A protective and broadly cross-neutralizing epitope of human papillomavirus L2

We generated a monoclonal antibody, RG-1, that binds to highly conserved L2 residues 17 to 36 and neutralizes human papillomavirus 16 (HPV16) and HPV18. Passive immunotherapy with RG-1 was protective in mice. Antiserum to the HPV16 L2 peptide comprising residues 17 to 36 (peptide 17-36) neutralized pseudoviruses HPV5, HPV6, HPV16, HPV 18, HPV31, HPV 45, HPV 52, HPV 58, bovine papillomavirus 1, and HPV11 native virions. Depletion of HPV16 L2 peptide 17-36-reactive antibodies from cross-neutralizing rabbit and human L2-specific sera abolished cross-neutralization and drastically reduced neutralization of the cognate type. This cross-neutralization of diverse HPVs associated with cervical cancer, genital warts, and epidermodysplasia verruciformis suggests the possibility of a broadly protective, peptide-based vaccine.     

State of Human Papilloma Virus DNA in Cervical Carcinomas

Cervical carcinoma is etiologically associated with the human papilloma virus (HPV), HPV 16 and HPV 18 being the most common. Viral DNA is thought to persist mostly in the episomal form in early tumor development, and in the integrated form in carcinomas. This assumption was checked with a new method that discriminated between RNAs transcribed from episomal and integrated HPV DNAs. Both forms were detected in carcinomas of Russian patients regardless of the disease stage. The data were verified by two other methods. RNA with sequences of the HPV transforming gene E7proved to be transcribed from either DNA form. The results suggest that HPV integration is not crucial for carcinoma progression.     

Characterization of a novel human papillomavirus DNA in the cervical carcinoma cell line ME180.

The human cervical carcinoma cell line ME180 was examined for human papillomavirus (HPV) DNA and RNA. The integrated DNA of a presumably new HPV type showing a relationship closer to HPV39 than to HPV18 was cloned and sequenced. HPV sequences from the E6-E7-E1 region are expressed as poly(A)+ RNAs.     

The cigarette smoke carcinogen benzo[a]pyrene enhances human papillomavirus synthesis

Epidemiological studies suggest that cigarette smoke carcinogens are cofactors which synergize with human papillomavirus (HPV) to increase the risk of cervical cancer progression. Benzo[a]pyrene (BaP), a major carcinogen in cigarette smoke, is detected in the cervical mucus and may interact with HPV. Exposure of cervical cells to high concentrations of BaP resulted in a 10-fold increase in HPV type 31 (HPV31) viral titers, whereas treatment with low concentrations of BaP resulted in an increased number of HPV genome copies but not an increase in virion morphogenesis. BaP exposure also increased HPV16 and HPV18 viral titers. Overall, BaP modulation of the HPV life cycle could potentially enhance viral persistence, host tissue carcinogenesis, and permissiveness for cancer progression.     

Hospital-based prevalence and sensitivity of high-risk human papillomavirus in Thai urban population

Background: To study the prevalence of high-risk human papillomavirus (HPV) types 16, 18, 31 and 33 in healthy Thai women using polymerase chain reaction (PCR) technique. Material and method: Two hundred and sixty three healthy urban women in Pathumthani, Thailand were recruited. Cervical cancer screening was performed and residual specimen from Pap smears was subjected to PCR to identify the presence of HPV types 16, 18, 31 and 33. Individuals’ demographic, health-specific and sexual behavior data were also collected. Results: Colposcopic biopsy revealed cases with high-grade squamous intraepithelial lesions (HSIL) and low-grade squamous intraepithelial lesions (LSIL). HPV 16 and 18 positive results were associated with abnormal Pap smears. Genotyping gave a 6.1%, 11.8%, 12.1%, and 14.1%, prevalence for HPV types 16, 18, 31 and 33, while 25% were infected with multiple HPV types. Discussion: High-risk HPV screening, used with abnormal pathology of HSIL gives 100% sensitivity and negative predictive value. Data from patient showed not significant correlation with neither different religion level of education, marital status, age of first sexual experience nor the number of sexual partners. Thus high-risk HPV screening is a recommended procedure with excellent sensitivity for detecting HSIL.     

Chlamydia trachomatis infection and human papillomavirus in women with cervical neoplasia in Pernambuco-Brazil

Chlamydia trachomatis (CT) is the most common bacterial cause of sexually transmitted disease. High-risk human papillomavirus (HR-HPV) is considered the main etiological agent for cervical neoplasia. Evidences showed that the presence of co-infection of CT and HR-HPV plays a central role in the etiology of cervical intraepithelial neoplasia (CIN) and cervical cancer. The goals of this study were: evaluate the human papillomavirus (HPV) and CT prevalence among Brazilian women with abnormal cytology and provide the effect of this association on the severity of cervical neoplasia. The population of this study was composed by 142 women with incident histological incidence of CIN grades I, II, III or cervical cancer from Recife, Northeast of Brazil. The polymerase chain reaction method on a cervical brush specimen was used to detect both agents and the automatic sequencing method was used for HPV genotyping assay. The prevalence of HPV and CT was 100 and 24.65 %, respectively. Thirteen types of HPV were detected; HPV 16, 18, 31 and 33 were the most common. The most prevalent HPV types were HPV 16 and 18. A significant association between CT positive and HPV 16 infection was found (p < 0.0106; OR = 5.31; 95 % IC 1.59–17.67). In the study population, there was diversity of HPV infections, with high-risk types being the most common. Also, the data collected suggest that CT infection may play an important role in the natural history of HPV infection.     

Genetic characteristics of human papillomavirus type 16, 18, 52 and 58 in southern China

Persistent infections of high-risk human papillomaviruses (HPVs) are the leading cause of cervical cancers. We collected cervical exfoliated cell samples from females in Changsha city, Hunan Province and obtained 338 viral genomes of four major HPV types, including HPV 16 (n = 82), 18 (n = 35), 52 (n = 121) and 58 (n = 100). The lineage/sublineage distribution of the four HPVs confirmed previous epidemiological reports, with the predominant prevailing sublineage as A4 (50%), A1 (37%) and A3 (13%) for HPV16, A1 (83%) for HPV18, B2 (86%) for HPV52 and A1 (65%), A3 (19%) and A2 (12%) for HPV58. We also identified two potentially novel HPV18 sublineages, i.e. A6 and A7. Virus mutation analysis further revealed the presence of HPV16 and HPV58 sublineages associated with potentially high oncogenicity. These findings expanded our knowledge of the HPV genetic diversity in China, providing valuable evidence to facilitate HPV DNA screening, vaccine effectiveness evaluation and control strategy development.     

Identification and Validation of Human Papillomavirus Encoded microRNAs

We report here identification and validation of the first papillomavirus encoded microRNAs expressed in human cervical lesions and cell lines. We established small RNA libraries from ten human papillomavirus associated cervical lesions including cancer and two human papillomavirus harboring cell lines. These libraries were sequenced using SOLiD 4 technology. We used the sequencing data to predict putative viral microRNAs and discovered nine putative papillomavirus encoded microRNAs. Validation was performed for five candidates, four of which were successfully validated by qPCR from cervical tissue samples and cell lines: two were encoded by HPV 16, one by HPV 38 and one by HPV 68. The expression of HPV 16 microRNAs was further confirmed by in situ hybridization, and colocalization with p16INK4A was established. Prediction of cellular target genes of HPV 16 encoded microRNAs suggests that they may play a role in cell cycle, immune functions, cell adhesion and migration, development, and cancer. Two putative viral target sites for the two validated HPV 16 miRNAs were mapped to the E5 gene, one in the E1 gene, two in the L1 gene and one in the LCR region. This is the first report to show that papillomaviruses encode their own microRNA species. Importantly, microRNAs were found in libraries established from human cervical disease and carcinoma cell lines, and their expression was confirmed in additional tissue samples. To our knowledge, this is also the first paper to use in situ hybridization to show the expression of a viral microRNA in human tissue.     

HPV-CCDC106 integration alters local chromosome architecture and hijacks an enhancer by three-dimensional genome structure remodeling in cervical cancer

Integration of human papillomavirus (HPV) DNA into the human genome is a reputed key driver of cervical cancer. However, the effects of HPV integration on chromatin structural organization and gene expression are largely unknown. We studied a cohort of 61 samples and identified an integration hot spot in the CCDC106 gene on chromosome 19. We then selected fresh cancer tissue that contained the unique integration loci at CCDC106 with no HPV episomal DNA and performed whole-genome, RNA, chromatin immunoprecipitation and high-throughput chromosome conformation capture (Hi-C) sequencing to identify the mechanisms of HPV integration in cervical carcinogenesis. Molecular analyses indicated that chromosome 19 exhibited significant genomic variation and differential expression densities, with correlation found between three-dimensional (3D) structural change and gene expression. Importantly, HPV integration divided one topologically associated domain (TAD) into two smaller TADs and hijacked an enhancer from PEG3 to CCDC106, with a decrease in PEG3 expression and an increase in CCDC106 expression. This expression dysregulation was further confirmed using 10 samples from our cohort, which exhibited the same HPV-CCDC106 integration. In summary, we found that HPV-CCDC106 integration altered local chromosome architecture and hijacked an enhancer via 3D genome structure remodeling. Thus, this study provides insight into the 3D structural mechanism underlying HPV integration in cervical carcinogenesis.     

Status of the human DNA papillomavirus in cervical tumors]

Cervical carcinoma is etiologically associated with the human papilloma virus (HPV), HPV 16 and HPV 18 being the most common. Viral DNA is thought to persist mostly in the episomal form in early tumor development, and in the integrated form in carcinomas. This assumption was checked with a new method that discriminated between RNAs transcribed from episomal and integrated HPV DNAs. Both forms were detected in carcinomas of Russian patients regardless of the disease stage. The data were verified by two other methods. RNA with sequences of the HPV transforming gene E7 proved to be transcribed from either DNA form. The results suggest that HPV integration is not crucial for carcinoma progression.     

Identification of the HPV-16 E6 protein from transformed mouse cells and human cervical carcinoma cell lines.

Human cervical carcinoma cell lines that harbor human papillomavirus (HPV) have been reported to retain selectively and express HPV sequences which could encode viral E6 and E7 proteins. The potential importance of HPV E6 to tumors is suggested further by the observation that bovine papillomavirus (BPV) E6 can induce morphologic transformation of mouse cells in vitro. To identify HPV E6 protein, a polypeptide encoded by HPV-16 E6 was produced in a bacterial expression vector and used to raise antisera. The antisera specifically immunoprecipitated the predicted 18-kd protein in two human carcinoma cell lines known to express HPV-16 RNA and in mouse cells morphologically transformed by HPV-16 DNA. The 18-kd E6 protein was distinct from a previously identified HPV-16 E7 protein. The HPV-16 E6 antibodies were found to be type specific in that they did not recognize E6 protein in cells containing HPV-18 sequences and reacted weakly, if at all, to BPV E6 protein. The results demonstrate that human tumors containing HPV-16 DNA can express an E6 protein product. They are consistent with the hypothesis that E6 may contribute to the transformed phenotype in human cervical cancers that express this protein.     

Evolutionary Dynamics of Variant Genomes of Human Papillomavirus Types 18, 45, and 97

Human papillomavirus type 18 (HPV18) and HPV45 account for approximately 20% of all cervix cancers. We show that HPV18, HPV45, and the recently discovered HPV97 comprise a clade sharing a most recent common ancestor within HPV α7 species. Variant lineages of these HPV types were classified by sequence analysis of the upstream regulatory region/E6 region among cervical samples from a population-based study in Costa Rica, and 27 representative genomes from each major variant lineage were sequenced. Nucleotide variation within HPV18 and HPV45 was 3.82% and 2.39%, respectively, and amino acid variation was 4.73% and 2.87%, respectively. Only 18 nucleotide variations, of which 10 were nonsynonymous, were identified among three HPV97 genomes. Full-genome comparisons revealed maximal diversity between HPV18 African and non-African variants (2.6% dissimilarity), whereas HPV18 Asian-American [E1 (AA)] and European (E2) variants were closely related (less than 0.5% dissimilarity); HPV45 genomes had a maximal difference of 1.6% nucleotides. Using a Bayesian Markov chain Monte Carlo (MCMC) method, the divergence times of HPV18, -45, and -97 from their most recent common ancestors indicated that HPV18 diverged approximately 7.7 million years (Myr) ago, whereas HPV45 and HPV97 split off around 5.7 Myr ago, in a period encompassing the divergence of the great ape species. Variants within the HPV18/45/97 lineages were estimated to have diverged from their common ancestors in the genus Homo within the last 1 Myr (<0.7 Myr). To investigate the molecular basis of HPV18, HPV45, and HPV97 evolution, regression models of codon substitution were used to identify lineages and amino acid sites under selective pressure. The E5 open reading frame (ORF) of HPV18 and the E4 ORFs of HPV18, HPV45, and HPV18/45/97 had nonsynonymous/synonymous substitution rate ratios (d_N/d_S) over 1 indicative of positive Darwinian selection. The L1 ORF of HPV18 genomes had an increased proportion of nonsynonymous substitutions (4.93%; average d_N/d_S ratio [M3] = 0.3356) compared to HPV45 (1.86%; M3 = 0.1268) and HPV16 (2.26%; M3 = 0.1330) L1 ORFs. In contrast, HPV18 and HPV16 genomes had similar amino acid substitution rates within the E1 ORF (2.89% and 3.24%, respectively), while HPV45 E1 was highly conserved (amino acid substitution rate was 0.77%). These data provide an evolutionary history of this medically important clade of HPVs and identify an unexpected divergence of the L1 gene of HPV18 that may have clinical implications for the long-term use of an L1-virus-like particle-based prophylactic vaccine.Papillomaviruses (PVs) are a large family of related viruses with circular double-stranded DNA genomes 8 kb in size. Some PV types cause epithelial hyperplasias ranging from benign exophytic warts to premalignant lesions that can progress to invasive cancer. Among the 61 currently designated alpha human PVs (HPVs), the majority have been isolated from the mucosal surface of the genital or oral region (8, 14). Of these, a select group have oncogenic potential and are associated with cervical cancer (11). Specifically, HPV type 16 (HPV16) and HPV18 have been identified in approximately two-thirds of cervical cancers, this tumor is the second most common cancer in women, and it is the principal cancer of women in developing countries (5, 24, 25, 30, 37).To date, studies of HPV18 variants have identified three lineages corresponding to the continental locations where the viral samples were obtained: European (E), Asian-American (AA), and African (Af) (29). The phylogeny of HPV18 variants is reflective of the migration patterns of Homo sapiens and suggests that HPV18 variant lineages might have diverged through genetic isolation at approximately the same time as Homo sapiens began establishing residence in different continental regions. Previous HPV18 intratypic phylogenetic analyses were limited to partial regions of the genome (3, 7, 29). Nevertheless, studies also suggest that HPV18 variants are associated with different levels of oncogenic potential and persistence and histological tumor types (1, 6, 35, 36, 46).HPV45 and HPV97 are the viral types most closely related to HPV18 and taken together form a clade and share a most recent common ancestor (MRCA). HPV97 is a recently described rare type (8, 17). HPV18 and HPV45 account for approximately 20% of all cervix cancers (25). Although HPV45 is a common type found in cervical cancer, its evolutionary history and sequence variability have not been extensively studied.In this report, 27 complete genomes representing the major variant lineages of HPV18, HPV45, and HPV97 were cloned and/or sequenced from clinical samples. Based on full genomes, the intratype/intertype evolutionary trees of HPV18, HPV45, and HPV97 were constructed. By examining the rate ratio of nonsynonymous (d_N) to synonymous (d_S) substitutions per site, diversifying selection acting on each of the eight protein-encoding regions of HPV18, HPV45, and HPV97 was evaluated. In addition, the times of divergence of HPV18/45/97 variants from their MRCA were investigated. These data provide an evolutionary history of this medically important clade of HPVs.