Current address: Joint DKFZ-EMBL Chemical Biology Core Facility, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany
Current address: University Children's Hospital, Heidelberg, Germany
Current address: Department of Dermatology und Allergology, Klinikum Augsburg Süd, Augsburg, Germany
Current address: Institute for Evolution and Biodiversity, Experimental Molecular Evolution Group, Westfälische Wilhelms University, Münster, Germany
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.
The authors have declared that no competing interests exist.
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.
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
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
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
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
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
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
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.
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.
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.
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).
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% (
seropositivity (%) | ||||||||||||||||||
adults | ||||||||||||||||||
children 1–14 years | all adults | 15–34 years (young adults) | >34 years (older adults) | |||||||||||||||
whole study | children | m | f | f/m | young adults | m | f | f/m | older adults | m | f | f/m | children vs young adults | young adults vs older adults | ||||
seropositive for | n = 1797 | n = 187 | n = 92 | n = 95 | prev. ratio | n = 1610 | n = 612 | n = 246 | n = 366 | prev. ratio | n = 998 | n = 420 | n = 578 | prev. ratio | p | p | ||
59.7 | 35.3 | 34.8 | 35.8 | 1.0 | 62.5 | 59.8 | 57.7 | 62.0 | 1.1 | 63.3 | 63.8 | 63.8 | 1.0 | |||||
55.5 | 32.1 | 29.3 | 34.7 | 1.2 | 58.3 | 54.7 | 54.9 | 54.6 | 1.0 | 60.4 | 62.6 | 58.8 | 0.9 | |||||
14.5 | 4.8 | 7.6 | 2.1 | 0.3 | 15.6 | 15.2 | 11 | 21 | 12.9 | 12.4 | 16.4 | 1.3 | ||||||
40.3 | 64.7 | 65.2 | 64.2 | 1.0 | 37.5 | 40.2 | 42.3 | 38.0 | 0.9 | 36.7 | 36.2 | 36.2 | 1.0 | **** | ||||
24.5 | 24.1 | 22.8 | 25.3 | 1.1 | 24.5 | 27.0 | 27.6 | 26.0 | 0.9 | 23.1 | 22.1 | 24.0 | 1.1 | |||||
11.5 | 4.8 | 6.5 | 3.2 | 0.5 | 12.3 | 11.6 | 11.4 | 12.0 | 1.1 | 12.4 | 12.9 | 12.5 | 1.0 | ** | ||||
7.4 | 4.3 | 3.3 | 5.3 | 1.6 | 7.8 | 7.5 | 5.7 | 9.3 | 1.6 | 7.9 | 7.4 | 8.0 | 1.1 | |||||
16.2 | 2.1 | 2.2 | 2.1 | 1.0 | 17.9 | 13.7 | 13.0 | 14.8 | 1.1 | 19.8 | 21.4 | 19.4 | 0.9 | **** | ** | |||
6.1 | 0.5 | 1.1 | 0.0 | 0.0 | 6.7 | 4.2 | 3.3 | 5.2 | 1.6 | 7.9 | 10.0 | 6.7 | 0.7 | * | ** | |||
1.9 | 0.0 | 0.0 | 0.0 | n/a | 2.2 | 1.3 | 0.8 | 1.6 | 2.0 | 2.7 | 2.9 | 2.6 | 0.9 | |||||
7 | 3.8 | 1.6 | 2.2 | 1.1 | 0.5 | 4.0 | 4.1 | 2.4 | 5.2 | 2.1 | 4.0 | 3.8 | 4.2 | 1.1 | ||||
9 | 7.1 | 0.5 | 1.1 | 0.0 | 0.0 | 7.8 | 7.8 | 2.0 | 11.7 | 7.8 | 4.3 | 10.4 | **** | |||||
9 | 4.1 | 2.1 | 4.3 | 0.0 | 0.0 | 4.3 | 5.1 | 3.7 | 6.0 | 1.6 | 3.9 | 4.0 | 3.8 | 0.9 | ||||
9 | 1.1 | 0.0 | 0.0 | 0.0 | n/a | 1.2 | 0.8 | 1.2 | 0.5 | 0.4 | 1.4 | 2.1 | 0.9 | 0.4 | ||||
9 | 4.1 | 1.1 | 1.1 | 1.1 | 1.0 | 4.4 | 6.2 | 6.5 | 6.0 | 0.9 | 3.3 | 3.8 | 2.9 | 0.8 | ** | ** | ||
4 | 4.1 | 1.1 | 1.1 | 1.1 | 1.0 | 4.5 | 3.1 | 1.2 | 4.4 | 5.3 | 6.4 | 4.5 | 0.7 | * | ||||
4 | 2.7 | 1.1 | 2.2 | 0.0 | 0.0 | 2.9 | 2.3 | 0.8 | 3.3 | 4.0 | 3.2 | 4.3 | 2.4 | 0.6 | ||||
2 | 6.0 | 8.6 | 7.6 | 9.5 | 1.2 | 5.7 | 7.2 | 6.9 | 7.4 | 1.1 | 4.8 | 3.8 | 5.5 | 1.5 | ||||
2 | 6.0 | 3.2 | 2.2 | 4.2 | 1.9 | 6.3 | 4.1 | 3.7 | 4.4 | 1.2 | 7.7 | 6.0 | 9.0 | 1.5 | ** | |||
2 | 6.7 | 2.1 | 0.0 | 4.2 | n/a | 7.3 | 6.4 | 4.1 | 7.9 | 1.9 | 7.8 | 6.9 | 8.5 | 1.2 | * | |||
17.1 | 11.2 | 9.8 | 12.6 | 1.3 | 17.8 | 16.0 | 13.0 | 18.0 | 1.4 | 18.8 | 18.3 | 19.2 | 1.0 | |||||
3 | 9.2 | 1.1 | 2.2 | 0.0 | 0.0 | 10.1 | 5.9 | 6.5 | 5.5 | 0.8 | 12.7 | 13.3 | 12.3 | 0.9 | ** | **** | ||
3 | 4.0 | 1.6 | 2.2 | 1.1 | 0.5 | 4.3 | 1.5 | 1.6 | 1.4 | 0.8 | 6.0 | 7.1 | 5.2 | 0.7 | **** | |||
3 | 5.0 | 1.1 | 1.1 | 1.1 | 1.0 | 5.5 | 2.6 | 2.4 | 2.7 | 1.1 | 7.2 | 9.8 | 5.4 | **** | ||||
2 | 7.6 | 0.5 | 1.1 | 0.0 | 0.0 | 8.4 | 4.6 | 4.1 | 4.9 | 1.2 | 10.8 | 12.1 | 9.9 | 0.8 | ** | **** | ||
2 | 3.5 | 0.5 | 1.1 | 0.0 | 0.0 | 3.8 | 2.0 | 1.6 | 2.2 | 1.3 | 4.9 | 6.2 | 4.0 | 0.6 | ** | |||
2 | 7.1 | 2.7 | 4.3 | 1.1 | 0.2 | 7.6 | 5.1 | 4.5 | 5.5 | 1.2 | 9.2 | 11.4 | 7.6 | ** | ||||
2 | 7.0 | 2.7 | 1.1 | 4.2 | 3.9 | 7.5 | 3.9 | 3.7 | 4.1 | 1.1 | 9.7 | 11.0 | 8.8 | 0.8 | **** | |||
2 | 7.1 | 0.5 | 0.0 | 1.1 | n/a | 7.9 | 5.1 | 3.3 | 6.3 | 1.9 | 9.6 | 11.7 | 8.1 | 0.7 | ** | *** | ||
4 | 5.2 | 0.5 | 0.0 | 1.1 | n/a | 5.8 | 3.4 | 1.6 | 4.6 | 2.9 | 7.2 | 8.3 | 6.4 | 0.8 | * | ** | ||
1 | 5.5 | 1.6 | 2.2 | 1.1 | 0.5 | 5.9 | 3.9 | 2.4 | 4.9 | 2.0 | 7.1 | 8.6 | 6.1 | 0.7 | ** | |||
1 | 0.6 | 0.0 | 0.0 | 0.0 | n/a | 0.7 | 0.5 | 0.4 | 0.5 | 1.3 | 0.8 | 1.2 | 0.5 | 0.4 | ||||
1 | 4.7 | 0.0 | 0.0 | 0.0 | n/a | 5.2 | 3.1 | 1.6 | 4.1 | 2.5 | 6.5 | 7.4 | 5.9 | 0.8 | * | ** | ||
1 | 5.5 | 1.1 | 2.2 | 0.0 | 0.0 | 6.0 | 3.6 | 2.8 | 4.1 | 1.4 | 7.4 | 10.5 | 5.2 | ** | ||||
1 | 3.8 | 1.1 | 0.0 | 2.1 | n/a | 4.2 | 3.3 | 2.4 | 3.8 | 1.6 | 4.7 | 5.7 | 4.0 | 0.7 | ||||
1 | 11.3 | 2.1 | 2.2 | 2.1 | 1.0 | 12.4 | 7.4 | 6.5 | 7.9 | 1.2 | 15.4 | 17.6 | 13.8 | 0.8 | ** | **** | ||
26.3 | 11.2 | 9.8 | 12.6 | 1.3 | 28.0 | 17.6 | 16.7 | 18.3 | 1.1 | 34.4 | 36.9 | 32.5 | 0.9 | * | **** | |||
1 | 7.7 | 1.6 | 2.2 | 1.1 | 0.5 | 8.4 | 6.4 | 5.3 | 7.1 | 1.3 | 9.6 | 10.2 | 9.2 | 0.9 | ** | * | ||
1 | 9.9 | 0.5 | 1.1 | 0.0 | 0.0 | 11.0 | 8.3 | 8.1 | 8.5 | 1.0 | 12.6 | 14.3 | 11.4 | 0.8 | **** | ** | ||
1 | 16.9 | 4.8 | 6.5 | 3.2 | 0.5 | 18.3 | 18.3 | 16.3 | 19.7 | 1.2 | 18.3 | 20.2 | 17.0 | 0.8 | **** | |||
3 | 4.1 | 1.1 | 0.0 | 2.1 | n/a | 4.4 | 2.8 | 1.2 | 3.8 | 3.1 | 5.4 | 6.4 | 4.7 | 0.7 | * | |||
2 | 7.4 | 1.6 | 1.1 | 2.1 | 1.9 | 8.1 | 5.7 | 6.5 | 5.2 | 0.8 | 9.5 | 10.5 | 8.8 | 0.8 | * | ** | ||
4 | 0.7 | 0.0 | 0.0 | 0.0 | n/a | 0.7 | 0.3 | 0.0 | 0.5 | n/a | 1.0 | 1.7 | 0.5 | 0.3 | ||||
26.8 | 9.1 | 9.8 | 8.4 | 0.9 | 28.9 | 25.2 | 23.6 | 26.2 | 1.1 | 31.2 | 34.3 | 28.9 | 0.8 | **** | * | |||
7.4 | 3.2 | 2.2 | 4.2 | 1.9 | 7.9 | 8.8 | 9.8 | 8.2 | 0.8 | 7.3 | 10.2 | 5.2 | * | |||||
1 | 17.0 | 6.4 | 4.3 | 8.4 | 1.9 | 18.2 | 21.9 | 23.2 | 21.0 | 0.9 | 15.9 | 14.8 | 16.8 | 1.1 | **** | ** | ||
2 | 10.6 | 2.7 | 2.2 | 3.2 | 1.5 | 11.5 | 12.3 | 13.0 | 11.7 | 0.9 | 11.0 | 11.4 | 10.7 | 0.9 | **** | |||
24.7 | 10.7 | 8.7 | 12.6 | 1.4 | 26.3 | 30.2 | 35.0 | 27.0 | 23.8 | 24.3 | 23.5 | 1.0 | **** | ** |
abbreviations: cut. cutaneous, f females, gen. genus, m males, muc. mucosal, n/a not available, prev. prevalence, sp. species.
female over male prevalence ratio;
p, two-sided Fisher's exact test;
p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.
HPV types 1 and 4 showed the highest type-specific seroprevalences (17.0 and 16.9%, respectively) (
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 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.
For statistical analysis of the age- and sex-dependent seroprevalence patterns, age groups were combined to increase statistical power (
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.
Antibody reactivity to more than one HPV type was frequent (
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 (
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
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%)
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
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%)
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
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
It has been shown that persistent infection is associated with higher seroprevalence
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
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
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
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
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
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
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
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.
HPV L1 open reading frames were expressed via the pGEX4T3 vector (GE Healthcare, München, Germany) in
HPV type | Accession number | Amplified region/cloning enzymes | Mismatches |
1a | V01116 | B-5431-6936-S | |
2a | X55964 | Sm-5742-7271-S | |
3 | X74462 | B-5751-7262-S | |
4 | X70827 | B-5345-6892-S | |
5 | AM922325 | B-1-1551-S | G1533A |
8 | M12737 | B-5851-7392-S | A7383G |
9 | X74464 | B-5745-7265-S | |
10 | X74465 | E-5823-7331-S | C7324G (T to S) |
15 | X74468 | B-5703-7223-S | |
16R | not yet entered | Sm-5669-7153-S | A6434G (T to A) |
17 | X74469 | B-5724-7244-S | |
18 | X05015 | Sm-5643-7133-S | C5701G (P to R) |
20 | U31778 | B-5893-7440-S | |
23 | U31781 | Sm-5660-7177-S | |
24 | U31782 | B-5713-7248-S | |
33 | M12732 | B-5594-7090-S | |
36 | U31785 | Sm-5891-7438-S | |
38 | U31787 | Sm-5661-7190-S | |
41 | X56147 | E-5546-7084-S | T5905C |
48 | U31789 | E5202-6740-S | |
49 | X74480 | B-5811-7337-S | |
50 | U31790 | E-5231-6775-S | |
52 | X74481 | B-5643-7151-S | |
57 | X55965 | B-5751-7232-S | T6180A (Y to N) |
58 | D90400 | B-5643-7136-S | |
60 | U31792 | E-5395-6918-S | |
63 | X70828 | E-5371-6891-S | |
65 | X70829 | B-5326-6873-S | |
75 | Y15173 | B-5771-7297-S | |
76 | Y15174 | Sm-5799-7325-S | |
77 | Y15175 | B-5789-7297-S | |
92 | AF531420 | B-5638-7173-S | |
93 | AY382778 | B-5640-7178-S | |
95 | AJ620210 | B-5368-6912-S |
source of genome sequences: GenBank.
B = BamHI, E = EcoRI, S = SalI, Sm = SmaI; first and last HPV nucleotide.
nucleotide changes and positions, amino acid changes are indicated in brackets.
PCR-generated mutation.
silent mutation.
described in Sehr
L1 lacks 10 N-terminal amino acids.
ftp://ftp-t10.lanl.gov/pub/papilloma/GenBank-files/Human-papilloma/HPV16R.gb.
mutations present in parental HPV plasmid.
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
Verification and concentration of full-length GST-L1-tag proteins in the lysates was determined by GST-capture ELISA
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,
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
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
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
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.