Analyses of human papillomavirus genotypes and viral loads in anogenital warts

Condylomata acuminata (genital warts) are the most common sexually transmitted viral diseases. These lesions are caused by infection with mucosal human papillomaviruses (HPVs). However, there is limited information on HPV strain distribution involved in the molecular pathogenesis of these lesions. To address this, the strain prevalence and the frequency of multiple HPV infections were determined in wart tissue obtained from 31 patients attending a wart clinic. These lesions were bisected and subjected to parallel DNA and mRNA extractions. HPV-type prevalence and incidence of multiple infections were determined by the Roche Linear Array assay. qPCR compared HPV 6, 11, 16, and 18 viral loads and RT-qPCR measured HPV 6 and 11 E6 genomic expression levels. Seventy-one percent of these samples were infected with multiple HPVs. Only one sample was negative for HPV 6 or 11 DNA. Forty-eight percent of samples were positive for a high risk (oncogenic) HPV. The results show that multiple infections in tissue are frequent and the subsequent analysis of HPV 6 and 11 E6 DNA viral loads suggested that other HPVs could be causing lesions. Further analysis of HPV 6/11 E6 mRNA levels showed that there was no discernable relationship between HPV 6 E6 DNA viral load and relative HPV 6 or 11 E6 mRNA levels thereby questioning the relevance of viral load to lesion causality.     

Developing HPV virus-like particle vaccines to prevent cervical cancer: a progress report.

BACKGROUND: the knowledge that sexually transmitted infection with one of a limited number of human papillomaviruses (HPVs) is a central cause of almost all cervical cancers affords the opportunity to prevent this common cancer through anti-viral vaccination. OBJECTIVE: the spectacular success of vaccines in preventing several other viral diseases offers hope that immunoprophylaxis against the relevant HPVs could lead to a major reduction in cervical cancer incidence. RESULTS AND CONCLUSION: the results of preclinical studies and early phase clinical trials of virus-like particle (VLP) based subunit vaccines have been very encouraging. However, unique aspects of papillomavirus biology and genital tract infections, and the lack of sexual a transmission model for papillomavirus, make it far from certain that effective prophylactic vaccination against genital HPV infection will be easily achieved. Future clinical efficacy trials will likely test the hypothesis that parenteral injection of VLPs can induce antibody mediated and type specific protection against genital tract HPV infection and subsequent development of premalignant neoplastic disease.     

The prevalence and genotype of human papillomavirus on cervical samples from an Irish female population with external genital warts

The aim of this study was to determine the cervical genotype profile of females who presented to an STI Clinic with external genital warts (EGW); and to determine the potential vaccine coverage prior to the uptake of the HPV vaccines. Sixty-one cervical scrapings were taken from females aged 18-35 y who had external genital warts or a history of external genital warts. The resulting 50 samples that were positive for HPV-DNA were subjected to genotype identification. Forty-six of these samples had detectable genotypes by LIPA analysis and most (78%, 36/46) had multiple low risk (LR) and high risk (HR) genotypes on the cervix. Twenty-five of these samples (54%) had more than 1 HR genotype. Of the 36 patients who had any HR genotypes, 18 (50%) were identified to have the most oncogenic HPV genotypes, namely 16 and 18. Three of these samples had both 16 and 18 on the cervix. The presence of multiple HR genotypes on the majority of cervical samples from a self-referred population of females with EGW is presented. This study is of importance since persistent HR-HPV is the necessary risk factor in the development of precancerous and cancerous lesions of the cervix. Gardisil, the quadrivalent HPV vaccine would have been useful in the prevention of 28% (13/46) of these infections.     

An overview of human papillomaviruses and current vaccine strategies

Cervical cancer is one of the most common cancers in women worldwide, particularly in developing countries. The viral origin of cervical cancer has been proven beyond any reasonable doubt. Persistent infection with certain subsets of human papillomaviruses is recognized as a necessary cause for the development of cervical cancer. Persistence of oncogenic HPVs, immunodeficiency, high HPV viral load and cofactors like smoking, multiple sex partners and poor nutrition predispose to cervical cancer. Prophylactic vaccines using HPV virus-like particles containing capsid protein L1 have shown protection against disease in animals and are currently undergoing clinical trials. Therapeutic vaccines using HPV E6 and E7 proteins are also being investigated for their ability to remove residual infection.     

Biology and pathological associations of the human papillomaviruses: a review

Historical cottontail rabbit papillomavirus studies raised early indications of a mammalian DNA oncogenic virus. Today, molecular cloning recognises numerous animal and human papillomaviruses (HPVs) and the development of in vitro transformation assays has escalated oncological research in HPVs. Currently, their detection and typing in tissues is usually by Southern blotting, in-situ hybridization and polymerase chain reaction methods. The complete papillomavirus virion constitutes a protein coat (capsid) surrounding a circular, double-stranded DNA organised into coding and non-coding regions. 8 early (E1-E8) open reading frames (ORFs) and 2 late (L1, L2) ORFs have been identified in the coding region of all papillomaviruses. The early ORFs encode proteins which interact with the host genome to produce new viral DNA while late ORFs are activated only after viral DNA replication and encode for viral capsid proteins. All papillomaviruses are obligatory intranuclear organisms with specific tropism for keratinocytes. Three possible courses of events can follow papillomaviruses entry into cells: (1) viral DNA are maintained as intranuclear, extrachromosomal, circular DNA episomes, which replicates synchronously with the host cell, establishing a latent infection; (2) conversion from latent into productive infection with assembly of complete infective virions; (3) integration of viral DNA into host cellular genome, a phenomenon seen in HPV infections associated with malignant transformation. Human papillomaviruses (HPVs) essentially induce skin and mucosal epithelial lesions. Various skin warts are well known to be HPV-associated (HPVs 1, 2, 3, 7 and 10). Besides HPVs 3 and 10, HPVs 5, 8, 17 and 20 have been recovered from Epidermodysplasia verruciformis lesions. Anogenital condyloma acuminatum, strongly linked with HPVs 6 and 11 are probably sexually transmitted. The same HPVs, demonstrable in recurrent juvenile laryngeal papillomas, are probably transmitted by passage through an infected birth canal. HPVs described in uterine cervical lesions are generally categorized into those associated with high (16, 18), intermediate (31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68) and low (6, 11, 26, 40, 42, 43, 44, 53, 54, 55, 62, 66) risk of cervical squamous carcinoma. Cervical adenocarcinoma, clear cell carcinoma and small cell neuroendocrine carcinoma have also been linked to HPVs, especially HPV18. Other lesions reported to be HPV-associated are: papillomas, dysplasia and carcinomas in the nasal cavity (HPV 6, 11, 57); squamous papilloma, condyloma acuminatum, and verruca vulgaris of the oral cavity (HPV 6, 11), oral focal epithelial hyperplasia (HPV 13, 32); warty lip lesions (HPV 2): and conjunctival papillomas (HPV 6, 11).     

Human papillomavirus infection and P53 codon 72 genotypes in a Hispanic population at high-risk for cervical cancer

Cervical cancer mortality is high in Texas, especially among Hispanic women living in south Texas and adjacent Mexico. Though human papillomavirus (HPV) infection has a causal role in the development of cervical cancer, there are no published data on the prevalence of HPV genotypes in this underscreened region. We studied 398 Hispanic women on both sides of the border along the lower Rio Grande River to determine the prevalence of HPV genotypes and risk factors for cervical cancer. Using a nested PCR system HPV was detected in 62% of cervical specimens, including all the known high-risk HPV genotypes, with HPV16 and HPV18 the most frequent (30.6% and 23.0%, respectively). Multiple infections were common (29.4% of the infected specimens), and where this occurred we were more likely to find high-risk HPV genotypes. We examined host p53 codon 72 genotype frequencies and found that patients with cervical abnormalities and women with HPV16 and HPV18 infections had a lower genotype frequency of the homozygous (AA) previously reported to be associated with cervical cancer, than uninfected women with no abnormalities. In this US/Mexico border population high rates of potentially oncogenic HPV viruses and multiple infections are consistent with observed elevated cervical cancer rates. These data are further evidence that in this underserved population HPV infections are associated with high rates of malignancy, but that host p53 genotypic variations are unlikely to be primary factors in oncogenesis.     

Human papillomavirus infection. Pathology and molecular pathology

Cervical cancer is the second most frequent female malignoma worldwide and accounts for about 500,000 cases every year. The peak incidence lies between 35 and 55 years of age. Persistent infections with a group of 15 so-called high-risk human papillomaviruses (HR-HPV) are the cause of cervical carcinogenesis of squamous cell carcinomas and for most of the adenocarcinomas. The transforming potential of HR-HPVs is based on the interaction of viral oncogene products E6 and E7 with the cellular tumor suppressor proteins p53 and pRB. The resulting loss of cell cycle control sets the basis for additional, as yet only incompletely discovered, genetic and epigenetic changes, finally leading to invasive growth. Preneoplastic changes, cervical intraepithelial neoplasias, can be identified morphologically, thus allowing for therapeutic interventions. Since November 2007, the St?ndige Impfkommission, the German standing committee on immunizations, has recommended the prophylactic use of vaccines against the two most frequent HR-HPV genotypes, HPV-16 and HPV-18, in women age 12-17 years before first sexual intercourse. In addition to cervical cancer, HPV infections are responsible for the development of genital warts (condyloma) and a number of vaginal, vulvar, and anal intraepithelial neoplasias. HPV infections are usually transmitted sexually.     

Human papillomavirus infection: Epidemiology,biology, host interactions,cancer development,prevention, and therapeutics

Human papillomavirus (HPV) infection is one of the most common sexually transmitted infections worldwide. It is caused by the HPV, a DNA virus that infects epithelial cells in various mucous membranes and skin surfaces. HPV can be categorised into high-risk and low-risk types based on their association with the development of certain cancers. High-risk HPV types, such as HPV-16 and HPV-18, are known to be oncogenic and are strongly associated with the development of cervical, anal, vaginal, vulvar, penile, and oropharyngeal cancers. These types of HPV can persist in the body for an extended period and, in some cases, lead to the formation of precancerous lesions that may progress to cancer if left untreated. Low-risk HPV types, such as HPV-6 and HPV-11, are not typically associated with cancer but can cause benign conditions like genital warts. Genital warts are characterised by the growth of small, cauliflower-like bumps on the genital and anal areas. Although not life-threatening, they can cause discomfort and psychological distress. HPV is primarily transmitted through sexual contact, including vaginal, anal, and oral sex. It can also be transmitted through non-penetrative sexual activities that involve skin-to-skin contact. In addition to sexual transmission, vertical transmission from mother to child during childbirth is possible but relatively rare. Prevention of HPV infection includes vaccination and safe sexual practices. HPV vaccines, such as Gardasil and Cervarix, are highly effective in preventing infection with the most common high-risk HPV types. These vaccines are typically administered to adolescents and young adults before they become sexually active. Safe sexual practices, such as consistent and correct condom use and limiting the number of sexual partners, can also reduce the risk of HPV transmission. Diagnosis of HPV infection can be challenging because the infection is often asymptomatic, especially in men. In women, HPV testing can be done through cervical screening programs, which involve the collection of cervical cells for analysis. Abnormal results may lead to further diagnostic procedures, such as colposcopy or biopsy, to detect precancerous or cancerous changes. Overall, HPV infection is a prevalent sexually transmitted infection with significant implications for public health. Vaccination, regular screening, and early treatment of precancerous lesions are key strategies to reduce the burden of HPV-related diseases and their associated complications. Education and awareness about HPV and its prevention are crucial in promoting optimal sexual health. This study aimed to carry out a literature review considering several aspects involving HPV infection: Global distribution, prevalence, biology, host interactions, cancer development, prevention, therapeutics, coinfection with other viruses, coinfection with bacteria, association with head and neck squamous cell carcinomas, and association with anal cancer.     

Denaturing high-performance liquid chromatography for detecting and typing genital human papillomavirus

Human papillomaviruses (HPVs) are important in the development of human cancers, including cervical and oral tumors. However, most existing methods for HPV typing cannot routinely distinguish among the more than 100 distinct types of HPV or the natural HPV intratypic variants that have also been documented. To address this problem, we developed a novel method, general primer-denaturing high-performance liquid chromatography (GP-dHPLC), for the detection and typing of genital HPV using an automated 96-well plate format. GP-dHPLC uses general primer PCR (GP-PCR) to amplify the viral DNA and then analyzes the GP-PCR products by denaturing high-performance liquid chromatography (dHPLC). A number of different primer pairs with homology to most known genital HPV types were tested, and the L1C1-L1C2M pair specific for the L1 region of the viral genome was chosen. A set of HPV standard control patterns, consisting of those for HPV types 16, 18, 31, 33, 39, 45, 51, 52, 56, 58, 59, 6, and 11, was established for genital HPV typing. One hundred eighty-six frozen and formalin-fixed cervical cancer tissue samples were analyzed for the presence of HPV and the HPV type by this method, and 95.8% of them were found to contain HPV DNA. GP-dHPLC accurately discriminated among HPV variants that differed by as little as one nucleotide. Several new variants of HPV types 16, 18, 39, 45, 52, and 59 were identified. Moreover, multiple HPV infections were detected in 26.6% of the samples. Our results indicate that HPV typing by GP-dHPLC permits discrimination of common genital HPV types, detection of multiple HPV infections, and identification of HPV variants in clinical samples.     

Search for cellular partners of human papillomavirus type 16 E2 protein

Human papillomaviruses (HPVs) are small, double-stranded DNA viruses that infect cutaneous and mucosal epithelia. Type 16 (HPV16) displays tropism to genital epithelia, giving rise to genital warts and cervical intraepithelial neoplasia (CIN), which is a precursor lesion to invasive carcinoma of the cervix. The great majority of human cervical cancers contain integrated HPV DNA where the E2 gene is usually disrupted, suggesting that the loss of the E2 protein is an important step in HPV-induced carcinogenesis. The HPV16 E2 protein is a regulatory protein that seems to be essential for creating favourable conditions for establishment of infection and proper completion of the viral life cycle. Recently, diverse activities of the E2 proteins have been described, but the molecular basis of these processes has not beenfully elucidated. Using a yeast two-hybrid system, we have identified epithelial cellular proteins that bind to the E2 protein of HPV16.     

Prevalence and distribution of single and multiple HPV infections in cytologically abnormal cervical samples from Italian women

The prevalence of single and multiple HPV infections was assessed over a cohort of 213 women with cytological abnormalities and its association with cervical neoplasia established. Roche linear array HPV genotyping test was used to identify HPV genotypes. The most prevalent HPV genotypes in cervical cancer samples were HPV16 (61.2%), HPV52 (16.1%), HPV18 (12.9%) and HPV 31 (9.6%). Multiple HR and LR HPV infections, comprising between two and 5+ HPV types, were identified in 49.7% of samples, with a significantly lower number in severe dysplasia and cervical cancer samples (p<0.05). These results seem to indicate that detection of multiple HPV infection with HR-HPV types is not significantly better as a predictor of cervical cancer than single HR-HPV infection, though further longitudinal studies are needed to better clarify the relevance of these infections to the progression of cervical neoplasia.     

Genital human papilloma virus infection in Oslo studied by dot blot DNA hybridization and the polymerase chain reaction.

Samples from patients with genital condyloma acuminata or with cervical condylomas and/or dysplasia and from women without cytological/clinical evidence of cervical affection were examined by dot blot DNA hybridization or the polymerase chain reaction (PCR). The PCR was much more sensitive than dot blot, more than doubling the human papilloma virus (HPV) findings. HPV DNA, mainly HPV 6/11, was detected in 18 of 19 biopsies of condyloma acuminata, whereas HPV 16 was most frequently detected in the 21 cervices (76%) with condyloma and/or dysplasia. HPV 16 was detected in eight of 103 cervical smears with no signs of infection. The prevalence of HPV 16 in cervical samples was somewhat higher than expected. This suggests that, in Oslo, HPV 16 is a common HPV type in women with cytologically normal cervices. HPV 18 was relatively rare and was detected only in combination with other HPVs.     

Natürlicher Verlauf der HPV-Infektion Nutzen der HPV-Analytik in der Zervixdiagnostik

Cervical carcinomas and their precursors (cervical dysplasia, CIN1-3) are associated with human papillomavirus (HPV) infections. Epidemiological and in vitro-studies have shown that some of the genital HPV types, the high risk-types 16, 18, 31 etc., code for proteins (E6/E7) which strongly influence the cell cycle and genome stability. Progression from weak to severe dysplasia and to invasive cancer is associated with increasing expression of these viral oncogenes. Which additional cofactors contribute to progression of some dysplasias to carcinomas is still a matter of investigation. Recent results point to genetic predisposition (p53 polymorphism), cellular immune reaction, and cytokine expression. For HPV detection in cervical swabs and biopsies two highly sensitive and reliable systems (PCR, Hybrid Capture system) are available. Although classical histological methods are sufficient for the diagnosis of high-grade lesions and invasive cancer, HPV testing might give valuable diagnostic and prognostic clues especially in cases of unclear cytology (ASCUS) or weak dysplasia.     

IgG antibodies to human papillomavirus 16, 52, 58, and 6 L1 capsids: case-control study of cervical intraepithelial neoplasia in Japan

In Japan, human papillomavirus (HPV) 16, 52, and 58 are most commonly associated with cervical intraepithelial neoplasia (CIN). By contrast, HPV6 is primarily associated with genital warts. This study was designed to evaluate the association between IgG antibody responses to common HPVs and the risk of CIN development within a Japanese population. CIN cases (n = 141) and controls (n = 109) were tested for cervical HPV DNA and serum IgG antibodies to L1 capsids of HPV16, 52, 58, and 6. Seropositivity to HPV16, 52, and 58 L1 capsids was significantly higher in CIN cases than in controls: 27%, 21%, and 31% versus 16%, 11%, and 11%, respectively (P < 0.05). HPV6 L1 seropositivity was not significantly associated with CIN lesions (P = 0.11). Presence of viral DNA for either HPV16, 52, or 58 correlated with a significant antibody response against the homologous L1 capsids but not heterologous L1 capsids. Furthermore, seropositivity to multiple types of HPV16, 52, and 58 was more strongly associated with an increased risk of CIN development than seropositivity to a single type (P for trend <0.001). These findings indicate that IgG antibodies to L1 capsids of HPV16, 52, and 58 represent an increased risk of CIN development, with antibodies to multiple types being indicative of a further increase in risk. The presence of CIN lesions in women with seropositivity to multiple types suggests that viral exposure to a given type may not be protective against infections by other types and subsequent CIN development.     

Patterns of genotype distribution in multiple human papillomavirus infections.

The relationship between severe-grade cervical lesions and clusters of human papillomavirus (HPV) genotypes in a taxonomic classification was surveyed in 232 women with previous abnormal cytology. HPV co-infections were clustered according to phylogenetic criteria. Multiple infections were detected in 22.0% of the entire sample. Clade A10 (represented by HPV-6 and HPV-11) appeared more frequently in multiple infections than clade A9, which was represented by five of the most common high-risk types, including HPV-16. Although HPV-16 was the most frequent genotype, it was not more prevalent in multiple infections. Abortion and two or more sexual partners were risk-factors associated with HPV co-infections. Severe cervical dysplasia was associated with co-infections with oncogenic types from different clades, with the association being significant for the high-risk clades A7 and A9.     

Recent developments in immunotherapy of cancers caused by human papillomaviruses

A subset of oncogenic human papillomaviruses (HPVs) is the main cause of genital cancers, most importantly cervical cancer and an increasing number of head and neck cancers. Despite the availability of prophylactic vaccines against the most prevalent oncogenic HPV types, HPV‐induced malignancies are still a major health and economic burden. Besides conventional treatment with surgery, chemotherapy and radiation, immunotherapy is emerging as an efficient adjuvant option. Here, we review relevant studies and ongoing clinical trials using immune checkpoint inhibitors, therapeutic vaccines, gene editing approaches and adoptive T cell therapies, with special focus on engineered TCR T cells, which are showing encouraging results and could lead to significant improvement in the treatment of HPV+‐infected cancer patients.     

Biologic characteristics of specific human papillomavirus types predicted from morphology of cervical lesions.

Human papillomavirus (HPV) DNA was detected by Southern blot hybridization in cervicovaginal lavage samples from 199 of 329 (60.5%) women attending a municipal hospital colposcopy clinic. Human papillomavirus was identified in 195 of 264 (73.9%) patients with a squamous intraepithelial lesion or cancer on biopsy or Papanicolaou smear (Bethesda system) compared with 11 of 65 (16.9%) without squamous intraepithelial lesion (P < .0001). The most common HPV type identified was HPV 16 (20.6% of positive samples), and 36.7% of isolates contained uncharacterized HPVs. Of women with cervical intraepithelial neoplasia (CIN) grade III or cancer, 23.4% were infected with HPV 16 compared with less than 4% with any other single HPV type. Based on biopsy diagnosis in patients infected with specific HPV types, HPVs 6 and 11 had low oncogenic potential; HPVs 18, 31, 35, and 45 had intermediate oncogenic potential; and HPVs 16 and 33 had high oncogenic potential. Hyperchromatic, unusually enlarged nuclei ("meganuclei"), and/or abnormal mitoses were found significantly more often in lesions infected with HPVs 16, 33, and 35 than in those infected with HPVs 6, 11, 18, 31, and 45, even in low-grade lesions, and may represent a histologic marker for HPVs with significant oncogenic potential. Human papillomavirus capsid protein was detected significantly less often by immunocytochemical staining in CIN I and CIN II lesions infected with HPVs 16 and 33 (8.3%) than in those infected with HPVs 6, 11, 18, and 31 (60%; P = .007), suggesting early abnormalities in cellular differentiation in lesions infected with highly oncogenic HPVs.     

Anal cancer. Microscopic condyloma and tissue demonstration of human papillomavirus capsid antigen and viral DNA

Sixteen cases of squamous cell carcinoma of the anus, including 4 incidentally discovered in situ lesions, and 3 anal condylomas, were examined for the presence of human papillomavirus (HPV). All in situ tumors and 6 of the invasive tumors were associated with histologic changes typical of condyloma, despite the absence of clinical anogenital warts. Immunohistochemical studies for viral capsid antigen gave positive reactions in two anal warts and in the condylomatous area associated with one invasive tumor. In situ hybridization was accomplished using isotopic DNA probes for HPV 6/11, 16, 18, and 31. Human papillomavirus 6/11 was expressed in the corresponding capsid-positive regions in the two warts and the wart-associated invasive carcinoma. Both HPV 6/11 and HPV 16 were associated with one carcinoma in situ, and HPV 16 was also found within two invasive anal carcinomas, one of which was associated with an extensive vulvar cancer. While these observations do not resolve the "passenger" or direct oncogenic role for HPV in anal carcinoma, the circumstantial evidence is that the oncogenic influence is similar to that accepted for female genital tract cancer.     

Human papillomavirus: current status and issues of vaccination

An association between human papillomavirus (HPV) infection and the development of cervical cancer was initially suggested over 30 years ago, and today there is clear evidence that certain subtypes of HPV are the causative agents of such malignancies. Papillomaviruses make up a vast family that comprises hundreds of different viruses. These viruses infect epithelia in humans and animals and cause benign hyperproliferative lesions, commonly called warts or papillomas, which can occasionally progress to squamous cell cancer. HPV infections are considered the most common among sexually transmitted diseases. One of the most prevalent cancer types induced by HPV (mostly types 16 and 18) is cervical cancer. Vaccination is the most effective means of preventing this infectious disease. These prophylactic vaccines, based on virus-like particles (VLPs), are extremely effective in providing protection from infection in almost 100 % of cases. VLP vaccines of HPV are subunit vaccines consisting only of the major viral capsid protein of HPV. There are two types of vaccine available: bivalent vaccine (against HPV-16/18) and quadrivalent vaccine (against HPV-6/11/16/18). Second-generation prophylactic HPV vaccines, currently in clinical trials, may hold several merits over the current bivalent and quadrivalent vaccines, such as protection against additional oncogenic HPV types, less dependence on cold-chain storage and distribution, and non-invasive methods of delivery.