The natural history of infections with many human papillomavirus (HPV) types is poorly understood. Here, we describe for the first time the age- and sex-dependent antibody prevalence for 29 cutaneous and five mucosal HPV types from 15 species within five phylogenetic genera (alpha, beta, gamma, mu, nu) in a general population. Sera from 1,797 German adults and children (758 males and 1,039 females) between 1 and 82 years (median 37 years) were analysed for antibodies to the major capsid protein L1 by Luminex-based multiplex serology. The first substantial HPV antibody reactions observed already in children and young adults are those to cutaneous types of the genera nu (HPV 41) and mu (HPV 1, 63). The antibody prevalence to mucosal high-risk types, most prominently HPV 16, was elevated after puberty in women but not in men and peaked between 25 and 34 years. Antibodies to beta and gamma papillomaviruses (PV) were rare in children and increased homogeneously with age, with prevalence peaks at 40 and 60 years in women and 50 and 70 years in men. Antibodies to cutaneous alpha PV showed a heterogeneous age distribution. In summary, these data suggest three major seroprevalence patterns for HPV of phylogenetically distinct genera: antibodies to mu and nu skin PV appear early in life, those to mucosal alpha PV in women after puberty, and antibodies to beta as well as to gamma skin PV accumulate later in life.
Papillomaviruses (PV) are a large and highly diverse group of DNA viruses that infect cutaneous and mucosal epithelia of warm-blooded vertebrates. Of the more than 100 identified human PV (HPV) types, many cause benign lesions like warts and papillomas, and some also cervical, other anogenital, and oral cancers. For most HPV, transmission routes, pathogenesis, and time and duration of infection are only poorly understood. In the German general population, we investigated the prevalence of antibodies to the capsid proteins of 34 HPV types representative of all five PV genera (alpha, beta, gamma, mu, and nu) that contain HPV. We provide evidence for different age- and sex-dependent seroprevalence patterns of phylogenetically related HPV: antibodies to cutaneous mu and nu PV appear early in life, those to mucosal alpha PV after puberty, and those to beta and gamma skin PV accumulate in adulthood.
Citation: Michael KM, Waterboer T, Sehr P, Rother A, Reidel U, et al. (2008) Seroprevalence of 34 Human Papillomavirus Types in the German General Population. PLoS Pathog 4(6): e1000091. doi:10.1371/journal.ppat.1000091
Editor: John T. Schiller, National Cancer Institute, United States of America
Received: February 5, 2008; Accepted: May 16, 2008; Published: June 20, 2008
Copyright: © 2008 Michael et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: We gratefully acknowledge the financial support from the European Commission for this study (QLK2-CT-2002-0117). Tim Waterboer was supported by the Peter und Traudl Engelhorn-Stiftung zur Förderung der Biotechnologie und Gentechnik. Ignacio G. Bravo is supported by the Initiative on Evolutionary Biology from the Volkswagen Foundation.
Competing interests: The authors have declared that no competing interests exist.
Papillomaviruses (PV) are non-enveloped DNA viruses infecting cutaneous or mucosal epithelia of warm-blooded vertebrates. So far at least 118 distinct PV types, more than 100 of them isolated from humans, have been completely described . In addition, about 130 L1 sequence fragments have been isolated by means of a broad spectrum polymerase chain reaction (PCR) representing putatively new cutaneous human papillomavirus (HPV) types . Based on the nucleotide sequence encoding the major capsid protein L1, PV systematics defines 16 genera (sharing less than 60% sequence identity) which encompass 44 species (sharing 60–70% sequence identity).
PV within the same genus may or may not show similar biological and pathological characteristics. Thus, cutaneous HPV are found among the five genera alpha (species 2, 4 and 8), beta (β), gamma (γ), mu (μ), and nu (ν), whereas the 48 HPV types infecting the mucosa belong exclusively to the genus alpha (α). HPV infections are widespread and can cause a variety of mostly benign tumours such as warts and condylomata. However, the infection with certain mucosal HPV types leads to malignant cell proliferation . Fifteen so-called high-risk (HR) and three putative HR mucosal HPV of genus α, most notably the two most prevalent HR types 16 and 18, are found in more than 90% of cervical tumours  and with lower frequency in other anogenital and oro-pharyngeal carcinomas . Thirteen of these HPV types have recently been classified as human carcinogens . The mucosal low-risk (LR) HPV types 6 and 11 cause benign genital lesions like condylomata acuminata and low-grade squamous intraepithelial lesions of the cervix. The cutaneous HPV types 1 (genus μ), 2, 3, 10, 57 (genus α) and HPV 4 (genus γ), although belonging to three different genera, are associated with benign plantar, common, and flat skin warts both in the general population and in renal transplant recipients ,. β PV are found in high copy numbers in benign macular skin lesions of patients with the rare hereditary disease Epidermodysplasia verruciformis (EV) . The same types are also found in non-melanoma skin cancer (NMSC), namely squamous cell carcinoma (SCC) and less frequently basal cell carcinoma (BCC) of the skin, but also in normal skin and plucked hairs of EV patients, immunosuppressed patients, e.g. transplant recipients, and less frequently immunocompetent patients ,. γ PV and HPV 41 (genus ν) cause benign skin lesions but have been also found in NMSC ,,,. Thus, infections with cutaneous HPV are discussed to play a role in the development of NMSC ,.
Besides the extensively studied HPV 16 and some closely related mucosal HR types, only little is known about the natural history of infections by other types. For both HR (e.g. HPV 16, 18, 31, 33, 35, 45, 52, 58) and LR (e.g. HPV 6 and 11) mucosal HPV, transmission occurs mainly via sexual intercourse . However, non-sexual routes of transmission, e.g. oral and perinatal transmission have been reported and cannot be entirely disregarded ,. The rare disease recurrent respiratory papillomatosis apparently is caused by perinatal transmission of the LR HPV types 6 and 11 . Infections with cutaneous HPV are assumed to occur via skin contact with contaminated material.
For mucosal HPV types, antibodies to the viral major capsid protein L1 have been shown to be markers for present and past infection. Thus serology provides a powerful epidemiological tool to investigate the wide variety of mucosal and cutaneous HPV types and their distribution in the population . DNA detection methods identify only current infections and are restricted to small samples of certain bodily sites. In contrast, for HPV 16 it has been shown that serology may be used as a proxy for lifetime cumulative exposure . However, only 50–60% of HPV 16 DNA positive women develop HPV antibodies and seroconversion can occur even several months after the infection ,. HPV 16 L1 antibodies have low but stable titres and can be detected decades after the infection . Antibodies to HPV 16 L1 have been shown to be type-specific ,, although some cross-reactivity with closely related types was reported ,. For the other HPV types, similar characteristics of L1 antibodies are assumed.
Seroepidemiological studies especially on cutaneous HPV infection are scarce and those performed in the past were restricted to a single or few HPV types. To our knowledge, mucosal HPV L1 antibody analyses have been performed so far for HR HPV types 16, 18, 31, 33, 39, 45, 52, 58, 59, 73, for LR HPV types 6 and 11 and for HPV types 13 and 32. The serologically best studied cutaneous types are HPV 1, 5, and 8 –, while little or nothing is known about the antibody response to other cutaneous types –.
HPV antibody analyses are complex because of the large number of HPV types, and conventional enzyme-linked immunosorbent assay (ELISA) methods allow analysing the reactivity of a serum sample to only one antigen per reaction. We recently developed multiplex serology , that allows the simultaneous determination of antibodies to a large number of HPV types and the analysis of more than 1,000 sera per day. Multiplex serology uses viral L1 proteins expressed in bacteria as glutathione S-transferase (GST) fusion proteins as antigens ,. Rizk et al.  demonstrated that these recombinant L1 proteins display conformational epitopes. Three first case-control studies on NMSC investigating the antibody response to a total of 16 , 31 , and 38  different HPV types have been reported. However, our knowledge about the natural history of HPV infections is still limited.
Here we describe for the first time the age- and sex-dependent antibody prevalence for 29 cutaneous and five mucosal HPV types representing 15 species within five phylogenetic genera (α, β, γ, μ, ν) in a general population.
HPV antibody reactivity patterns
To obtain an unbiased (cut-off free) impression of HPV antibodies, the strength of the antibody reactions was plotted against the percentile for each HPV type and age and sex group. Figure 1 shows the plots for HPV types 1, 16 and 8, which are representative for the three major antibody patterns found among the 34 types analysed.
Figure 1. Distribution of antibody reactivity for HPV types 1, 16 and 8 in the German population.
Antibodies against HPV L1 proteins were analysed in 1797 sera from the German general population and stratified by sex and age. The strength of the antibody reactions expressed as median fluorescence intensity (MFI) on the y-axis is plotted against the percentile on the x-axis. Data points were connected by spline curves. Colour code, age groups in years and size of the groups are indicated in the inserts. Dashed black lines indicate the cut-off of 200 MFI. The figure reads as follows (upper left panel, HPV 1 antibodies among females): In the 45–54 years age group, approximately 40% showed antibody reactions above 50 MFI, approximately 30% above 100 MFI, and approximately 20% above 200 MFI. For all HPV types and in both genders antibody reactivity was lowest in children. The biggest difference in antibody reactivity was observed between children and young adults (15–24 years) against HPV 1. In comparison to children, antibody levels against HPV 16 were substantially higher in young women but not in men. Antibody reactivity to HPV 8 became stronger with age in both genders and peaked in women at about 40 years and in men at about 50 years. Especially among men, a second peak was found in the oldest age group. These antibody patterns were present also for weak responses well below the chosen cut-off.doi:10.1371/journal.ppat.1000091.g001
For all HPV types and in both sexes, antibody reactivity was lowest in children. The biggest difference in antibody reactivity was observed between children and young adults (15–24 years) against HPV 1, which is representative for the HPV types within the μ and ν genera. HPV 1 antibody reactivities were similar in adults aged 25–54 years and gradually declined thereafter.
Antibody levels against HPV 16, which is representative for genital/mucosal HPV types within the α PV, changed with age in women but hardly in men. In comparison to children, antibody reactivities were substantially higher in young women and peaked in the 25–34 years age group.
Antibody reactivity to HPV 8, which is representative for HPV types within the β and γ PV, became stronger with age in both genders. A first peak in antibody reactivity was observed in women at about 40 years and in men at about 50 years. Especially among men, a second peak was found in the oldest age group.
These age-dependent antibody patterns were present for both strong and weak responses, down to reactions as low as 50 median fluorescence intensities (MFI).
HPV antibody prevalence
The analysis of the complex antibody patterns was simplified by the generation of seroprevalence values. HPV seroprevalence is defined here for all HPV types and age and sex strata as percent of sera reacting with a given HPV type above a cut-off value of 200 MFI.
In all 1797 sera, the overall seroprevalence for any of the 34 HPV types analysed was 59.7% (Table 1). However, the overall type-specific seroprevalences strongly depended on age and sex.
Table 1. Type-specific age- and sex-stratified HPV antibody prevalence in the German population.doi:10.1371/journal.ppat.1000091.t001
HPV types 1 and 4 showed the highest type-specific seroprevalences (17.0 and 16.9%, respectively) (Table 1). Among children, HPV antibody prevalences were mostly low, with the highest seroprevalences for types 3 (8.6%), 1 (6.4%), and 4 (4.8%). Among adults (individuals post-puberty, ≥15 years), seroprevalence was highest again for HPV types 4 (18.3%) and 1 (18.2%), followed by types 8 (12.4%), 63 (11.5%), 65 (11.0%), and 49 (10.1%), and lowest (<1.5%) for types 52, 60 and 93.
Seroprevalence patterns were similar for μ and ν PV, for high-risk mucosal α PV in females, and for β and γ PV, respectively (Figure 2). Most HPV showed two seroprevalence peaks. Age at the first peak varied from about 20 to 50 years for the different genera, but the second prevalence peak rather uniformly occurred at old age, in females at about 60 years and in males at above 64 years.
Figure 2. Prevalence of antibodies to 34 HPV types in the German population.
Antibodies against HPV L1 proteins of 29 cutaneous and 5 mucosal HPV types were analysed in 1797 sera from the German general population and stratified by sex and age. The type-specific seroprevalences in seven age groups are shown for females and males separately. Colour code, age groups in years and size of the groups are indicated in the inserts. HPV types are specified below each graph, and genus, species, and tropism are shown between the graphs. Seroprevalence patterns were similar for μ and ν PV, for high-risk mucosal α PV in females, and for β and γ PV, respectively. Antibodies to cutaneous μ and ν PV were already present in children with substantial prevalences in young adults (15–24 years). Antibodies to mucosal high-risk HPV appeared after puberty and peaked in the 25–34 years group mainly in women. Seroprevalence for β and γ PV accumulated and peaked late in life in both genders. Most HPV showed two seroprevalence peaks. Age at the first peak varied from about 20 to 50 years for the different genera, but the second prevalence peak rather uniformly occurred at old age, in females at about 60 years and in males at above 64 years.doi:10.1371/journal.ppat.1000091.g002
For statistical analysis of the age- and sex-dependent seroprevalence patterns, age groups were combined to increase statistical power (Table 1). Highly significant seroprevalence increases (p<0.0001) from children to younger adults (15–34 years) (but not from younger to older adults >34 years) were seen for mucosal α (HPV 16), γ (HPV 4, 65) and both μ PV (HPV 1, 63). In contrast, none of the 15 β PV types showed highly significant seroprevalence increases from children to younger adults, but 6 types from younger to older adults (HPV 49, 76, 75, 38, 17 and 8). Multiple seropositivity (>3, >8 types) increased significantly (at least p<0.05) from children to younger adults but also from younger to older adults.
The strongest sex difference was observed for the mucosal high-risk HPV type 16. Seroprevalence in younger women was 5.8-fold and in older women still 2.4-fold higher than in men of the same age. For two other mucosal high-risk types, seroprevalence was also slightly elevated in younger women (HPV 18, 2.1-fold and HPV 33, 1.6-fold), however without statistical significance. Seroprevalence for cutaneous α, β and γ PV tended to be higher in younger women than in younger men (median 1.3-fold), but HPV 2 was the only individual type for which this difference was statistically significant. In contrast, women >34 years showed a lower seroprevalence for these genera than men (median 0.8-fold), with significant differences for the HPV types 75, 9, and 5.
Multiple HPV seropositivity
Antibody reactivity to more than one HPV type was frequent (Table 1). While 24.5% of the sera (41.0% of the antibody positives) were single positive, 11.5% reacted with two, 7.4% with three and 16.2% with more than three HPV types. Even seropositivities to more than 8 (6.1%) and more than 16 (1.9%) types were observed. Multiple seropositivity (Figure 3), here defined operationally as antibody positivity to at least half of the HPV types analysed within a species or genus, followed in general the patterns seen for type-specific seroprevalences (Figure 2). Multiple seroreactions were rare in children (<15 years) and were most prevalent in the second half of life (>34 years). Mucosal and cutaneous α PV showed substantially less multiple seropositivity than β, γ, μ and ν types.
Figure 3. Multiple HPV seropositivity stratified by age and sex.
Frequency of multiple seropositivity (seropositivity to at least half of the HPV types analysed within a species or genus) in seven age groups are shown for females and males separately. Colour code, age groups in years and size of the groups are specified in the inserts. Phylogenetic groups (genera and species) and their specific multiple seropositivity definitions (e.g. seropositive for ≥8 out of 15 β PV types) are indicated. muc., mucosal; cut., cutaneous. Multiple seropositivity was lowest for mucosal and cutaneous α PV and in general followed the patterns seen for type-specific seroprevalences (Fig. 2). For μ and ν PV, it was the earliest to occur in young adults of both genders. For mucosal α PV, it was present mainly in women and peaked at 25–34 years. For cutaneous α PV, multiple seropositivity showed an isolated peak in the oldest age group. For β and γ PV, both genders showed two prevalence peaks, in women at around 35 and 60 years and in men some 5 to 10 years later.doi:10.1371/journal.ppat.1000091.g003
Multiple seropositvity to μ and ν PV showed a steep increase already in young adults (15–24 years) and thus was the earliest to occur in both genders. Multiple seropositivity to high-risk mucosal α PV in women (but not in men) first occurred and also peaked at 25 to 34 years. For cutaneous α PV, multiple seropositivity showed an isolated peak in the oldest age group in both genders. For β and γ PV, both genders showed two prevalence peaks, in women at around 35 and 60 years and in men some 5 to 10 years later.
Fundamental data on the humoral immune response to HPV infections is scarce. Most serological analyses performed in the past were case-control studies that mainly focussed on mucosal HR HPV types highly prevalent in cervical cancer and mostly on women at productive age. Little is known about the age distribution of HPV infections, especially in children, men and in old-aged individuals, and for many HPV types no serological data is available at all. To understand the natural history of HPV infections, investigations of a broad range of HPV types belonging to different species and genera are needed. This is the first large study to analyse simultaneously antibody reactions to 34 HPV types in a general population. The cross-sectional data presented here allow a detailed and comprehensive assessment of the seroprevalence in the adult German general population by HPV type (and higher taxonomic order), sex and age. The data for the children originating from two hospitalized groups may be less representative for the German population. The conclusions of our study results might also be limited due to the use of unadjusted seroprevalence values. However, when age standardization was applied seroprevalence estimates changed only marginally.
HPV of the same genus, with the exception of cutaneous α PV, showed similar antibody patterns and are therefore discussed as group. For comparisons with previously published prevalence data across different laboratories it is important to keep in mind that different assay formats and, probably even more important, different cut-off definitions may greatly influence absolute prevalence figures.
Genus μ PV are associated with distinct common warts, HPV 1 with deep palmoplantar warts (myrmecia) and HPV 63 with cystic or punctate, mainly plantar warts . Warts , and especially HPV 1 positive warts mostly occur at the age of 5 to 20 years , , , – while patients with HPV 63-induced warts were 10 to 39 years old ,. HPV 41 of genus ν originally isolated from a facial wart of a 15 years old girl was found mainly in plane hand warts but also in some skin carcinomas and actinic keratoses ,,,.
In our study, seroprevalence for HPV 1 (17.0%) was the highest among all types analysed, antibodies to HPV 63 (10.6%) were less prevalent. These findings are in agreement with a study reporting HPV 1 as the most prevalent type in warts from Germany (27.3%)  and with other non-German studies reporting higher prevalences in warts for HPV 1 (44.1% and 31.5%) than for HPV 63 (0.9% and 16.4%), respectively ,. Serological studies using virus particles purified from warts as antigens also frequently found antibodies to HPV 1 among mostly adults in 10%  and 19.3%  of the control sera. However, HPV 1 antibody prevalences of 32% and higher in both patients with and without a history of foot warts have been reported –,. The substantial seroprevalence found in all studies indicates a high frequency of infections with HPV 1 in the normal population. In addition, HPV 1-induced warts contain high viral loads , likely to result in more antigen presentation and thus in increased antibody production.
In both sexes, antibodies against μ and ν PV were the first to be seen. Seroprevalence peaked after the age of 14 and declined only slightly thereafter. In line with this observation, Hamsikova et al.  and Pfister et al.  reported a seroprevalence peak for HPV 1 at 13–30 years and 11–20 years, respectively. Two other studies reported seroprevalence to peak in children younger than 15 years ,. Assuming no newly acquired foot wart in adults and thus no new seroconversion, the data suggest a delayed but long lasting antibody induction detectable even decades after the initial infection.
In our study, type-specific seroprevalences for HR mucosal HPV in adult women (>14 years) were highest for HPV 16 (10.9%) followed by HPV 33 (4.7%), 18 (4.6%), 58 (4.1%), and 52 (0.7%). DNA prevalences for HR types in German women without cervical abnormalities were concordantly also highest for HPV 16 (14.6%) followed by HPV 58 (2.7%), 52 (2.5%), 33 (2.2%), and 18 (1.7%) . Another study on type-specific HPV DNA prevalence in West German women also found HPV 16 (26.2%) as the most frequent type, followed by HPV 31 (10.1%), 18 (5.3%), 58 (4.5%), 33 and 52 (both <4%) . In adult men, we found the highest seroprevalence for HPV 58 (4.8%), inconsistent with DNA data identifying HPV 16 as the most prevalent HR type in men .
Antibodies to HR mucosal α PV were rare in children (HPV 16 0.5%, other HR types 0.0–2.1%), which is consistent with seroprevalence rates for HPV 16 in children ranging from 1.5–7.6% reported by other studies . In women, these antibodies increased after puberty (reflecting the start of sexual activity) and peaked at 25–34 years, whereas in men seroprevalence did not increase strongly until the second half of life. This antibody increase and peak in young women was reported by several other studies – and is in agreement with a DNA prevalence peak for mucosal (predominantly HR) HPV frequently found in women younger than 30 years. With increasing age, HPV DNA prevalence decreases –, however a second peak in women around 50 years  or 60 years and older – has been reported. Consistently with the latter studies, we found a second seroprevalence peak for HR HPV in women older than 45 years.
HPV 16 serology in comparison to the other HR HPV was unique. Only for HPV 16, seroprevalence was significantly higher in women than in men both among younger and older adults, while seroprevalence for the other mucosal HR types showed only a non-significant increase in women. Although studies on HPV seroprevalence in men are rare, the lower seroprevalence for mucosal HPV types 6, 11, and 16 assessed by virus-like particle (VLP) serology is well known , , –. Several biological and anatomical sex-dependent differences were discussed as possible explanations: HPV infections of the penis involve rather keratinized than mucosal epithelium which might be less susceptible for the virus, less productive, and less accessible for the immune system. Men might have rather transient infections with lower viral loads that induce a weaker antibody response than in women. Overall DNA prevalence of mucosal HPV types in cytologically normal cervical smears ranges from 1.4% to 44% ,. In healthy men, overall HPV DNA prevalence values of 3.5% to 45% for mucosal types in exfoliated cells of the penis are in the same range . In view of this similar prevalence in penile versus cervical samples, the assumption of lower susceptibility of men cannot be held up.
It has been shown that persistent infection is associated with higher seroprevalence ,. Thus, HPV 16 might not only be more prevalent in women compared with men but also more persistent, with higher viral DNA loads, and with a greater amount of intact virions. HPV 16 is the most prevalent HPV type in low-grade squamous intraepithelial lesions , and these lesions are known to express L1 . In addition, HPV 16 is possibly more immunogenic than other HR types ,.
The most heterogeneous antibody distribution with regard to both age and type was seen for cutaneous α PV, although they are closely sequence-related. However, due to the overall low seroprevalence, the power of this pattern analysis is low. While antibodies to HPV 3 were frequent in children and decreased with age, seroprevalences for HPV types 2, 57, 10, and 77 were low in children and increased with age. These findings may suggest different natural histories of the individual wart-associated α PV types. HPV 3 and 10 are associated with plane, HPV 2 and 57 with common, and HPV 27 with intermediate warts . HPV 77 has been found in tumours and warts of immunosuppressed but not in lesions of immunocompetent individuals . HPV 2-associated warts have been most frequently found in the 20–40 years age group ,,, and in another study the prevalence of HPV 2/27/57-induced warts peaked at the age of 21–25 years .
For β and γ PV, age distribution patterns were very homogeneous. Seroprevalence for these types was low in children and increased with age. In middle-aged women, seroprevalence tended to be higher than in men of the same age. In older adults, this sex ratio shifted to a higher seroprevalence for men. We observed two seroprevalence peaks, both occurring slightly earlier in women than in men. At present, we can only speculate about potential reasons for the sex-associated serological differences for β and γ PV. In men, body hair is more abundant, the use of chemicals on the skin (cosmetics) is probably reduced, and for the population studied here sun exposure for large parts of the body during outdoor work might have been more frequent.
Serological studies showed elevated prevalences of antibodies to β PV, mainly HPV 5 and 8, in EV and in immunosuppressed patients, in patients with dermatological diseases like squamous cell carcinoma of the skin , –, – or psoriasis , and in patients with second degree burns . Feltkamp et al. found a statistically significant association of seropositivity with age and male sex for HPV 24 but not for HPV 5, 8, 15, 20 and 38  while in two other studies HPV 8 seropositivity was not correlated with age and/or sex ,.
DNA prevalence data indicate an ubiquitous distribution of these types in the population. Cutaneous HPV mainly of genus β are found in normal skin and plucked hairs of different body sites from healthy individuals, with up to 96% overall DNA prevalence in adults ,,. In samples from different skin regions of an individual, the same types are frequently found ,. Type patterns in plucked eye brow hairs and on healthy skin of an individual frequently persist ,. DNA from β and γ PV has been found in specimens taken with wet cotton-tipped swabs from the foreskin of infants within days after birth  thus demonstrating exposure. However, in the absence of additional data showing active virus infection in the infant's cells, it remains unclear whether these findings show infection to occur early in life. In adults, HPV DNA prevalence increases with age in both genders ,. We observed increasing seroprevalence in females until around 40 and in males until around 50 years. This suggests that seroconversion for HPV of these two genera in contrast to μ and ν PV is extremely slow and occurs only years or even decades after the initial infection. Alternatively, this might indicate that despite early exposure active infection occurs much later. DNA of these types in immunocompetent individuals is present only in very low copy numbers . A single contact with these types might therefore not result in antibody induction. However, accumulation of cutaneous HPV infections over lifetime due to an increased exposure via intense skin contacts could lead to higher overall viral loads and thus to an increasing seroprevalence with age. In addition, the weakening of the immune system in older age and/or perhaps increased sun-exposure might be responsible for the inability to control viral replication which consequently leads to higher viral loads and higher seroprevalences.
For most of the analysed HPV types, seroprevalence peaked twice. A possible explanation for the second peak at ages beyond 55 years is a reactivation of latent infections perhaps by reduction of immune surveillance with increasing age followed by increasing viral load and antibody induction. For mucosal α PV in women, it might in addition be caused by changes in the genital epithelium associated with menopause. Alternatively, the seroprevalence in older age groups of this serum collection might be due to a cohort effect. Possibly varying behaviour in different birth cohorts might have influenced exposure, such as sexual behaviour for genital HPV. We speculate that also environmental factors, such as sun exposure or nutrition, may have influenced the extent of HPV replication and/or the functionality of the immune system. The sera of this study were collected in 1987 and 1988. It remains to be examined whether this age-dependent distribution will also be present in sera collected 20 years later.
We observed a substantial frequency of multiple seropositive reactions, e.g. 48% of HPV 5 positive sera reacted also with HPV 8. Multiple infections with mucosal HPV , and even more often with cutaneous HPV both in immunosuppressed as well as immunocompetent individuals are common ,. Multiple seropositivity may result from type-specific reactions to multiple infections and/or could be due to cross-reactive antibodies induced by an infection with only one or few HPV types. The antibody detection assay used here is not able to distinguish these two possibilities. To address this issue, we investigated i) whether double seropositivity for a given pair of HPV types was observed more often than expected by chance and ii) whether double seropositivity was correlated with sequence identity of the respective L1 proteins (shown in supplementary material Figure S1 and Text S1). If multiple seropositivity was due to cross-reactivity, it should be explainable by the degree of relatedness of HPV types. Our results showed that multiple seropositivity was not or only weakly correlated with L1 amino acid sequence relatedness, and thus we consider the measured antibodies to be mainly type-specific. However, cross-reactivity can only be appropriately investigated by means of absorption experiments or monospecific antisera which are currently not available for most HPV types.
One limitation of this study is the use of a uniform, arbitrarily defined cut-off for seroprevalence calculations. For assays of antibodies to sexually transmitted genital HPV, cut-off definitions can be based on seroreactivities in groups of virgins  while for the cutaneous types representing the vast majority of HPV types analysed here the definition of a mostly uninfected and thus mostly seronegative group is not possible. In the absence of defined reference sera that could be used as international standards, any cut-off definition has an arbitrary component and seroprevalence values obtained by different laboratories in general should only be compared with caution.
In our study all antigens were identically constructed L1 fusion proteins expressed in the same bacterial expression system. The full-length L1 fusion protein density on the beads for different HPV types was very similar, since MFI values obtained after staining of the carboxy (C)-terminal tag epitope with a monoclonal antibody varied less than two-fold. Thus, given the similar properties of the antigens the use of an uniform cut-off appears justified. To avoid false-positivity by low-level cross-reactivity and to increase type specificity of the seroprevalence values, a cut-off well above background levels was chosen. Thus, the cut-off is rather stringent and probably underestimates the true seroprevalence of cutaneous HPV infections.
However, as shown in Figure 1, the major conclusions are independent of the chosen cut-off. The age-dependent distribution of skin HPV antibodies as well as the unique pattern of HPV 16 antibodies in women are also present at very low seroreactivity levels (MFI values well below the chosen cut-off).
Direct comparison of GST-L1 fusion protein- and VLP-based ELISA with human sera has been performed for HPV 16 and 18, and showed good correlation ,. Of 46 monospecific monoclonal antibodies detecting conformational epitopes on VLP of 9 mucosal types of which 4 (HPV 16, 18, 33, and 52) are also used here all reacted with the GST-L1 fusion protein of the same type  demonstrating the ability of GST-L1 to bind type-specific antibodies. Based on these observations, it is reasonable to assume similar properties for the other 30 HPV types investigated here. We cannot entirely exclude cross-reactivity and thus unspecific antibody reactions. Like VLP, GST fusion proteins present conformational and neutralising epitopes but display a higher amount of linear/cross-reacting epitopes than VLP . Thus, under the condition that only highly purified, intact VLP preparations are used as antigens, VLP-based assays may yield a higher ratio of specific to unspecific reactions than with GST-L1-based serology.
In conclusion, the serological results presented here suggest different seroprevalence patterns of phylogenetically related HPV: Antibodies to cutaneous μ and ν PV appear late in childhood, those to mucosal high-risk HPV after puberty mainly in women, and seroprevalence for β and γ PV peaked late in life in both genders.
The assays developed here allow further serological investigations to enlarge our still scarce knowledge on the natural history of especially cutaneous HPV infections. Interesting issues are e.g. effect of sun exposure on HPV prevalence, and the immune response in EV patients and patients with cutaneous warts in comparison to healthy people. In addition, seroepidemiological case-control studies might help to understand the potential role of cutaneous HPV in the development of non-melanoma skin cancer.
Materials and Methods
Recombinant HPV L1 proteins
HPV L1 open reading frames were expressed via the pGEX4T3 vector (GE Healthcare, München, Germany) in E.coli Rosetta cells (Merck, Darmstadt, Germany) as double fusion proteins with amino (N)-terminal GST and a C-terminal peptide (tag) consisting of the last 11 amino acids from the large T antigen of simian virus 40. The expression constructs for the L1 proteins of HPV types 16 and 18 have been described . Constructs for full-length L1 of HPV types 1a, 2a, 3, 4, 5, 8, 9, 10, 15, 17, 20, 23, 24, 33, 36, 38, 41, 48, 49, 50, 52, 57, 58, 60, 63, 65, 75, 76, 77, 92, 93, and 95 were generated in the same fashion. HPV genomes used as PCR templates, amplified regions, and cloning enzymes are listed in Table 2. HPV sequences were verified by commercial sequence analyses and aligned by HUSAR (Heidelberg Unix Sequence Analysis Resources, http://genome.dkfz-heidelberg.de/). Sequences of 27 of the 34 L1 constructs were identical with the published HPV nucleotide sequence. Seven of the 11 sequence variations in L1 of HPV 5, 8, 10, 16, 18, 41, and 57 resulted in amino acid changes (Table 2). Eight of the mismatches were present already in the parental plasmids, indicating possibly mistakes in the published sequences. Of the three PCR-generated mismatches, only one lead to a threonine (T) to serine (S) amino acid change in HPV 10 L1. This error was not corrected since T and S are functionally very similar amino acids. The sequence of the parental HPV 5 L1 ORF used here , was newly determined and submitted to GenBank (AM922325). In comparison to the most closely related published HPV 5 sequence (HPV 5b , D90252), it showed 17 mismatches and a 27 nucleotide deletion (2.9% sequence variation) and therefore probably represents a new subtype.
Table 2. Characteristics of expressed HPV L1 sequences.doi:10.1371/journal.ppat.1000091.t002
Fusion protein expression was induced at room temperature by 0.25 mM isopropyl-β-D-thiogalactoside (IPTG) and six h after induction bacteria were harvested. The pellet from a 1 liter culture was resuspended in 10 mL of 40 mM Tris, 200 mM NaCl, 1 mM EDTA, pH 8.0, 2 mM DTT containing complete protease inhibitor cocktail (Roche, Mannheim, Germany), and lysed with a high pressure homogenizer (Avestin, Ottawa, Canada). After incubation with 2 mM ATP and 5 mM MgCl2 for 1 h at room temperature, lysates were cleared from insoluble components (e.g. cell membranes) by centrifugation (14,000 rpm, 4°C, 30 min). For all HPV types, the proportion of insoluble fusion protein was low. Supernatants were stored with 50% glycerol at −20°C.
Fusion protein expression was characterised by Coomassie-stained sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as described . Western blot analyses using GST and tag specific antibodies showed full-length fusion proteins of approximately 84 kDa and some additional minor bands of shorter fragments. N-terminal (GST-reactive) fragments were more prevalent than C-terminal (tag-reactive) fragments indicating incomplete translation rather than proteolysis as cause for the smaller fragments (data not shown).
Verification and concentration of full-length GST-L1-tag proteins in the lysates was determined by GST-capture ELISA  using a tag-specific antibody for fusion protein detection. For all constructs, glutathione-casein saturation with fusion protein was reached with 200 µg total lysate protein/mL or less. Half maximal binding ranged from 16.5 µg/mL (HPV 3 L1) to 0.3 µg/mL (HPV 48 L1) (median 2.6 µg/mL) indicating about 55-fold variation in GST-L1-tag concentrations in the lysates.
Anonymous sera from 1797 individuals (age 1–82 years, median 37 years; 758 males and 1039 females) were analysed. These sera originated from three serum collections. The largest contribution is from the population-based German Nutrition Survey (Nationale Verzehrstudie NVS, ,) collected in the German population aged 4–94 years between October 1985 and January 1989 (n = 23,209). The VERA study (Verbundstudie Ernährungserhebung und Risikofaktorenanalytik) is a random subsample of the NVS population aged 18 years and older collected in 1987 and 1988 (n = 1988) ,. We tested the available 1573 (79.1%) VERA serum samples (age 18–82 years, median 41 years; 651 males and 922 females). Sera from children (below 19 years) were obtained from two other collections: (i) 175 sera from children (age 2–18 years, median 9 years; 71 males and 104 females) treated in 2002 at the university hospital in Homburg (Germany) were randomly selected from a collection of stored diagnostic sera and (ii) 49 sera (age 1–10 years, median 5 years; 36 boys and 13 girls) collected for a previous HPV serology study  in 1991 and 1992 from children treated at the university hospital in Heidelberg (Germany).
The age structure of the tested VERA samples is indifferent from the age structure of the NVS population aged 18 years and older (p = 0.9808), and the age structure of our total study (1573 sera from VERA, 175 children sera from Homburg, and 49 children sera from Heidelberg, total n = 1797) is characteristic for the age structure of the total NVS population (p = 0.9629). This allows the conclusion that our study is likely to be representative of the German population.
Sera were analysed simultaneously for antibodies to 34 HPV types by multiplex serology as described ,. Briefly, GST-L1-tag fusion proteins from cleared lysates were affinity-purified in situ through binding to the glutathione casein-coated fluorescence-labelled polystyrene beads. Each fusion protein was bound to a spectrally distinct bead set. A total lysate protein concentration of 1 mg/mL was used for all lysates, an at least five-fold excess to saturate GST fusion protein binding on the beads. Monoclonal anti-tag antibody binding to the different antigen-loaded bead sets varied less than two-fold, indicating similar full-length L1 fusion protein density for the different HPV types. Fusion protein-loaded bead sets were mixed. Sera were pre-incubated at 1:50 dilution in PBS containing 1 mg/mL casein, 2 mg/mL lysate from bacteria expressing GST-tag alone to block antibodies directed against residual bacterial proteins and GST-tag, 0.5% polyvinylalcohol (PVA, Sigma-Aldrich), 0.8% polyvinylpyrrolidone (PVP, Sigma-Aldrich) and 2.5% Superchemiblock (Millipore, Billerica, MA, USA) to suppress unspecific binding of antibodies to the beads themselves . Serum dilutions were incubated with the same volume of mixed bead sets, resulting in a final serum dilution of 1:100. Bound antibodies were detected with biotinylated goat-anti-human IgG (H+L) secondary antibody and streptavidin-R-phycoerythrin. A Luminex analyser (xMAP, Luminex Corp., Austin, Texas, USA) was used to identify the internal colour of the individual beads and to quantify their reporter fluorescence (expressed as median fluorescence intensity (MFI) of at least 100 beads per set per serum). A fusion protein consisting of GST and tag without intervening viral antigen served for background determination.
Assay design and data processing
The glutathione-casein coupled bead sets were loaded with their respective antigen in one batch. The efficiency of antigen loading on beads was quantified via the C-terminal tag as described . Values (mean MFI of 3 wells) for the different antigens ranged from 4604 (HPV 58 L1) to 9065 (HPV 95 L1) with a median of 6180 MFI. Correct antigen loading was verified with 25 reference sera with known HPV antibody pattern. These sera originated from two earlier studies analysing the antibody reactivity to 16  and 31 HPV types  by multiplex serology. All types except HPV 33, 52, 58, and 60 were represented in the reference sera.
Study sera were analysed once on three consecutive days. A quality control panel (QC) of 46 sera was included each day resulting in three QC data sets to determine inter-day variation. For inter-day variations of raw MFI values, Pearson correlation coefficients (R2) for the individual antigens ranged from 0.761 to 0.986 (median 0.963) for day 2 versus 1 and from 0.691 to 0.989 (median 0.947) for day 3 versus 1. The raw data of days 2 and 3 for each antigen were divided by the slopes of the regression lines of the QC data pairs of days 1 and 2 or days 1 and 3, respectively, to correct for inter-day variation.
The QC sera were also pooled and included as positive standard on each plate. Inter-plate variation coefficients (CV) calculated from these data for the various antigens ranged from 13.0% to 23.4% with a median of 16.6%.
Auto-fluorescence of each bead set and background reactions resulting from binding of secondary reagents to the antigen-loaded beads were determined in one well per plate without human serum. After correction for inter-day variation, mean background values (range 4 to 27 MFI, except for unusually high bead backgrounds of 150 and 350 MFI for HPV 4 L1 and HPV 33 L1, respectively) were subtracted from the raw MFI values and then antigen-specific reactivity was determined by subtraction of the MFI of GST-tag from the MFI of the specific antigen. Cut-off values to define seropositivity for all antigens were arbitrarily set to 200 MFI.
Serological data were stratified by sex and age. Since sexual transmission is known to play a major role in mucosal HPV infection, the first age group encompassed children (14 years and younger), followed by age groups in 10 year intervals. The age distribution of our study and the German standard population as reported by  is similar (p = 0.695), and we present data stratified by age. Age standardization was applied but changed seroprevalence estimates only marginally. Therefore, unadjusted seroprevalences are reported throughout this manuscript. Statistical significance of differences in seroprevalence was assessed by two-sided Fisher's exact test. P-values below 0.05 were considered statistically significant.
Double seropositivity in relation to amino acid sequence identity of the paired HPV L1 proteins
(0.57 MB TIF)
Double seropositivity in relation to amino acid sequence identity of the paired HPV L1 proteins
(0.03 MB DOC)
We thank Ute Koch and Monika Oppenländer (DKFZ) for excellent technical assistance, and E.-M. de Villiers (HPV 1, 2a, 3, 4, 5, 8, 16, 18, 38, 41, 48, 57, 63, 65, 75, 76, 77, 95), W. Lancaster (HPV 52), T. Matsukura (HPV 58, 60), G. Orth (HPV 9, 10a, 15, 17, 20, 23, 24, 33, 36), M. Favre (HPV 49, 50), and O. Forslund (HPV 92, 93) for the gift of HPV plasmids.
Conceived and designed the experiments: JS MP. Performed the experiments: KM TW. Analyzed the data: KM TW PS IB MP. Contributed reagents/materials/analysis tools: KM TW PS AR UR HB IB BG. Wrote the paper: KM. Drafted the article: KM MP. Critically revised the article: TW PS AR UR HB IB JS BG MP.
- 1. de Villiers EM, Fauquet C, Broker TR, Bernard HU, zur Hausen H (2004) Classification of papillomaviruses. Virology 324: 17–27.
- 2. Forslund O (2007) Genetic diversity of cutaneous human papillomaviruses. J Gen Virol 88: 2662–2669.
- 3. zur Hausen H (1996) Papillomavirus infections-a major cause of human cancers. Biochim Biophys Acta 1288: F55–78.
- 4. Munoz N, Bosch FX, de Sanjose S, Herrero R, Castellsague X, et al. (2003) Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med 348: 518–527.
- 5. IARC (2007) IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, Vol. 90. Human Papillomaviruses (ISBN 978 92 832 1290 4), Lyon.
- 6. Jablonska S, Majewski S, Obalek S, Orth G (1997) Cutaneous warts. Clin Dermatol 15: 309–319.
- 7. Gassenmaier A, Fuchs P, Schell H, Pfister H (1986) Papillomavirus DNA in warts of immunosuppressed renal allograft recipients. Arch Dermatol Res 278: 219–223.
- 8. Jablonska S, Majewski S (1994) Epidermodysplasia verruciformis: immunological and clinical aspects. Curr Top Microbiol Immunol 186: 157–175.
- 9. Akgul B, Cooke JC, Storey A (2006) HPV-associated skin disease. J Pathol 208: 165–175.
- 10. Nindl I, Gottschling M, Stockfleth E (2007) Human papillomaviruses and non-melanoma skin cancer: basic virology and clinical manifestations. Dis Markers 23: 247–259.
- 11. Grimmel M, de Villiers EM, Neumann C, Pawlita M, zur Hausen H (1988) Characterization of a new human papillomavirus (HPV 41) from disseminated warts and detection of its DNA in some skin carcinomas. Int J Cancer 41: 5–9.
- 12. Hirt L, Hirsch-Behnam A, de Villiers EM (1991) Nucleotide sequence of human papillomavirus (HPV) type 41: an unusual HPV type without a typical E2 binding site consensus sequence. Virus Res 18: 179–189.
- 13. Dillner J, Andersson-Ellstrom A, Hagmar B, Schiller J (1999) High risk genital papillomavirus infections are not spread vertically. Rev Med Virol 9: 23–29.
- 14. Cason J, Mant CA (2005) High-risk mucosal human papillomavirus infections during infancy & childhood. J Clin Virol 32: Suppl 1S52–58.
- 15. Syrjanen S, Puranen M (2000) Human papillomavirus infections in children: the potential role of maternal transmission. Crit Rev Oral Biol Med 11: 259–274.
- 16. Kashima HK, Mounts P, Shah K (1996) Recurrent respiratory papillomatosis. Obstet Gynecol Clin North Am 23: 699–706.
- 17. Dillner J (1999) The serological response to papillomaviruses. Semin Cancer Biol 9: 423–430.
- 18. Coursaget P (2003) Serology for human papillomavirus. Salud Publica Mex 45: Suppl 3S361–366.
- 19. Iftner T, Villa LL (2003) Chapter 12: Human papillomavirus technologies. J Natl Cancer Inst Monogr 80–88.
- 20. Favre M, Majewski S, Noszczyk B, Maienfisch F, Pura A, et al. (2000) Antibodies to human papillomavirus type 5 are generated in epidermal repair processes. J Invest Dermatol 114: 403–407.
- 21. Favre M, Orth G, Majewski S, Baloul S, Pura A, et al. (1998) Psoriasis: A possible reservoir for human papillomavirus type 5, the virus associated with skin carcinomas of epidermodysplasia verruciformis. J Invest Dermatol 110: 311–317.
- 22. Majewski S, Jablonska S (2003) Possible involvement of epidermodysplasia verruciformis human papillomaviruses in the immunopathogenesis of psoriasis: a proposed hypothesis. Exp Dermatol 12: 721–728.
- 23. Steger G, Olszewsky M, Stockfleth E, Pfister H (1990) Prevalence of antibodies to human papillomavirus type 8 in human sera. J Virol 64: 4399–4406.
- 24. Stark S, Petridis AK, Ghim SJ, Jenson AB, Bouwes Bavinck JN, et al. (1998) Prevalence of antibodies against virus-like particles of Epidermodysplasia verruciformis-associated HPV8 in patients at risk of skin cancer. J Invest Dermatol 111: 696–701.
- 25. Bouwes Bavinck JN, Stark S, Petridis AK, Marugg ME, Ter Schegget J, et al. (2000) The presence of antibodies against virus-like particles of epidermodysplasia verruciformis-associated humanpapillomavirus type 8 in patients with actinic keratoses. Br J Dermatol 142: 103–109.
- 26. Weber F, Fuchs PG, Pfister HJ, Hintner H, Fritsch P, et al. (2001) Human papillomavirus infection in Netherton's syndrome. Br J Dermatol 144: 1044–1049.
- 27. Pfister H, Fuchs PG, Majewski S, Jablonska S, Pniewska I, et al. (2003) High prevalence of epidermodysplasia verruciformis-associated human papillomavirus DNA in actinic keratoses of the immunocompetent population. Arch Dermatol Res 295: 273–279.
- 28. Pfister H, zur Hausen H (1978) Seroepidemiological studies of human papilloma virus (HPV-1) infections. Int J Cancer 21: 161–165.
- 29. Pfister H, zur Hausen H (1978) Characterization of proteins of human papilloma viruses (HPV) and antibody response to HPV 1. Med Microbiol Immunol (Berl) 166: 13–19.
- 30. Kienzler JL, Lemoine MT, Orth G, Jibard N, Blanc D, et al. (1983) Humoral and cell-mediated immunity to human papillomavirus type 1 (HPV-1) in human warts. Br J Dermatol 108: 665–672.
- 31. Steele JC, Gallimore PH (1990) Humoral assays of human sera to disrupted and nondisrupted epitopes of human papillomavirus type 1. Virology 174: 388–398.
- 32. Viac J, Chomel JJ, Chardonnet Y, Aymard M (1990) Incidence of antibodies to human papillomavirus type 1 in patients with cutaneous and mucosal papillomas. J Med Virol 32: 18–21.
- 33. Carter JJ, Hagensee M, Taflin MC, Lee SK, Koutsky LA, et al. (1993) HPV-1 capsids expressed in vitro detect human serum antibodies associated with foot warts. Virology 195: 456–462.
- 34. Carter JJ, Hagensee MB, Lee SK, McKnight B, Koutsky LA, et al. (1994) Use of HPV 1 capsids produced by recombinant vaccinia viruses in an ELISA to detect serum antibodies in people with foot warts. Virology 199: 284–291.
- 35. Anisimova E, Bartak P, Hamsikova E, Vonka V (1994) Comparative sensitivity of the immunoelectronmicroscopy test and ELISA with intact virions in detecting HPV-1 antibodies and age distribution of these antibodies among general population and in children's homes. Arch Virol 137: 157–160.
- 36. Cubie HA, Plumstead M, Zhang W, de Jesus O, Duncan LA, et al. (1998) Presence of antibodies to human papillomavirus virus-like particles (VLPs) in 11–13-year-old schoolgirls. J Med Virol 56: 210–216.
- 37. Hamsikova E, Ludvikova V, Smahel M, Sapp M, Vonka V (1998) Prevalence of antibodies to human papillomaviruses in the general population of the Czech Republic. Int J Cancer 77: 689–694.
- 38. Feltkamp MC, Broer R, di Summa FM, Struijk L, van der Meijden E, et al. (2003) Seroreactivity to epidermodysplasia verruciformis-related human papillomavirus types is associated with nonmelanoma skin cancer. Cancer Res 63: 2695–2700.
- 39. Karagas MR, Nelson HH, Sehr P, Waterboer T, Stukel TA, et al. (2006) Human papillomavirus infection and incidence of squamous cell and basal cell carcinomas of the skin. J Natl Cancer Inst 98: 389–395.
- 40. Masini C, Fuchs PG, Gabrielli F, Stark S, Sera F, et al. (2003) Evidence for the association of human papillomavirus infection and cutaneous squamous cell carcinoma in immunocompetent individuals. Arch Dermatol 139: 890–894.
- 41. Struijk L, Hall L, van der Meijden E, Wanningen P, Bavinck JN, et al. (2006) Markers of cutaneous human papillomavirus infection in individuals with tumor-free skin, actinic keratoses, and squamous cell carcinoma. Cancer Epidemiol Biomarkers Prev 15: 529–535.
- 42. Termorshuizen F, Feltkamp MC, Struijk L, de Gruijl FR, Bavinck JN, et al. (2004) Sunlight exposure and (sero)prevalence of epidermodysplasia verruciformis-associated human papillomavirus. J Invest Dermatol 122: 1456–1462.
- 43. Wieland U, Ritzkowsky A, Stoltidis M, Weissenborn S, Stark S, et al. (2000) Communication: papillomavirus DNA in basal cell carcinomas of immunocompetent patients: an accidental association? J Invest Dermatol 115: 124–128.
- 44. Casabonne D, Michael KM, Waterboer T, Pawlita M, Forslund O, et al. (2007) A prospective pilot study of antibodies against human papillomaviruses and cutaneous squamous cell carcinoma nested in the Oxford component of the European Prospective Investigation into Cancer and Nutrition. Int J Cancer 121: 1862–1868.
- 45. Waterboer T, Abeni D, Sampogna F, Rother A, Masini C, et al. (2008) Serological association of beta and gamma human papillomaviruses with squamous cell carcinoma of the skin. British Journal of Dermatology. In press.
- 46. Waterboer T, Sehr P, Michael KM, Franceschi S, Nieland JD, et al. (2005) Multiplex human papillomavirus serology based on in situ-purified glutathione s-transferase fusion proteins. Clin Chem 51: 1845–1853.
- 47. Waterboer T, Sehr P, Pawlita M (2006) Suppression of non-specific binding in serological Luminex assays. J Immunol Methods 309: 200–204.
- 48. Sehr P, Muller M, Hopfl R, Widschwendter A, Pawlita M (2002) HPV antibody detection by ELISA with capsid protein L1 fused to glutathione S-transferase. J Virol Methods 106: 61–70.
- 49. Sehr P, Zumbach K, Pawlita M (2001) A generic capture ELISA for recombinant proteins fused to glutathione S-transferase: validation for HPV serology. J Immunol Methods 253: 153–162.
- 50. Rizk RZ, Christensen ND, Michael KM, Muller M, Sehr P, et al. (2008) Reactivity pattern of 92 monoclonal antibodies with 15 human papillomavirus types. J Gen Virol 89: 117–129.
- 51. Williams HC, Pottier A, Strachan D (1993) The descriptive epidemiology of warts in British schoolchildren. Br J Dermatol 128: 504–511.
- 52. Larsson PA, Liden S (1980) Prevalence of skin diseases among adolescents 12–16 years of age. Acta Derm Venereol 60: 415–423.
- 53. Chen SL, Tsao YP, Lee JW, Sheu WC, Liu YT (1993) Characterization and analysis of human papillomaviruses of skin warts. Arch Dermatol Res 285: 460–465.
- 54. Hagiwara K, Uezato H, Arakaki H, Nonaka S, Nonaka K, et al. (2005) A genotype distribution of human papillomaviruses detected by polymerase chain reaction and direct sequencing analysis in a large sample of common warts in Japan. J Med Virol 77: 107–112.
- 55. Rubben A, Kalka K, Spelten B, Grussendorf-Conen EI (1997) Clinical features and age distribution of patients with HPV 2/27/57-induced common warts. Arch Dermatol Res 289: 337–340.
- 56. Egawa K (2005) Eccrine-centred distribution of human papillomavirus 63 infection in the epidermis of the plantar skin. Br J Dermatol 152: 993–996.
- 57. Iftner A, Klug SJ, Garbe C, Blum A, Stancu A, et al. (2003) The prevalence of human papillomavirus genotypes in nonmelanoma skin cancers of nonimmunosuppressed individuals identifies high-risk genital types as possible risk factors. Cancer Res 63: 7515–7519.
- 58. Egawa K, Honda Y, Inaba Y, Ono T (1998) Pigmented viral warts: a clinical and histopathological study including human papillomavirus typing. Br J Dermatol 138: 381–389.
- 59. Gissmann L, Pfister H, Zur Hausen H (1977) Human papilloma viruses (HPV): characterization of four different isolates. Virology 76: 569–580.
- 60. Sotlar K, Diemer D, Dethleffs A, Hack Y, Stubner A, et al. (2004) Detection and typing of human papillomavirus by e6 nested multiplex PCR. J Clin Microbiol 42: 3176–3184.
- 61. Speich N, Schmitt C, Bollmann R, Bollmann M (2004) Human papillomavirus (HPV) study of 2916 cytological samples by PCR and DNA sequencing: genotype spectrum of patients from the west German area. J Med Microbiol 53: 125–128.
- 62. Partridge JM, Koutsky LA (2006) Genital human papillomavirus infection in men. Lancet Infect Dis 6: 21–31.
- 63. Dunne EF, Karem KL, Sternberg MR, Stone KM, Unger ER, et al. (2005) Seroprevalence of human papillomavirus type 16 in children. J Infect Dis 191: 1817–1819.
- 64. Marais D, Rose RC, Williamson AL (1997) Age distribution of antibodies to human papillomavirus in children, women with cervical intraepithelial neoplasia and blood donors from South Africa. J Med Virol 51: 126–131.
- 65. Shin HR, Lee DH, Herrero R, Smith JS, Vaccarella S, et al. (2003) Prevalence of human papillomavirus infection in women in Busan, South Korea. Int J Cancer 103: 413–421.
- 66. Stone KM, Karem KL, Sternberg MR, McQuillan GM, Poon AD, et al. (2002) Seroprevalence of human papillomavirus type 16 infection in the United States. J Infect Dis 186: 1396–1402.
- 67. Sukvirach S, Smith JS, Tunsakul S, Munoz N, Kesararat V, et al. (2003) Population-based human papillomavirus prevalence in Lampang and Songkla, Thailand. J Infect Dis 187: 1246–1256.
- 68. Sun Y, Eluf-Neto J, Bosch FX, Munoz N, Walboomers JM, et al. (1999) Serum antibodies to human papillomavirus 16 proteins in women from Brazil with invasive cervical carcinoma. Cancer Epidemiol Biomarkers Prev 8: 935–940.
- 69. Wang SS, Schiffman M, Herrero R, Carreon J, Hildesheim A, et al. (2004) Determinants of human papillomavirus 16 serological conversion and persistence in a population-based cohort of 10 000 women in Costa Rica. Br J Cancer 91: 1269–1274.
- 70. Wang SS, Schiffman M, Shields TS, Herrero R, Hildesheim A, et al. (2003) Seroprevalence of human papillomavirus-16, -18, -31, and -45 in a population-based cohort of 10000 women in Costa Rica. Br J Cancer 89: 1248–1254.
- 71. Kreimer AR, Alberg AJ, Viscidi R, Gillison ML (2004) Gender differences in sexual biomarkers and behaviors associated with human papillomavirus-16, -18, and -33 seroprevalence. Sex Transm Dis 31: 247–256.
- 72. Jacobs MV, Walboomers JM, Snijders PJ, Voorhorst FJ, Verheijen RH, et al. (2000) Distribution of 37 mucosotropic HPV types in women with cytologically normal cervical smears: the age-related patterns for high-risk and low-risk types. Int J Cancer 87: 221–227.
- 73. Melkert PW, Hopman E, van den Brule AJ, Risse EK, van Diest PJ, et al. (1993) Prevalence of HPV in cytomorphologically normal cervical smears, as determined by the polymerase chain reaction, is age-dependent. Int J Cancer 53: 919–923.
- 74. Burk RD, Kelly P, Feldman J, Bromberg J, Vermund SH, et al. (1996) Declining prevalence of cervicovaginal human papillomavirus infection with age is independent of other risk factors. Sex Transm Dis 23: 333–341.
- 75. Schiffman MH (1992) Recent progress in defining the epidemiology of human papillomavirus infection and cervical neoplasia. J Natl Cancer Inst 84: 394–398.
- 76. Klug SJ, Hukelmann M, Hollwitz B, Duzenli N, Schopp B, et al. (2007) Prevalence of human papillomavirus types in women screened by cytology in Germany. J Med Virol 79: 616–625.
- 77. Munoz N, Mendez F, Posso H, Molano M, van den Brule AJ, et al. (2004) Incidence, duration, and determinants of cervical human papillomavirus infection in a cohort of Colombian women with normal cytological results. J Infect Dis 190: 2077–2087.
- 78. Cuzick J, Beverley E, Ho L, Terry G, Sapper H, et al. (1999) HPV testing in primary screening of older women. Br J Cancer 81: 554–558.
- 79. Herrero R, Hildesheim A, Bratti C, Sherman ME, Hutchinson M, et al. (2000) Population-based study of human papillomavirus infection and cervical neoplasia in rural Costa Rica. J Natl Cancer Inst 92: 464–474.
- 80. Herrero R, Schiffman MH, Bratti C, Hildesheim A, Balmaceda I, et al. (1997) Design and methods of a population-based natural history study of cervical neoplasia in a rural province of Costa Rica: the Guanacaste Project. Rev Panam Salud Publica 1: 362–375.
- 81. Lazcano-Ponce E, Herrero R, Munoz N, Cruz A, Shah KV, et al. (2001) Epidemiology of HPV infection among Mexican women with normal cervical cytology. Int J Cancer 91: 412–420.
- 82. Eisemann C, Fisher SG, Gross G, Muller M, Gissmann L (1996) Antibodies to human papillomavirus type 11 virus-like particles in sera of patients with genital warts and in control groups. J Gen Virol 77 (Pt 8): 1799–1803.
- 83. Slavinsky J 3rd, Kissinger P, Burger L, Boley A, DiCarlo RP, et al. (2001) Seroepidemiology of low and high oncogenic risk types of human papillomavirus in a predominantly male cohort of STD clinic patients. Int J STD AIDS 12: 516–523.
- 84. Svare EI, Kjaer SK, Nonnenmacher B, Worm AM, Moi H, et al. (1997) Seroreactivity to human papillomavirus type 16 virus-like particles is lower in high-risk men than in high-risk women. J Infect Dis 176: 876–883.
- 85. Thompson DL, Douglas JM Jr, Foster M, Hagensee ME, Diguiseppi C, et al. (2004) Seroepidemiology of infection with human papillomavirus 16, in men and women attending sexually transmitted disease clinics in the United States. J Infect Dis 190: 1563–1574.
- 86. van Doornum G, Prins M, Andersson-Ellstrom A, Dillner J (1998) Immunoglobulin A, G, and M responses to L1 and L2 capsids of human papillomavirus types 6, 11, 16, 18, and 33 L1 after newly acquired infection. Sex Transm Infect 74: 354–360.
- 87. Carter JJ, Wipf GC, Hagensee ME, McKnight B, Habel LA, et al. (1995) Use of human papillomavirus type 6 capsids to detect antibodies in people with genital warts. J Infect Dis 172: 11–18.
- 88. Greer CE, Wheeler CM, Ladner MB, Beutner K, Coyne MY, et al. (1995) Human papillomavirus (HPV) type distribution and serological response to HPV type 6 virus-like particles in patients with genital warts. J Clin Microbiol 33: 2058–2063.
- 89. Strickler HD, Kirk GD, Figueroa JP, Ward E, Braithwaite AR, et al. (1999) HPV 16 antibody prevalence in Jamaica and the United States reflects differences in cervical cancer rates. Int J Cancer 80: 339–344.
- 90. Bosch FX, de Sanjose S (2003) Chapter 1: Human papillomavirus and cervical cancer-burden and assessment of causality. J Natl Cancer Inst Monogr 3–13.
- 91. Clifford GM, Gallus S, Herrero R, Munoz N, Snijders PJ, et al. (2005) Worldwide distribution of human papillomavirus types in cytologically normal women in the International Agency for Research on Cancer HPV prevalence surveys: a pooled analysis. Lancet 366: 991–998.
- 92. Carter JJ, Koutsky LA, Wipf GC, Christensen ND, Lee SK, et al. (1996) The natural history of human papillomavirus type 16 capsid antibodies among a cohort of university women. J Infect Dis 174: 927–936.
- 93. Wideroff L, Schiffman MH, Nonnenmacher B, Hubbert N, Kirnbauer R, et al. (1995) Evaluation of seroreactivity to human papillomavirus type 16 virus-like particles in an incident case-control study of cervical neoplasia. J Infect Dis 172: 1425–1430.
- 94. Clifford GM, Rana RK, Franceschi S, Smith JS, Gough G, et al. (2005) Human papillomavirus genotype distribution in low-grade cervical lesions: comparison by geographic region and with cervical cancer. Cancer Epidemiol Biomarkers Prev 14: 1157–1164.
- 95. Middleton K, Peh W, Southern S, Griffin H, Sotlar K, et al. (2003) Organization of human papillomavirus productive cycle during neoplastic progression provides a basis for selection of diagnostic markers. J Virol 77: 10186–10201.
- 96. Molano M, Van den Brule A, Plummer M, Weiderpass E, Posso H, et al. (2003) Determinants of clearance of human papillomavirus infections in Colombian women with normal cytology: a population-based, 5-year follow-up study. Am J Epidemiol 158: 486–494.
- 97. Schiffman M, Kjaer SK (2003) Chapter 2: Natural history of anogenital human papillomavirus infection and neoplasia. J Natl Cancer Inst Monogr 14–19.
- 98. Shamanin V, Glover M, Rausch C, Proby C, Leigh IM, et al. (1994) Specific types of human papillomavirus found in benign proliferations and carcinomas of the skin in immunosuppressed patients. Cancer Res 54: 4610–4613.
- 99. de Villiers EM (1989) Heterogeneity of the human papillomavirus group. J Virol 63: 4898–4903.
- 100. Antonsson A, Forslund O, Ekberg H, Sterner G, Hansson BG (2000) The ubiquity and impressive genomic diversity of human skin papillomaviruses suggest a commensalic nature of these viruses. J Virol 74: 11636–11641.
- 101. de Koning MN, Struijk L, Bavinck JN, Kleter B, ter Schegget J, et al. (2007) Betapapillomaviruses frequently persist in the skin of healthy individuals. J Gen Virol 88: 1489–1495.
- 102. Kohler A, Forschner T, Meyer T, Ulrich C, Gottschling M, et al. (2007) Multifocal distribution of cutaneous human papillomavirus types in hairs from different skin areas. Br J Dermatol 156: 1078–1080.
- 103. Hazard K, Karlsson A, Andersson K, Ekberg H, Dillner J, et al. (2007) Cutaneous human papillomaviruses persist on healthy skin. J Invest Dermatol 127: 116–119.
- 104. Antonsson A, Karanfilovska S, Lindqvist PG, Hansson BG (2003) General acquisition of human papillomavirus infections of skin occurs in early infancy. J Clin Microbiol 41: 2509–2514.
- 105. Boxman IL, Russell A, Mulder LH, Bavinck JN, ter Schegget J, et al. (2001) Association between epidermodysplasia verruciformis-associated human papillomavirus DNA in plucked eyebrow hair and solar keratoses. J Invest Dermatol 117: 1108–1112.
- 106. Weissenborn SJ, Nindl I, Purdie K, Harwood C, Proby C, et al. (2005) Human papillomavirus-DNA loads in actinic keratoses exceed those in non-melanoma skin cancers. J Invest Dermatol 125: 93–97.
- 107. Berkhout RJ, Bouwes Bavinck JN, ter Schegget J (2000) Persistence of human papillomavirus DNA in benign and (pre)malignant skin lesions from renal transplant recipients. J Clin Microbiol 38: 2087–2096.
- 108. Clifford GM, Shin HR, Oh JK, Waterboer T, Ju YH, et al. (2007) Serologic response to oncogenic human papillomavirus types in male and female university students in Busan, South Korea. Cancer Epidemiol Biomarkers Prev 16: 1874–1879.
- 109. Gassenmaier A, Lammel M, Pfister H (1984) Molecular cloning and characterization of the DNAs of human papillomaviruses 19, 20, and 25 from a patient with epidermodysplasia verruciformis. J Virol 52: 1019–1023.
- 110. Pfister H, Gassenmaier A, Nurnberger F, Stuttgen G (1983) Human papilloma virus 5-DNA in a carcinoma of an epidermodysplasia verruciformis patient infected with various human papillomavirus types. Cancer Res 43: 1436–1441.
- 111. Yabe Y, Sakai A, Hitsumoto T, Kato H, Ogura H (1991) A subtype of human papillomavirus 5 (HPV-5b) and its subgenomic segment amplified in a carcinoma: nucleotide sequences and genomic organizations. Virology 183: 793–798.
- 112. Hepp KD (1995) Food consumption habits in Germany-the clinician's point of view. Metabolism 44: 14–17.
- 113. Heseker H, Hartmann S, Kubler W, Schneider R (1995) An epidemiologic study of food consumption habits in Germany. Metabolism 44: 10–13.
- 114. Mund K, Han C, Daum R, Helfrich S, Muller M, et al. (1997) Detection of human papillomavirus type 16 DNA and of antibodies to human papillomavirus type 16 proteins in children. Intervirology 40: 232–237.
- 115. Böhning D (1998) Allgemeine Epidemiologie. München, Wien: Oldenbourg Verlag.