 vaccinated and 179 unvaccinated women were tested by E7-MPG for 19 HR or possible or probable HR types and two LR types (HPV6 and HPV11), and the results are shown in Table 2. The age-standardized proportion (ASP) of HPV16/18 infections was 2.0 % (95 % CI, 1.0–3.0 %) in vaccinated women and 4.2 % (95 % CI, 1.2–7.2 %) in unvaccinated women, with a non-significant difference between the two groups (OR = 0.4; 95 % CI, 0.2–1.0). Specifically, HPV16 was detected in 3.5 % of single-dose recipients, 1.2 % of two-dose recipients (days 1 and 180), and 1.5 % of three-dose recipients (days 1, 60, and 180), whereas 3.3 % of the unvaccinated women tested positive for HPV16. The prevalence of HPV18 was higher in unvaccinated women (0.8 %) than in vaccinated women (0.4 %), although this difference was not statistically significant. Similarly, the prevalence of HPV11 was higher in unvaccinated women (3.3 %) than in vaccinated women (2.9 %), but the difference was not significant (OR = 0.8; 95 % CI, 0.3–1.9). Only one vaccinated woman tested positive for HPV6, and none of the unvaccinated women tested positive for HPV6. The frequency of prevalent infections with non-vaccine-targeted HPV types was similar in the vaccinated and unvaccinated groups (2.1 % in each) (Table 2). The HPV types not targeted by the quadrivalent vaccine detected in the samples were HPV types 33, 39, 51, 52, 56, 58, 66, 70, 73, and 82.
Table 2.
Oral HPV infections prevalence by the vaccine dose received.
| HPV type | Vaccine dose received |
Vaccinated women | Unvaccinated women | |||
|---|---|---|---|---|---|---|
| 3 doses (Days 1, 60 and 180) | 2 doses (Days 1 and 180) | 2 doses (Days 1 and 60) | A single dose | |||
| Women assessed | 323 | 190 | 101 | 204 | 818 | 179 |
| Hisk-risk types | ||||||
| HPV 16, - n | 6 | 2 | 0 | 5 | 13 | 6 |
| CP, - % | 1.9 | 1.1 | 2.5 | 1.6 | 3.4 | |
| ASP, - % | 1.5 | 1.2 | 3.5 | 1.5 | 3.3 | |
| (95 % CI), - % | (0.3–2.7) | (−0.6–3.1) | (0.2–6.7) | (0.7–2.4) | (0.5–6.2) | |
| Odds ratio* | 0.5 | 1.0 | ||||
| (95 % CI), - % | (0.2–1.4) | |||||
| HPV 18, - n | 2 | 0 | 1 | 0 | 3 | 2 |
| CP, - % | 0.6 | 1.0 | 0.4 | 1.1 | ||
| ASP, - % | 0.6 | 1.1 | 0.4 | 0.8 | ||
| (95 % CI), - % | (−0.3–1.6) | (−1.1–3.3) | (−0.1–1.0) | (−0.3–1.9) | ||
| Odds ratio* | 0.2 | 1.0 | ||||
| (95 % CI), - % | (0.0–2.2) | |||||
| HPV 31, - n | 0 | 0 | 0 | 0 | 0 | 0 |
| HPV 33, - n | 0 | 0 | 0 | 1 | 1 | 0 |
| CP, - % | 0.5 | 0.1 | ||||
| HPV 35, - n | 0 | 0 | 0 | 0 | 0 | 0 |
| HPV 39, - n | 0 | 1 | 0 | 1 | 2 | 0 |
| CP, - % | 0.5 | 0.5 | 0.2 | |||
| HPV 45, - n | 0 | 0 | 0 | 0 | 0 | 0 |
| HPV 51, - n | 1 | 0 | 0 | 1 | 2 | 0 |
| CP, - % | 0.3 | 0.5 | 0.2 | |||
| HPV 52, - n | 0 | 0 | 0 | 1 | 1 | 1 |
| CP, - % | 0.5 | 0.1 | 0.6 | |||
| HPV 56, - n | 0 | 0 | 0 | 1 | 1 | 0 |
| CP, - % | 0.5 | 0.1 | ||||
| HPV 58, - n | 0 | 1 | 0 | 2 | 3 | 0 |
| CP, - % | 0.5 | 1.0 | 0.4 | |||
| HPV 59, - n | 0 | 0 | 0 | 0 | 0 | 0 |
| Probably high-risk types | ||||||
| HPV 26, - n | 0 | 0 | 0 | 0 | 0 | 0 |
| HPV 53, - n | 0 | 0 | 0 | 0 | 0 | 0 |
| HPV 66, - n | 1 | 2 | 0 | 1 | 4 | 2 |
| CP, - % | 0.3 | 1.1 | 0.5 | 0.5 | 1.1 | |
| HPV 68, - n | 0 | 0 | 0 | 0 | 0 | 0 |
| HPV 70, - n | 0 | 0 | 0 | 1 | 1 | 0 |
| CP, - % | 0.5 | 0.1 | ||||
| HPV 73, - n | 1 | 0 | 0 | 1 | 2 | 0 |
| CP, - % | 0.3 | 0.5 | 0.2 | |||
| HPV 82, - n | 1 | 1 | 0 | 0 | 2 | 0 |
| CP, - % | 0.3 | 0.5 | 0.2 | |||
| Low-risk types | ||||||
| HPV 6, - n | 1 | 0 | 0 | 0 | 1 | 0 |
| CP, - % | 0.3 | 0.1 | ||||
| HPV 11, - n | 7 | 4 | 3 | 10 | 24 | 7 |
| CP, - % | 2.2 | 2.1 | 3.0 | 4.9 | 2.9 | 3.9 |
| ASP, - % | 2.3 | 2.0 | 2.6 | 5.1 | 2.9 | 3.3 |
| (95 % CI), - % | (0.6–4.1) | (−0.1–4.0) | (−0.3–5.5) | (1.6–8.6) | (1.7–4.1) | (0.8–5.8) |
| Odds ratio* | 0.8 | 1.0 | ||||
| (95 % CI), - % | (0.3–2.1) | |||||
| Type combinations | ||||||
| HPV 16/18, - n | 8 | 2 | 1 | 5 | 16 | 8 |
| CP, - % | 2.5 | 1.1 | 1.0 | 2.5 | 2.0 | 4.5 |
| ASP, - % | 2.1 | 1.2 | 1.1 | 3.5 | 2.0 | 4.2 |
| (95 % CI), - % | (0.7–3.6) | (−0.6–3.1) | (−1.1–3.3) | (0.2–6.7) | (1.0–3.0) | (1.2–7.2) |
| Odds ratio* | 0.4 | 1.0 | ||||
| (95 % CI), - % | (0.2 -1.0) | |||||
| HPV 16/18/6/11, - n | 15 | 6 | 4 | 14 | 39 | 15 |
| CP, - % | 4.6 | 3.2 | 4.0 | 6.9 | 4.8 | 8.4 |
| ASP, - % | 4.3 | 3.2 | 3.7 | 8.2 | 4.7 | 7.5 |
| (95 % CI), - % | (2.1–6.5) | (0.4–5.9) | (0.1–7.4) | (3.6–12.8) | (3.2–6.2) | (3.6–11.3) |
| Odds ratio* | 0.6 | 1.0 | ||||
| (95 % CI), - % | (0.3 -1.1) | |||||
| Non HPV 16/18/6/11, - n | 3 | 4 | 0 | 9 | 16 | 3 |
| CP, - % | 0.9 | 2.1 | 4.4 | 2.0 | 1.7 | |
| ASP, - % | 1.0 | 2.5 | 4.7 | 2.1 | 2.1 | |
| (95 % CI), - % | (−0.2–2.2) | (−0.1–5.0) | (1.3–8.2) | (1.0–3.1) | (−0.3–4.6) | |
| Odds ratio* | 1.3 | 1.0 | ||||
| (95 % CI), - % | (0.4 -5.8) | |||||
| Any HPV, - n | 16 | 10 | 4 | 21 | 51 | 17 |
| CP, - % | 5.0 | 5.3 | 4.0 | 10.3 | 6.2 | 9.5 |
| ASP, - % | 4.7 | 5.7 | 3.7 | 11.7 | 6.2 | 8.7 |
| (95 % CI), - % | (2.4–7.0) | (2.0–9.4) | (0.1–7.4) | (6.4–17.0) | (4.4–7.9) | (4.5–13.0) |
| Odds ratio* | 0.7 | 1.0 | ||||
| (95 % CI), - % | (0.4 -1.2) | |||||
HPV: human papillomavirus; CP: crude proportion; ASP: age standardized proportion; CI: confidence interval; * Adjusted for age at oral sample collection.
A total of 648 (82.5 %) vaccinated and unvaccinated participants provided a cervical sample on the same day as the oral sampling, and an additional 137 (17.5 %) participants provided a cervical sample within 6 months of the oral sampling. Of the 785 participants with both cervical and oral samples, 87 (11.1 %) were positive for any HPV type in the cervix, and 55 (7.0 %) had any HPV infection in the oral cavity. For the vaccine-targeted HPV types, the prevalence was 2.0 % (16/785) in the cervix compared with 5.5 % (43/785) for the oral cavity. No infections with the same HPV types were detected in the oral cavity and the cervix of the same woman. However, HPV infections of different types were detected in both anatomical sites of six participants: HPV11 vs HPV52/53/58 (n = 1), HPV11 vs HPV58 (n = 2), HPV11 vs HPV66 (n = 1), HPV11/16 vs HPV52/82 (n = 1), and HPV56 vs HPV51 (n = 1). Among these 785 participants, a total of 20 different HPV types were found in the cervix compared with 14 in the oral cavity, out of the 21 HPV types assessed. HPV16 (2.0 %, n = 16) and HPV11 (3.1 %, n = 24) appeared to be the most common HPV types in the oral cavity, whereas HPV58 (3.2 %, n = 25) and HPV52 (2.0 %, n = 16), followed by HPV16 (1.4 %, n = 11) were the most common HPV types in the cervix (data not shown).
Discussion
Unlike for the cervix, the natural history of HPV infection in the oral cavity is still poorly studied. Several studies have shown that HPV colonizes the oral mucosa through sexual activities or autoinoculation; however, the possibility of autoinoculation from the genitals to the oral site still remains unclear [24]. Sexual behavior is a well-established determinant of oral infection by mucosal HPV types. The number of oral sex partners is a strong risk factor for HPV-related OPC [25], [26], [27], [28]. In addition, tobacco consumption has been identified as a strong risk factor for HPV-related OPC [25], [29], [30]. Age and cigarette smoking were also independently associated with oral HPV infection [31]. However, in the present study, the role of tobacco and sexual behaviors could not be evaluated.
In our study, 3.3 % of the unvaccinated women tested positive for HPV16, which is almost three times the prevalence that has previously been shown elsewhere. Kreimer et al. (2010) [32] showed that HPV16 was present in the oral cavity of 1.3 % of 3977 healthy subjects. Moreover, a meta-analysis showed that 0.8 % of women have oral infection with HPV16 [33]. Similarly, a meta-analysis performed by Mena et al. (2019) [34] estimated the oral prevalence of HPV16 to be 1.0 %. Other studies reported that HPV prevalence in the oral cavity of healthy adults can vary, from 0 % in southern India to 0.6 % in Japan and China [35], [36], 3.5 % in the United States [30], 4.0 % in Italy [37], and 7.3 % in Brazil [38], with a sex-ratio imbalance in favor of men [30]. HPV DNA positivity in the oral cavity could be affected by the sampling method (e.g., saliva or oral rinses) as well as by the sensitivity of the HPV genotyping assay used [26], [39]. The highest prevalence in our study might be explained by the ultra-sensitivity of the well-validated E7-MPG assay [22], [40].
Our findings show that vaccinated women had a reduced prevalence of HPV16 and/or HPV18 oral viral infection compared with unvaccinated women, with a non-significant difference between the two groups (OR = 0.4; 95 % CI, 0.2–1.0), although no difference was observed between single-dose recipients and unvaccinated women.
In addition, in our study, HPV infection appears to be less common in the oral cavity than in the cervix, as reported elsewhere [41]. Only 14 of the 20 HPV types found to infect the cervix were also found within the oral cavity, although not concurrently. However, an opposite trend was observed for vaccine-targeted HPV types (i.e., HPV11, HPV16, and HPV18). For vaccine-targeted HPV types, the prevalence was lower in the cervix than in the oral cavity (2.0 % vs 5.5 %) of the same participants who provided both oral and cervical samples within a 6-month period, and no concurrent HPV infections were detected, which is consistent with other reports, particularly in US studies where type-specific concordance at the two sites was low [42], [43].
In our study, HPV16 made up half of all HPV infections of the oral cavity of both vaccinated and unvaccinated women. The remaining HR HPV types were HPV18, 33, 39, 51, 52, 56, and 58. A slightly different distribution was reported by Kreimer et al. (2010) [32]: HPV16 was present in only 28 % of all HPV detected in the oral region from US participants. In our study, the non-vaccine-targeted HPV types (HPV types 33, 39, 51, 52, 56, 58, 66, 70, 73, and 82) were equally distributed in unvaccinated and vaccinated women.
A persistent HPV infection is a prerequisite for the development of cervical cancer and OPC; however, most people clear the infection within 2 years [44], [45]. In a 7-year natural history study of oral HPV infection, 70 % of oral HPV infections were cleared within 2 years. One third of the HPV16 infections persisted for at least 5 years, making this HPV type less likely to clear than other HR HPV types [44].
Our findings agree with those of other previous studies on the efficacy of HPV vaccination in reducing oral HPV infections. In recent studies performed in the United States and Colombia [46], [47], the prevalence of vaccine-targeted HPV types was significantly lower in participants who had received the HPV vaccine than in those who were unvaccinated. As a consequence of the HPV vaccination efforts in the United States, the prevalence of vaccine-targeted HPV types in oral rinses was on a declining trend from 2009 to 2016 in unvaccinated men and women, suggesting herd protection against oral HPV infection [48].
The observation from our study that a single dose of HPV vaccine may be less protective than two or three doses against oral HPV infection is contrary to the high protective efficacy of a single dose observed against persistent HPV16 or HPV18 infection in the cervix [21]. Studies have shown that a small amount of vaccine-induced antibody can efficiently neutralize HPV before the virus gets internalized to the basal layer of cells of the cervical epithelium [49].
However, the mechanism of oral infection with HPV and the possible role of virus-induced antibodies in preventing the infection are much less studied.
Limitations of our study are that we collected a single oral cavity sample and we were not able to discriminate between persistent and transitory HPV infections, as well as the imbalance between the sizes of the vaccinated and unvaccinated groups.
Conclusions
In our study, we performed for the first time a comparison of the impact of HPV vaccine protocols and doses on the HPV DNA prevalence in the oral cavity, providing indications that a single vaccine dose may be less efficient than two or three doses in preventing oral HPV infection. To better evaluate the efficacy of HPV vaccine in the upper respiratory tract, future studies are required, in which multiple oral specimens are collected and the presence of viral DNA is determined at different times after vaccination, to establish persistent infection.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
Acknowledgments
We are grateful to Dr. Karen Müller and Ms. Nicole Suty for their help with the preparation of this manuscript.
Funding sources
This study was funded by the European Commission HPV-AHEAD (grant number FP7-HEALTH-2011-282562) and partially funded by the Italian Ministry of Health (“Ricerca Corrente 2022” Del. 219/2022). This work was also financially supported by the Bill & Melinda Gates Foundation (grant number OPP48979).
Ethics approval
The original study was approved by IARC Ethics Committee and the ethics committees of all participating institutions. The substudy was approved by ethics committees from the participating study sites.
Disclaimer
Where authors are identified as personnel of the International Agency for Research on Cancer/World Health Organization and the Istituto Tumori Giovanni Paolo II, the authors alone are responsible for the views expressed in this article and they do not necessarily represent the decisions, policy, or views of the respective institutes.
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