Biology of papillomavirus infections. IV. Sero-epidemiological data]

Serological assays using synthetic peptides or recombinant proteins to identify specific HPV infection either fail to correlate with the type of infection or are only able to identify a small percentage of infected individuals. But genetically engineered Virus Like Particles (VLP) seem useful to develop Elisas for serological diagnosis of HPV infection and antibodies to VLP appear to correlate well with HPV DNA presence. The levels of secretory immunoglobulins in genital secretions would provide a better indicator of HPV infection, but reproducibility and standardization of the detection methods are unresolved. The clinical relevance of serologic responses is still questionable, since frequency and titer of several types of antibodies generated against HPV show a great variability which is dependent on the HPV type specificity, on the recognized epitopes and on the type of samples. However the detection of neutralizing antibodies associated with disease recurrence and antibodies raised against HPV16 E6 and E7 peptides found to react with sera of cancer patients, needs to pay attention.     

Human papillomavirus update with a particular focus on cervical disease

Human papillomavirus (HPV) is a common viral infection of squamous epithelial tissues, but its importance has only recently been recognised by the medical community. HPVs are now realised to consist of many genotypes and are associated with a diverse spectrum of clinical manifestations. Within the genital tract, some diseases have been recognised since antiquity; for example, genital warts which are caused by HPV types distinct from those causing genital cancer. However, others (such as cervical cancer), although recognised centuries ago as linked to sexual activity, have only been associated with oncogenic HPVs relatively recently, with the tools of molecular biology. We now understand that genital HPV infections are the most common sexually transmitted viral infections, are largely transient, asymptomatic and of no consequence. This virus manifests as more than just benign warts. Chronic carriage of with oncogenic genotypes (over years and in a minority of patients), together with other cofactors (host and/or exogenous) in complex pathways not totally understood, result in severe dysplasia or, ultimately, carcinogenesis. As it takes time for precursor lesions to develop and there are effective screening programmes for their detection and treatment, HPV-related neoplastic disease of the cervix is largely a preventable reproductive health issue of women. Yet, on a global scale, cervical cancer is the second most common cancer of women, with the majority of cases occurring in developing countries. Although HPV is noncultivatable by traditional diagnostic virological methods, successfully applied molecular biology techniques have underpinned development of vaccines which are now in phase II/III clinical trials. Successful vaccination ultimately has the greatest potential to impact upon the global burden of disease from genital HPV infection. However, the outcome from reduction in incidence of dysplasia and neoplasia will take years to eventuate; consequently, various cervical cancer prevention strategies still need to be endorsed and maintained in the meantime.     

Use of PGMY primers in L1 consensus PCR improves detection of human papillomavirus DNA in genital samples

The novel PGMY L1 consensus primer pair is more sensitive than the MY09 and MY11 primer mix for detection and typing with PCR of human papillomavirus (HPV) DNA in genital specimens. We assessed the diagnostic yield of PGMY primers for the detection and typing of HPV by comparing the results obtained with PGMY09/PGMY11 and MY09/MY11/HMB01 on 299 genital samples. Amplicons generated with PGMY primers were typed with the line blot assay (PGMY-line blot), while HPV amplicons obtained with the degenerate primer pool MY09/MY11/HMB01 were detected with type-specific radiolabeled probes in a dot blot assay (standard consensus PCR test). Cervicovaginal lavage samples (N = 272) and cervical scrape samples (N = 27) were tested in parallel with both PCR tests. The PGMY-line blot test detected the presence of HPV DNA more frequently than the standard consensus PCR assay. The concordance for HPV typing between the two assays was 84.3% (214 of 255 samples), for a good kappa value of 0.69. Of the 177 samples containing HPV DNA by at least one method, 40 samples contained at least one HPV type detected only with PGMY-line blot, whereas positivity exclusively with the standard consensus PCR test was found for only 7 samples (P < 0.001). HPV types 45 and 52 were especially more frequently detected with PGMY than MY primers. However, most HPV types were better amplified with PGMY primers, including HPV-16. Samples with discordant results between the two PCR assays more frequently contained multiple HPV types. Studies using PGMY instead of MY primers have the potential to report higher detection rates of HPV infection not only for newer HPV types but also for well-known genital types.     

Basic concepts and practical applications of recombinant DNA techniques in detection of human papillomavirus (HPV) infection. Review article

Recent studies on the role of infectious agents in the pathogenesis of malignancy have demonstrated a strong association between HPV and several human benign and malignant epithelial neoplasms. There are 60 distinct types of HPV, of which HPV 16, 18, 31, 33, 35 and 39 have been associated with squamous cell neoplasia of the genital tract. Rapid progress in the field of recombinant DNA technology with the availability of specific probes has enabled the detection of HPV genomic sequences in characteristic HPV lesions. In addition, HPV sequences have been found in malignant squamous cell lesions, and even in normal tissues lacking the morphologic signs of HPV infection. Currently, hybridization analysis of the nucleic acid is the most reliable method for diagnosis of HPV infections, also permitting the genotyping of these viruses. A variety of hybridization procedures have been developed with different sensitivities and specificities. Despite the divergent technical modifications, however, all hybridization tests working according to the same basic principles. The double helix of DNA composed of two complementary polynucleotide chains can be opened by heating or by increasing pH. Cooling of the two strands allows reassociation. Labeled HPV DNA or RNA probe hybridizes with the complementary sequences allowing the detection of HPV sequences in the samples. Hybridization assays can be conducted under conditions in which virtually all HPVs will be detected, but not specifically typed (low stringency) or under conditions in which the type can be identified (high stringency). Widely divergent results have been reported both in prevalence of HPV infection and distribution of different HPV types in the genital tract. These discordant results have been explained on the basis of sampling effects, differences in histopathological diagnosis, geographical variations in HPV types and interlaboratory variation in HPV detection and typing techniques. In this review the various procedures for detecting HPV sequences by hybridization and related techniques are shortly described.     

Epitope specificity and longevity of a vaccine-induced human T cell response against HPV18

Persistent human papillomavirus (HPV) type 16 and 18 infection can lead to pre-malignant and malignant diseases of the lower genital tract. Several lines of evidence suggest that T cell responses can control HPV infection. However, relative to other human viruses, strong effector memory T cell responses against HPV have been difficult to detect. We used an in vitro stimulation step prior to enzyme-linked immunospot assays to identify IFN-gamma-secreting T cells specific for HPV16 and 18 E6/E7 peptides. This allowed the detection of HPV-specific CD4+ T cells that were not evident in direct ex vivo assays. T cell responses against HPV16 or 18 peptides were detected in healthy volunteers (7/9) and patients with lower genital tract neoplasia (10/20). Importantly, this assay allowed tracking of vaccine-induced T cell responses in nine patients, following inoculation with a live recombinant vaccinia virus (HPV16 and 18 E6/E7, TA-HPV). Novel vaccine-induced T cell responses were demonstrated in five patients, but no clinical responses (lesion regressions) were seen. For one vaccinated patient, the T cell response was mapped to a single dominant HPV18 E7 epitope and this response was sustained for >3 years. Our data suggest that systemic memory T cells against HPV16 and 18, induced naturally or by TA-HPV vaccination, are relatively rare. Nevertheless, the assay system developed allowed estimation of magnitude, epitope specificity, and longevity of vaccine-induced CD4+ T cell responses. This will be useful for vaccine design and measurement of immunological endpoints in clinical trials.     

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.     

Spotlight on Quadrivalent Human Papillomavirus (Types 6, 11, 16, 18) Recombinant Vaccine (Gardasil®) in the Prevention of Premalignant Genital Lesions, Genital Cancer, and Genital Warts in Women

Quadrivalent human papilloma virus (HPV) [types 6, 11, 16, 18] recombinant vaccine (Gardasil®; Silgard®) is composed of virus-like particles (VLPs) formed by self-assembly of recombinant L1 capsid protein from each of HPV types 6, 11, 16, and 18. The VLPs are noninfectious, containing no DNA, and are highly immunogenic, inducing high levels of neutralizing antibodies against the particular HPV types when administered to animals or humans. Quadrivalent HPV vaccine is indicated for use from the age of 9 years for the prevention of premalignant genital lesions (cervical, vulvar, and vaginal), cervical cancer, and external genital warts (condyloma acuminata) causally related to certain oncogenic or specific HPV types. In placebo-controlled clinical trials, quadrivalent HPV vaccine administered as three doses over 6 months provided high-level protection against infection or disease caused by the vaccine HPV types over 2–4 years of follow-up in females aged 15–45 years who were naive to the vaccine HPV types. A degree of cross-protection against certain other non-vaccine high-risk HPV types was also observed. The vaccine is not effective against current infection with a vaccine HPV type. Girls or women with current infection with one or more of the vaccine HPV types gained protection from infection or disease caused by the remaining vaccine HPV types and they were also protected against reinfection with the same HPV type after clearance of an infection caused by a vaccine HPV type. High seroconversion rates and high levels of anti-HPV antibodies were observed in all vaccinated individuals of all age ranges from 9 to 45 years. No correlation was found between antibody levels and protective efficacy of the vaccine. Rechallenge with quadrivalent HPV vaccine produced a potent anamnestic humoral immune response. The vaccine is generally well tolerated and is projected to be cost effective in most pharmacoeconomic models. Therefore, quadrivalent HPV vaccine offers an effective means, in combination with screening programs, to substantially reduce the burden of HPV-related precancerous lesions and cancer, particularly cervical cancer, as well as anogenital warts.     

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.     

Results of the first World Health Organization international collaborative study of detection of human papillomavirus DNA

Twenty-nine laboratories in 12 countries participated in a study to assess the performance of various human papillomavirus (HPV) detection assays through the use of a recombinant HPV DNA standard reagent panel. The panel was designed by a group of HPV experts, and samples were prepared and distributed by the World Health Organization International Laboratory for Standards and Biologicals in The Netherlands. Each panel consisted of 24 coded samples including a dilution series for HPV types 16 and 18, alone or in combination with five other high-risk (HR) HPV types including HPV types 31, 33, 35, 45, and 52, the low-risk HPV type 6, and a negative control. Qualitative assays were generally consistent across laboratories, and most invalid results reflected a lack of HPV test sensitivity. The combined data sets had a proficiency for HPV 16 of 62.5% (15/24) and for HPV 18 of 73.9% (17/23). HPV 31 was the least accurately detected by participating laboratories. Approximately half of participating laboratories failed to detect high concentrations of HPV 31 and, to a lesser extent, to detect HPV types 35, 52, and 6. The panel sample materials offer a source of renewable and reproducible material that could be used in the future development of international standard reagents for calibration of HPV DNA assays and kits.     

Human papillomavirus (HPV) DNA in penile carcinomas in Argentina: analysis of primary tumors and lymph nodes

Among sexually transmitted diseases, infection by human papillomavirus (HPV) has become one of the most important. On the other hand, though epidemiological data show that some HPV types are closely associated with cervical cancer, few reports have been found with reference to penile carcinoma because of its rare occurrence. The aim of this study was to investigate the relationship between HPV infection and penile cancer in Argentina. A retrospective study was carried out on 38 white men with penile squamous-cell carcinoma. Sixty-five archival fixed biopsies taken from 34 primary penile tumors, 25 nodal metastases, 1 skin "satellite" metastasis and 5 histologically normal lymph nodes were used as specimens. HPV detection and typing were carried out by the polymerase chain reaction (PCR) using generic primers, combined with single-stranded conformational polymorphism (SSCP) analysis. HPV DNA was found in 71% patients, corresponding 81% of them to "high risk" types, with predominance of HPV 18. Both primary tumors and metastases showed concordance of HPV occurrence and type in both lesions. In 3 patients, HPV 16 was detected not only in primary tumors and metastases, but also in histologically normal lymph nodes. Our data indicate that most penile carcinomas in Argentine patients are etiologically related to HPV, especially to "high risk" genital types. The agreement in HPV detection between primary tumors and metastases suggests a potential viral role in tumor progression. HPV detection in otherwise histologically normal lymph nodes might be useful as early marker of a metastatic process.     

Human papillomaviruses’ proteins with clinical utility

ABSTRACT

Cervical cancer, the fourth leading cause of cancer-associated deaths among women worldwide, is associated with human papilloma virus (HPV) infection. Despite the prophylactic HPV vaccination and the implementation of cervical and HPV-based screening programs, a significant increase in cervical cancer incidence is estimated by the year 2020. Thus, further development of diagnostic tools that allow detection and risk assesment in genital HPV infection is necessary. A special interest is focused on the HPV viral proteins whose expression might be of use either as primary screening tool or in conjunction with other markers (cellular proteins, HPV DNA, PAP test).     

Detection of human papillomaviruses and other agents causing sexually transmitted diseases with molecular diagnosis methods

Causative agents of sexually transmitted diseases (STD) are different types of bacteria, viruses, fungi and protozoa. The last two decades of the twentieth century were marked with a sudden rise in the number of cases of STDs. Human immunodeficiency virus (HIV), which emerged in the 1980s, is the most prominent STD agent because of its fast spread and severity of the disease it causes, acquired immunodeficiency syndrome (AIDS). Beside HIV, human papillomaviruses (HPVs), herpes simplex viruses (HSVs) and Chlamydia trachomatis are nowadays among most health-threatening STD pathogens. In order to stop the spread of infection, apart from education about precautions, early detection of the disease is essential. Although most STD pathogens can be detected by classical methods of cultivation, biochemical and/or serologic methods, molecular diagnosis of infectious diseases has largely simplified and accelerated their detection. For instance, HPVs that cause benign and malignant tumors of genital skin and mucosa cannot be routinely detected on cell culture, whereas serologic analysis is not sensitive and informative enough. Moreover, cytologic (Pap smear) and histologic analyses can indicate changes associated with HPV infection, but neither of these methods can prove the presence of HPV. That is why the molecular methods are essential to demonstrate the presence of the infection and, even more important, to determine the type of the virus, which is associated either with low-grade or high-grade genital lesions. There are numerous methods based on hybridization with DNA or RNA probes, some of them are suitable for detecting wide range of types and screening of large collection of samples. However, the most sensitive and informative methods are based on polymerase chain reaction (PCR), and they have the advantage of being able to determine the type of the virus and distinguishing between multiple infections. Herein, we present when and why molecular analysis is useful and necessary for the detection of STD agents.     

Mucosal vaccination with a recombinant Salmonella typhimurium expressing human papillomavirus type 16 (HPV16) L1 virus-like particles (VLPs) or HPV16 VLPs purified from insect cells inhibits the growth of HPV16-expressing tumor cells in mice

Human papillomaviruses, mainly type 16 (HPV16), are responsible for cervical intraepithelial neoplasia, which can lead, in association with other factors, to cervical cancer. Both Salmonella recombinant vaccine strains assembling HPV16 virus-like particles (VLPs) and HPV16 VLPs purified from insect cells are able to induce HPV16 neutralizing antibodies in genital secretions of mice after nasal immunization. Anti-HPV16-specific antibodies in cervical secretions of women may prevent genital infection with HPV16, although this cannot be critically evaluated in the absence of an experimental model for genital papillomavirus infection. Induction of HPV16-specific cell-mediated immunity in the genital mucosa could improve the efficacy of a vaccine and a mucosal route of immunization might be necessary to do so. It has been shown that systemic immunization of mice with purified HPV16 VLPs confers protection against an HPV16-expressing tumor cell challenge through the induction of cytotoxic T-lymphocytes. Using the same C3 tumor model, we show that intranasal immunization of mice with purified HPV16 VLPs in a prophylactic setting also induces anti-tumor immunity. More interestingly, mucosal vaccination of mice with a Salmonella recombinant strain stably expressing HPV16 L1 VLPs also induces anti-tumor immunity in prophylactic as well as in therapeutic settings. Our data suggest that attenuated Salmonella strains expressing chimeric VLPs containing nonstructural viral proteins might be a promising candidate vaccine against cervical cancer by inducing both neutralizing antibodies and cell-mediated immunity.     

Type-specific and cross-reactive epitopes in human papillomavirus type 16 capsid proteins.

Genital human papillomavirus (HPV) 16 infection is frequently associated with cancer of the uterine cervix, as well as with precancerous lesions. In order to generate serologic reagents which might be useful in the diagnosis of HPV 16 infection, rabbit polyclonal and mouse monoclonal antisera were raised to carboxy terminal peptides from the HPV 16 L1 and L2 open reading frames (ORFs). Anti-L1 and -L2 peptide sera recognized HPV 16 L1 and L2 fusion proteins in Western blots and by immunoprecipitation. In Western blot analysis of L1 proteins from different HPV types, antisera to the L1 peptide reacted only with HPV 16, thus identifying an HPV 16 type-specific linear epitope. Anti-L2 peptide sera reacted with L2 fusion proteins from HPVs 6 and 16, but not from BPV, thus identifying a partially cross-reactive epitope in the HPV 16 L2. Computer analysis of carboxy terminal amino acid sequences of the L1 and L2 ORFs of multiple HPV types supported the Western blot findings. Despite the HPV 16 type specificity found in Western blots, anti-L1 peptide sera identified nuclear antigen by immunocytochemistry in cervical biopsies infected with HPV 16, as well as other genital HPV types. Anti-L2 peptide sera failed to recognize antigen in infected tissue.     

Evaluation of a novel microplate colorimetric hybridization genotyping assay for human papillomavirus

Persistent infection with high-risk human papillomavirus (HR-HPV) has been associated with cervical cancer. Developing assays for the identification of these viral types is of great importance for monitoring patients and controlling strategies. The development of the MCHA (microplate colorimetric hybridization assay), a PCR-based method for identifying six of the most common HR-HPV types (HPV 16, 18, 31, 33, 39 and 45) is described. The MCHA combines the amplification with the GP5+/GP6+ consensus primers followed by PCR reverse hybridization with specific probes and detection through a colorimetric assay. The performance of the MCHA was evaluated using 108 DNA samples typed previously by the PapilloCheck^®. The agreement between both methods was 69.4% for HPV 16; 79.1% for HPV 45; 82.4% for HPV 18; 93.6% for HPV 31; 87.9% for HPV 33, and 17.6% for HPV 39. The assay had higher sensitivity than the Papillocheck^®, particularly for identifying HPV 16 and 18. The MCHA seemed to be sensitive and specific for the identification of the most prevalent HPV types in invasive cervical cancer, HPV 16, 18, 45, 33 and 31. It requires low-cost reagents and common laboratory apparatus.