Cervical human papillomavirus (HPV) infection and HPV type 16 antibodies in South African women

There is a high incidence of cervical cancer in South African women. No large studies to assess human papillomavirus virus (HPV) infection or HPV type 16 (HPV-16) exposure have occurred in the region, a requirement for policy making with regards to HPV screening and the introduction of vaccines. Control women (n = 1,003) enrolled in a case control study of hormonal contraceptives and cervical cancer were tested for 27 cervical HPV types by reverse line blot analysis. The seroprevalence of HPV-16 immunoglobulin G (IgG) and IgA antibodies was assessed by a virus-like particle-based enzyme-linked immunoassay of 908 and 904 control women, respectively, and of 474 women with cervical cancer. The cervical HPV prevalence was 26.1%. The HPV-16 IgG seroprevalence was 44.4% and the HPV-16 IgA seroprevalence was 28.7% in control women, and these levels were significantly higher (61.8% and 52.7%, respectively) for women with cervical cancer (odds ratio [OR], 2.1 and 2.8, respectively). The cervical HPV prevalence showed an association with cervical disease, and the HPV-16 IgG prevalence decreased while the HPV-16 IgA prevalence increased with increasing age (P < 0.05). The prevalence of oncogenic HPV types (including HPV-16) decreased with age, whereas nononcogenic HPV types showed limited association with age. Multivariate analysis revealed cervical HPV infection to be associated with herpes simplex virus type 2 infection (OR, 1.7) and increasing years of education (OR, 1.9). HPV-16 IgG antibodies were inversely associated with current smoking status (OR, 0.6), and the presence of HPV-16 IgA antibodies was inversely associated with the use of alcohol (OR, 2.1) and inversely associated with the use of oral contraceptives (OR, 0.6). High levels of exposure to HPV, and particularly HPV-16, were evident in this population. The apparent increase of serum HPV-16 IgA with increasing age requires further investigation.     

Memory T cells specific for novel human papillomavirus type 16 (HPV16) E6 epitopes in women whose HPV16 infection has become undetectable

Human papillomavirus (HPV)-specific T-cell response to the HPV type 16 (HPV16) E6 protein has been shown to be associated with successful viral clearance. The patterns of CD8 T-cell epitopes within HPV16 E6 protein were previously studied in two women with HPV16 clearance. The goal of this study was to characterize these epitopes in terms of their minimal and optimal amino acid sequences and the human leukocyte antigen (HLA) restriction molecules. The presence of the epitope-specific memory T cells after viral clearance was also examined. In subject A, the dominant epitope was characterized to be E6 75-83 (KFYSKISEY), restricted by the HLA-B62 molecule, while that of subject B was E6 133-142 (HNIRGRWTGR), restricted by the HLA-A6801 molecule. Homologous epitopes were identified in five other high-risk HPV types for both of these epitopes, but they were not recognized by respective T-cell clone cells. An enzyme-linked immunospot assay or tetramer analysis was performed on peripheral blood mononuclear cells from blood samples collected after viral clearance but prior to isolation of the T-cell clones. The presence of epitope-specific memory T cells was demonstrated. These data suggest that HPV-specific memory T cells were generated in vivo and that they may remain in circulation many months, if not years, after viral clearance. Our findings broaden the spectrum of the CD8 T-cell epitopes of the HPV16 E6 protein. The characterization of novel T-cell epitopes and long-lasting epitope-specific memory T cells may be useful for the development of a potential epitope-based vaccine.     

Polymer-based enzyme-linked immunosorbent assay using human papillomavirus type 16 (HPV16) virus-like particles detects HPV16 clade-specific serologic responses

Human papillomavirus type 16 (HPV16) virus-like particles (VLP) were used as antigen in a polymer enzyme-linked immunosorbent assay (ELISA) to measure antibodies to HPV capsid proteins. Serum samples from 575 college women, previously tested for the presence of cervicovaginal HPV DNA, were analyzed. The prevalences of anti-HPV16 VLP antibodies at baseline were 14.1% for immunoglobulin G (IgG) and 6.4% for IgA. The seroprevalences of IgG in women with cervicovaginal HPV16, HPV16-related types, and other HPV types were 55, 33, and 19%, respectively (P < 0.001), compared to the prevalence in women without an HPV infection (10%). HPV VLP IgA seropositivity was associated with high HPV16 VLP IgG optical density values. The seropositivity of IgG antibodies was independently associated with infection with HPV16 or HPV16-related types, increased number of lifetime male partners for vaginal sex, having sex with men >/= 5 years older, history of abnormal PAP smear, older age, and living separately from parents. Use of HPV16 VLP polymer ELISA detects clade-specific responses and suggests an HPV16 VLP vaccine may have broader protection that initially anticipated.     

Oncogenic human papillomavirus (HPV) type distribution and HPV type 16 E6 variants in two Spanish population groups with different levels of HPV infection risk

The aim of this study is to determine oncogenic human papillomavirus (HPV) types and HPV type 16 (HPV16) variant distribution in two Spanish population groups, commercial sex workers and imprisoned women (CSW/IPW) and the general population. A multicenter cross-sectional study of 1,889 women from five clinical settings in two Spanish cities was conducted from May to November 2004. Oncogenic HPV infection was tested by an Hybrid Capture II (HC2) test, and positive samples were genotyped by direct sequencing using three different primer sets in L1 (MY09/11 and GP5+/GP6+) and E6/E7. HPV16 variants were identified by sequencing the E6, E2, and L1 regions. Four hundred twenty-five samples were positive for the HC2 test, 31.5% from CSW/IPW and 10.7% from the general population. HPV16 was the most frequent type. Distinct profiles of oncogenic HPV type prevalence were observed across the two populations. In order of decreasing frequency, HPV types 16, 31, 58, 66, 56, and 18 were most frequent in CSW/IPW women, and types 16, 31, 52, 68, 51, and 53 were most frequent in the general population. We analyzed HPV16 intratype variants, and a large majority (78.7%) belonged to the European lineage. AA variants were detected in 16.0% of cases. African variants belonging to classes Af1 (4.0%) and Af2 (1.3%) were detected. Different HPV types and HPV16 intratype variants are involved in oncogenic HPV infections in our population. These results suggest that HPV type distribution differs in CSW/IPW women and in the general population, although further analysis is necessary.     

Candidate human papillomavirus (HPV) type 27b: nucleotide sequence and heterogeneity with HPV 27

More than 100 human papillomavirus (HPV) types have been reported as tumorigenic agents. HPV types 2 and 27 were identified in benign tumors and classified closely according to the sequence homology. An HPV type 2 related sequence was identified previously in an oral papilloma. However, the correct typing and sequencing was impossible at that time. A candidate HPV was cloned by the long-PCR technique from an oral papilloma and characterized by nucleotide sequence. This virus was basically HPV type 27, but there was some heterogeneity in comparison with the prototype. Most differences were nucleotide-sequence variations. Two of four-nucleotide insertions were frame shift, resulting in longer L1 protein than that of HPV 27.     

Analysis of human papillomavirus type 16 (HPV16) DNA load and physical state for identification of HPV16-infected women with high-grade lesions or cervical carcinoma

Integration of human papillomavirus (HPV) DNA into the host cell genome is a frequent event in cervical carcinogenesis, even though this phenomenon does not seem to be mandatory for cervical cancer development. Our objective was to describe the load and physical state of HPV type 16 (HPV16) DNA in a series of cervical samples representative of the natural history of cervical cancer. We used a combination of three real-time PCR assays targeting E6, E2, and albumin genes to calculate HPV16 load (E6 and albumin) and the E2/E6 ratio as a surrogate of integration. This method was applied to 173 HPV16-positive cervical samples. Results show that viral load increases with the lesion grade (from 102 HPV16 DNA copies per 10³ cells in normal samples up to 56,354 copies per 10³ cells in cancers), while E2/E6 ratio decreases (from 1 in normal samples down to 0.36 in cancers). We propose that, according to this technique, an HPV16 viral load of higher than 22,000 copies/10³ cells or an E2/E6 ratio of lower than 0.50 allows the identification of women with prevalent high-grade lesions or worse with a high specificity. In conclusion, both viral load and E2/E6 ratio, used in combination with an appropriate cutoff value, are suitable to screen women with prevalent cervical intraepithelial neoplasia grade 2 or 3 or cancer. Therefore, these assays would be useful in addition to routine HPV testing to more accurately identify women with (pre)cancerous lesions.     

Patterns of CD8 T-cell epitopes within the human papillomavirus type 16 (HPV 16) E6 protein among young women whose HPV 16 infection has become undetectable

The patterns of CD8 T-cell epitopes recognized within the E6 protein in women who had cleared their human papillomavirus 16 infection were examined. T-cell lines were established using autologous dendritic cells infected with a recombinant vaccinia virus. Evidence of potential antigenic epitopes was shown in 8 of 23 (34.8%) women.     

Detection of antibodies against human papillomavirus (HPV) type 16 virions by enzyme-linked immunosorbent assay using recombinant HPV 16 L1 capsids produced by recombinant baculovirus.

The L1 major capsid protein of human papillomavirus type 16 (HPV-16) was expressed in Sf-21 insect cells with a recombinant baculovirus. Virus-like particles obtained were purified and used to develop an enzyme-linked immunosorbent assay for detection of anti-HPV-16 antibodies in sera from 76 women with evidence of genital HPV infection and 79 controls. HPV-16-infected individuals developed antibodies directed at HPV-16 virions since reactivity against recombinant HPV-16L1 capsids was observed in 50% of them compared with only 6% in the general adult population. However, some cross-reactivities with sera from women infected with others HPV types were observed.     

Human papillomavirus (HPV) DNA copy number is dependent on grade of cervical disease and HPV type

The association between human papillomavirus (HPV) DNA copy number and cervical disease was investigated. Viral DNA copy number for the most common high-risk HPV types in cervical cancer (types 16, 18, 31, and 45) was determined in cervical cytobrush specimens from 149 women with high-grade cervical intraepithelial neoplasias (CIN II-CIN III), 176 with low-grade CIN (CIN I), and 270 with normal cytology. Quantitative, PCR-based fluorescent assays for each of the HPV genotypes and for the beta-globin gene were used. The amount of cellular DNA increased significantly with increasing disease; thus, HPV was expressed as copies per microgram of cellular DNA. The assay had a dynamic range of >10(7), allowing documentation for the first time of the wide range of HPV copy numbers seen in clinical specimens. Median HPV DNA copy number varied by more than 10(4) among the viral types. HPV16 was present in the highest copy number; over 55% of HPV16-positive samples contained more than 10(8) copies/microgram. Median copy number for HPV16 showed dramatic increases with increasing epithelial abnormality, an effect not seen with the other HPV types. HPV16 increased from a median of 2.2 x 10(7) in patients with normal cytology, to 4.1 x 10(7) in CIN I patients, to 1.3 x 10(9) copies/microgram in CIN II-III patients. Even when stratified by cervical disease and viral type, the range of viral DNA copies per microgram of cellular DNA was quite large, precluding setting a clinically significant cutoff value for "high" copy numbers predictive of disease. This study suggests that the clinical usefulness of HPV quantitation requires reassessment and is assay dependent.     

Detection of human papillomavirus (HPV) DNA type 6/11 in a conjunctival papilloma by in situ hybridization with biotinylated probes.

Four cases of conjunctival papilloma in two different patients were examined by in situ hybridization for HPV DNA type 6/11, 16/18 and 31/33/51. Formalin-fixed and paraffin-embedded tissues were hybridized by biotinylated probes. One tumor and one of its recurrences showed nuclear positivity for HPV 6/11 in the superficial cells of the epithelium. The results suggest that HPV type 6/11 may be etiologic agent of conjunctival papillomas. The benign behavior of these neoplasms may be related to the etiologic role of this type of HPV.     

Prevaccination genomic diversity of human papillomavirus genotype 6 (HPV 6)

Prevaccination genomic diversity of human papillomavirus genotype 6 (HPV 6) was established by sequencing 3798 bp of 77 clinically important HPV 6 isolates obtained from 45 and 32 patients with genital warts and laryngeal papillomas, respectively. By analyzing pooled L1, LCR, E6, E2, and E5 nucleotide data of an individual isolate, a total of 36 different genomic variants were identified, of which six (12 isolates), one (one isolate) and 29 (64 isolates) corresponded to HPV 6b, HPV 6a, and HPV 6vc genetic lineages, respectively. Several novel, potentially important mutations were identified. Non-prototypic HPV 6vc genomic variants were found in the majority of genital warts and laryngeal papillomas included in the study. The presence of serious HPV 6 genome sequence errors was confirmed and novel sequence errors were identified in sequence repositories.     

Detection of human papillomavirus (HPV) type 16 and 52b in cervical cancer tissues by Southern blot hybridization and polymerase chain reaction (PCR)

DNA samples from cancer tissues diagnosed histologically as squamous cell carcinoma of the uterine cervix were examined for the presence of human papillomavirus (HPV) genome DNA by Southern blot hybridization. Of 63 specimens, 32 were found to hybridize with HPV DNA probes; 23 specimens (35%) with HPV type 16 (HPV16), one (2%) with HPV type 18 (HPV18), four (7%) with HPV type 52b (HPV52b), and four others (7%) weakly with HPV52b. Specimens negative for HPV DNA with Southern blot hybridization were subjected to polymerase chain reaction (PCR) to determine the presence of HPV DNA under more sensitive conditions. After PCR using one set of primers specific for HPV16 and HPV52b, 7 out of 31 specimens were found to have HPV16 DNA. None was positive for HPV52b DNA. Our results indicate that HPV52b, as well as HPV16 and HPV18, is associated with squamous cell carcinoma of uterine cervix, and more sensitive determination of HPV infection can be made by amplification of the viral genome by PCR.     

A prospective study of antibody responses to defined epitopes of human papillomavirus (HPV) type 16 in relationship to genital and anorectal presence of HPV DNA.

The aim of this study was to investigate whether antibody responses against synthetic peptides derived from genital human papillomavirus (HPV) proteins are associated with laboratory-proven genital and anorectal HPV infection. In this study, 158 heterosexual patients (110 women and 48 men) were followed prospectively. At each visit we collected serum samples as well as specimens from several sites in the anogenital area for detection of HPV type 6/11 (HPV-6/11), -16, -18, and -33 DNAs by PCR. Immunoglobulin A (IgA) and IgG responses against disrupted bovine papilloma virions and eight different synthetic peptides derived from HPV-6/11, -16, and -18 were determined for serum samples from the first and the last visits. The subjects attended the Municipal Sexually Transmitted Disease Clinic in Amsterdam, The Netherlands, two to seven times (mean, four times) at approximately 4-month intervals. Women were monitored over a period of 155 person-years, and men were monitored over 65 person-years. The magnitudes of the IgA responses against HPV-16 late protein epitopes L1:13, L1:31, and L2:49 were significantly higher in the sera from the last visit among the currently HPV DNA-positive participants than in HPV DNA-negative persons (P = 0.02). When the persons positive for any HPV type at any time during the follow-up period were compared with those who were negative at all times during the follow-up period, we also found a significant elevation of IgA responses against L1:31 and L2:49 (P = 0.04 and 0.01, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

RU486 inhibits glucocorticoid hormone-dependent oncogenesis by human papillomavirus type 16 DNA.

We have previously reported (Pater et al., Nature 335, 832-835, 1988) the glucocorticoid hormone-dependent oncogenic transformation of primary baby rat kidney (BRK) cells by a combination of human papillomavirus (HPV) type 16 DNA and the activated form of the human Ha-ras-1 (ras) oncogene. In this study we provide evidence for the inhibition of such hormone-dependent transformation by the hormone antagonist, RU486. The level of inhibition was dependent on the ratio of hormone to antagonist and severe inhibition was observed at a 1:5 ratio. The effect of RU486 on the growth of HPV16-transformed cells was also studied. Severe growth inhibition and cell death occurred when cells transformed in the absence of RU486 were subsequently grown in the presence of either a 1:2.5 or 1:5 ratio of hormone to hormone antagonist. RU486 did not alter the level or electrophoretic mobility pattern of HPV16 mRNA expressed in transformed cells, when antihormone was applied either continuously or subsequent to transformation by HPV. These results indicate the importance of continuous viral gene expression for initiation and maintenance of the transformed phenotype.     

人乳头状瘤病毒16型结构蛋白L1和L2在大肠埃希菌 …

目的　研究人乳头状瘤病毒(HPV)主要结构蛋白L1(HPV16L1)和次要结构蛋白L2(HPV16L2)在原核系统的表达。方法　采用pET30a载体分别构建pET30aL1和pET30aL2质粒,并分别转入大肠埃希菌BL21菌株,经IPTG(异丙基硫代βD半乳糖苷)诱导,表达融合蛋白6×HisL1和6×HisL2,用SDSPAGE和Westernblot方法进行检测。结果　6×HisL1和6×HisL2融合蛋白在BL21菌中高效表达,约2～3mgml,其相对分子质量分别约为60000和97000;6×HisL1降解较6×HisL2多。结论　HPV16结构蛋白L1和L2能在原核表达系统高效表达;6×HisL2稳定性高于6×HisL1融合蛋白。     

Prevalence of antibodies to human papillomavirus (HPV) type 16 virus-like particles in relation to cervical HPV infection among college women.

A human papillomavirus type 16 (HPV-16) virus-like particle (VLP)-based enzyme-linked immunosorbent assay (ELISA) was used to measure serum antibody to capsid proteins in 376 sexually active college women who were also screened for the presence of genital HPVs by PCR and interviewed for demographic and behavioral risk factors for HPV infection. The seroprevalence was 46% in women with HPV-16 DNA in the genital tract. The corresponding values for women who harbored other HPV types or no HPV in the genital tract were 30 and 19%, respectively (HPV-16 group versus no-HPV group; odds ratio [OR], 3.7; 95% confidence interval [CI], 1.5 to 8.9). The antibody response was significantly higher among women with a high viral load than among those with a low viral load (median optical density value, 0.838 versus 0.137, P = 0.009). Comparable levels of seroreactivity were observed among women infected with HPV types distantly or closely related genetically to HPV-16. Seroreactivity was significantly associated with an age of 25 to 30 years (OR, 2.3; 95% CI, 1.2 to 4.4), three or more lifetime sexual partners (OR, 2.9; 95% CI, 1.1 to 10), and history of a sexually transmitted disease other than HPV (OR, 3.1; 95% CI, 1.5 to 6.3). The percent seropositivity increased linearly with number of lifetime sexual partners until reaching a plateau at 35% for women with more than six partners (chi for linear trend, P < 0.001). The low sensitivity of HPV-16 VLP-based ELISA may limit the usefulness of the assay as a diagnostic test for HPV-16 infection. However, the assay appears to have adequate specificity and should be useful as an epidemiological marker of HPV-16 infection and sexual behavior.     

Human papillomavirus (HPV) E6/E7 mRNA as a triage test after detection of HPV 16 and HPV 18 DNA

High‐risk human papillomavirus (HPV) DNA detection provides high sensitivity but low specificity for moderate‐grade cervical intraepithelial neoplasia or worse histological identification. A prospective study evaluated mRNA testing efficacy for predicting this histological diagnosis in case of HPV 16 and/or 18 DNA detection. A total of 165 endocervical samples harboring HPV 16 and/or 18 DNA were tested with NucliSENS‐EasyQ® HPV E6/E7‐mRNA‐assay (Biomerieux, Marcy l´Etoile, France). Women with cytological alterations were referred to colposcopy (n = 111). Moderate‐grade cervical intraepithelial neoplasia or worse was diagnosed in 25.8% of women presenting atypical squamous cells of undetermined significance or low‐grade squamous intraepithelial lesions and in 89.8% of women with high‐grade squamous intraepithelial lesions. mRNA sensitivity was 81.3% and 84.1%, respectively. Specificity was 52.2%, and 80.0%, respectively. Negative predictive value (NPV) was 88.9% in undetermined or low‐grade squamous lesions. Positive predictive value (PPV) was 97.4% in high‐grade squamous lesions. mRNA reduced colposcopies by 44.3% in undetermined or low‐grade squamous lesions. Direct treatment of mRNA‐positive cases reduced 77.5% of colposcopies in high‐grade squamous lesions. Women without cytological alterations were followed for 18 months (n = 35), and moderate‐grade cervical intraepithelial neoplasia or worse was diagnosed in 34.3%; mRNA sensitivity and specificity were 83.3% and 86.9%, respectively. PPV and NPV were 76.9% and 90.9%, respectively for predicting moderate‐grade cervical intraepithelial neoplasia or worse in 18 months. mRNA reduced the number of visits for follow‐up in 62.2%. In conclusion, NucliSENS‐EasyQ® HPV E6/E7‐mRNA‐assay (Biomerieux) can serve as a triage test in case of HPV 16 and/or 18 DNA detection. J. Med. Virol. 85: 1063–1068, 2013. © 2013 Wiley Periodicals, Inc.