Vaccination to prevent and treat cervical cancer

Human papillomaviruses (HPVs) are the primary etiologic agents of cervical cancer. Thus, cervical cancer and other HPV-associated malignancies might be prevented or treated by HPV vaccines. Transmission of papillomavirus may be prevented by the generation of antibodies to capsid proteins L1 and L2 that neutralize viral infection. However, because the capsid proteins are not expressed at detectable levels by infected basal keratinocytes or in HPV-transformed cells, therapeutic vaccines generally target nonstructural early viral antigens. Two HPV oncogenic proteins, E6 and E7, are critical to the induction and maintenance of cellular transformation and are coexpressed in the majority of HPV-containing carcinomas. Thus, therapeutic vaccines targeting E6 and E7 may provide the best option for controlling HPV-associated malignancies. Various candidate therapeutic HPV vaccines are currently being tested whereby E6 and/or E7 are administered in live vectors, as peptides or protein, in nucleic acid form, as components of chimeric virus-like particles, or in cell-based vaccines. Encouraging results from experimental vaccination systems in animal models have led to several prophylactic and therapeutic vaccine clinical trials. If these preventive and therapeutic HPV vaccines prove successful in patients, as they have in animal models, then oncogenic HPV infection and its associated malignancies may be controllable by vaccination.     

International standard reagents for HPV detection

Human papillomavirus is the commonest genital viral infection in healthy sexually active subjects, and the presence of chronic or persistent HPV types in genital cells may constitute a prognostic marker of underlying, or predict future HPV-associated diseases. A variety of novel tests for detecting the presence of oncogenic HPV types in biological specimens have been reported. These are based on the various stages of infection and viral life cycle. HPV infects squamous epithelium with expression of various gene products intimately linked to epithelial cell differentiation. Hence, there are basically three classes of detectable markers directly derived from HPVs: molecular markers based on detection of nucleic acid sequences, serological markers based on detection of antibodies against viral proteins, and cellular markers based on detection of proteins expressed intracellularly, upon either infection or carcinogenesis. The nature of various assays and the development of international standard reagents for qualitative and quantitative assessment of assay performance are outlined. There is an increasing demand to develop standard tools to assess the quality of HPV detection systems, for regulatory and clinical management purposes. International standard reagents for HPV will help defining the analytical sensitivity and specificity of various detection methods, and will allow assuring that laboratory services used to evaluate disease burden, HPV vaccines, and cancer prevention strategies are accurate and comparable worldwide. The advancement of prophylactic vaccine candidates against HPV infections and related diseases stresses the increasing importance of HPV assays in monitoring the impact of HPV vaccination on disease burden.     

Inhibition of gamma secretase blocks HPV infection

Human papillomaviruses (HPV) are common sexually transmitted pathogens that predispose women to cervical and other anogenital cancers. HPV vaccines can prevent infection by some but not other sexually transmitted HPVs but are too costly for use in much of the world at greatest risk to HPV-associated cancers. Microbicides provide an inexpensive alternative to vaccines. In a high throughput screen, drugs that inhibit the cellular protein complex known as gamma secretase were identified as potential HPV microbicides. gamma Secretase inhibitors (GSIs) inhibited the infectivity of HPV pseudoviruses both in human keratinocytes and in mouse cells, with IC₅₀ values in the picomolar to the nanomolar range. Using a mouse model, we observed that a GSI could inhibit HPV infection to the same degree as its effectiveness in inhibiting gamma secretase activity in vivo. We conclude that gamma secretase activity is required for HPV infection and that GSIs are effective microbicides against anogenital HPVs.     

Preventative and therapeutic vaccines for cervical cancer

"High-risk" genotypes of the human papillomavirus (HPV), most commonly HPV genotype 16, are the primary etiologic agents of cervical cancer. Indeed HPV DNA is detected in 99% of cervical carcinomas. Thus, cervical cancer and other HPV-associated malignancies might be prevented or treated by the induction of the appropriate viral-antigen-specific immune responses. Transmission of papillomavirus may be prevented by the generation of antibodies to capsid proteins L1 and L2 that neutralize viral infection. HPV L1 virus-like particles (VLPs) show great promise as prophylactic HPV vaccines in ongoing clinical trials but L2-based preventative vaccines have yet to be tested in patients. Since the capsid proteins are not expressed at detectable levels by infected basal keratinocytes or in HPV-transformed cells, therapeutic vaccines generally target the nonstructural early viral antigens. Two HPV oncogenic proteins, E6 and E7, are critical to the induction and maintenance of cellular transformation and are co-expressed in the majority of HPV-containing carcinomas. Although other early viral antigens show promise for vaccination against papillomas, therapeutic vaccines targeting E6 and E7 may provide the best opportunity to control HPV-associated malignancies. Various candidate therapeutic HPV vaccines are currently being tested whereby E6 and/or E7 are administered in live vectors, as peptides or proteins, in nucleic acid form, as components of chimeric VLPs, or in cell-based vaccines. Encouraging results from experimental vaccination systems in animal models have led to several prophylactic and therapeutic vaccine clinical trials. Should this new generation of HPV preventative and therapeutic vaccines function in patients as demonstrated in animal models, oncogenic HPV infection and its associated malignancies could be controlled by vaccination. Importantly, recent advances in HPV detection and continued improvements in screening further enhance our opportunities to systematically eradicate HPV-associated malignancy.     

Human papillomavirus vaccines for the treatment of cervical cancer

Human papillomavirus (HPV) infection is a major cause of cervical cancer, the second most common cancer in women worldwide. Currently, a HPV L1-based virus-like particle has been approved as a prophylactic vaccine against HPV infection, which will probably lead to a reduction in cervical cancer incidence within a few decades. Therapeutic vaccines, however, are expected to have an impact on cervical cancer or its precursor lesions, by taking advantage of the fact that the regulatory proteins (E6 and E7) of HPV are expressed constantly in HPV-associated cervical cancer cells. Vaccine types targeting these regulatory proteins include the recombinant protein and DNA vaccines, peptide vaccines, dendritic-cell vaccines, and viral and bacterial vector deliveries of vaccines, and these may provide an opportunity to control cervical cancer. Further approaches incorporating these vaccine types with either conventional therapy modalities or the modulation of CD4(+) regulatory T cells appear to be more promising in achieving increased therapeutic efficacy. In this review, we summarize current and future therapeutic vaccine strategies against HPV-associated malignancies at the animal and clinical levels.     

Biology of the papillomavirus infections: V. Vaccine developments]

Several genotypes of human papillomaviruses (HPV) are recognised as aetiologic factors for cervical cancer, and viral DNA account for more 99% of cases. Thus, prevention of HPV infection by, for example, types 16 and 18, should reduce the world-wide incidence of cervical cancer. Many strategies are being developed for the control of HPV-associated lesions of the uterine cervix: prophylactic vaccines which elicit neutralizing antibodies to prevent HPV infection, and therapeutic vaccines which induce a T-cytotoxic response to early viral oncoproteins. Experimental trials are being conducted to test mucosal immunization with an ideal antigen delivery system. Vaccination strategies elicit a protective antibody response in animal species, but in humans, strategies which are likely to be effective in the control of HPV-associated preneoplastic and neoplastic lesions of the uterine cervix are still under investigation.     

Three novel papillomaviruses (HPV109, HPV112 and HPV114) and their presence in cutaneous and mucosal samples

To expand our knowledge of the genomic diversity of human papillomaviruses (HPVs), we searched for new HPVs in squamous cell carcinomas of the skin (SCC) and seemingly HPV-negative, otherwise typically HPV-associated lesions. We describe the characterization of three novel HPV types. HPV109 was isolated from an SCC, HPV112 from a condyloma and HPV114 from a low-grade cervical lesion. Pairwise alignment of the L1 sequences classified HPV114 to genus alpha species 3, whereas HPV112 defined a new species in the genus gamma. HPV109 had uncertain classification because of a low and about equal similarity in the L1 gene (between 60% and 65%) to different genera. Type-specific real-time PCRs of cervical samples, a majority from women with low grade atypical cytology, (n = 2856) and various cutaneous samples (n = 538), found HPV114 in 1.7% (48/2856) of the genital samples, whereas both HPV109 and 112 were rare viruses found at high viral loads only in their index samples.     

Human papillomavirus vaccines

A wealth of epidemiological and molecular evidence has led to the conclusion that virtually all cases of cervical cancer and its precursor intra-epithelial lesions are a result of infection with one or other of a subset of genital human papillomaviruses (HPVs) suggesting that prevention of infection by prophylactic vaccination would be an effective anti-cancer strategy. The papillomaviruses cannot be grown in large amounts in culture in vitro, but the ability to generate HPV virus like particles (VLPs) by the synthesis and self-assembly in vitro of the major virus capsid protein L1 provides for a potentially effective sub unit vaccine. HPV L1 VLP vaccines are immunogenic and have a good safety profile. Published data from proof of principle trials and preliminary reports from large Phase III efficacy trials suggest strongly that they will protect against persistent HPV infection and cervical intra epithelial neoplasia. However, the duration of protection provided by these vaccines is not known, the antibody responses induced are probably HPV type specific and immunisation should occur pre-exposure to the virus. Second generation vaccines could include an early antigen for protection post-exposure and alternative delivery systems may be needed for the developing world.     

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).     

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.     

Distribution of human papillomavirus genotypes in Paraguayan women according to the severity of the cervical lesion

The incidence of cervical cancer in Paraguay is among the highest in the world. This study aimed to determine the distribution of human papillomavirus (HPV) genotypes in Paraguayan women, according to the severity of the cervical lesion. This cross-sectional study included 207 women without a squamous intraepithelial lesion, 164 with low-grade squamous intraepithelial lesions, 74 with high-grade squamous intraepithelial lesions, and 41 with cervical cancer. Type-specific HPV was determined by the polymerase chain reaction with MY9/11 L1 and GP5+/GP6+ L1 primers, followed by restriction fragment length polymorphism and reverse line blotting hybridization, respectively. In total, 12 high-risk and 24 low-risk HPVs types were detected. HPV 16 was the most prevalent, followed by HPV 18 in cervical cancer (14.6%), HPV 31 in high-grade squamous intraepithelial lesions (14.9%), HPVs 58/42 in low-grade squamous intraepithelial lesions (9.1% each), and HPVs 31/58 (2.4% each) in women without squamous intraepithelial lesions. Among 285 positive samples, 24.2% harbored multiple HPV types, being this more prevalent in women with squamous intraepithelial lesions (30.8% in low-grade squamous intraepithelial lesions, 22.5% in high-grade squamous intraepithelial lesions, and 22.0% in cervical cancer) than in women without lesions (9.3%). The higher prevalence of HPV 16 and other high-risk HPVs in women both with and without cervical lesions may explain the high incidence of cervical cancer in Paraguay. This information may be of importance for local decision makers to improve prevention strategies. In addition, these results may be useful as baseline pre-vaccination data for a future virological surveillance in Paraguay.     

HPV-assoziierte Plattenepithelkarzinogenese

About 7-8% of all human cancers are thought to be related to infections with high-risk (HR) human papilloma virus (HPV). Besides cervical cancer, especially squamous cell carcinomas of the anogenital and oropharyngeal regions are associated with HR-HPV. Transmission of HPV is due to sexual activity. Harald zur Hausen was awarded in 2008 with the Nobel price in medicine for the establishment of a causal link between certain HPV infections and cervical cancer. Meanwhile potent prophylactic vaccines are available to prevent infections with HPV-16 and HPV-18, the two most frequently observed HR HPV types worldwide. On molecular grounds a persistent HPV infection is the central risk factor for the development of HPV-associated neoplasias. Continued expression of the viral E6 and E7 oncogenes disrupts cell cycle control mechanisms in infected cells, thereby gaining limitless proliferative capacity and resistance against apoptotic signals. However acquisition of mutations and genomic instability might cause malignant transformation in these cells.     

Prevention of cancer by prophylactic human papillomavirus vaccines

Oncogenic human papillomaviruses (HPVs) are exclusively mucosal pathogens that are noncytopathic and the basal epithelial cells harboring and maintaining an infection do not produce either capsid antigen or virus. The efficacy of the licensed L1 virus-like particle (VLP) vaccines has encouraged development of several second generation vaccines aimed at expanding the coverage to all oncogenic HPV types and reducing barriers to global implementation. Currently there is no defined immune correlate of protection that can be used to determine if an individual patient is protected and for the evaluation of these second generation vaccines. Surprisingly, passive transfer of neutralizing serum antibody is protective in animal models. Recent studies suggest how neutralizing antibody mediates immunity against mucosal HPV and the possible impact of memory B cells.     

Molecular mechanisms of cervical carcinogenesis by high‐risk human papillomaviruses: novel functions of E6 and E7 oncoproteins

Over the last two decades, since the initial discovery of human papillomavirus (HPV) type 16 and 18 DNAs in cervical cancers by Dr. Harald zur Hausen (winner of the Nobel Prize in Physiology or Medicine, 2008), the HPVs have been well characterised as causative agents for cervical cancer. Viral DNA from a specific group of HPVs can be detected in at least 90% of all cervical cancers and two viral genes, E6 and E7, are invariably expressed in HPV‐positive cervical cancer cells. Their gene products are known to inactivate the major tumour suppressors, p53 and retinoblastoma protein (pRB), respectively. In addition, one function of E6 is to activate telomerase, and E6 and E7 cooperate to effectively immortalise human primary epithelial cells. Though expression of E6 and E7 is itself not sufficient for cancer development, it seems to be either directly or indirectly involved in every stage of multi‐step carcinogenesis. Epidemiological and biological studies suggest the potential efficacy of prophylactic vaccines to prevent genital HPV infection as an anti‐cancer strategy. However, given the widespread nature of HPV infection and unresolved issues about the duration and type specificity of the currently available HPV vaccines, it is crucial that molecular details of the natural history of HPV infection as well as the biological activities of the viral oncoproteins be elucidated in order to provide the basis for development of new therapeutic strategies against HPV‐associated malignancies. This review highlights novel functions of E6 and E7 as well as the molecular mechanisms of HPV‐induced carcinogenesis. Copyright © 2009 John Wiley & Sons, Ltd.     

Prophylactic human papillomavirus vaccines: potential for sea change

Persistent human papillomavirus (HPV) infection is the central cause in the development of anogenital warts, precancers and cancers of uterine cervix, and a major factor in the genesis of other malignancies of the lower anogenital and upper aerodigestive tracts. The burden of disease carries very high medical, financial and psychosocial costs. The role of prophylactic HPV vaccines in reducing the burden of disease is discussed in light of the results of multiple randomized, controlled trials conducted worldwide in thousands of young females. The review discusses some of the issues that are still unknown, with respect to long-term vaccine performance, challenges to be overcome to achieve universal, mass prophylactic HPV vaccination, as well as the potential impact of the vaccines on primary screening for, and management of, HPV-related anogenital infection and disease.     

Loss of cytokeratin 14 expression is related to human papillomavirus type and lesion grade in squamous intraepithelial lesions of the cervix

In a recent study of low-grade cervical squamous intraepithelial lesions (SILs), we reported that infection with both low- and high-risk human papillomaviruses (HPVs) upregulated cyclin A, B, E, and Ki67 expression in basal and suprabasal cells. In view of the intricate link between cell cycle exit, proliferation, and differentiation, we examined the morphologic distribution of cytokeratins 13 and 14 and involucrin expression in 49 low-grade SILs infected with HPV types 6, 11, 16, 18, 31, 33, 39, 42, 43, 44, 45, 51, 52, 56, 58, and 66; 2 lesions contained both low- and high-risk HPVs. The findings were compared with 30 high-grade SILs infected with HPV types 16, 31, 33, 51, 58, 66, and 67; 3 of these were infected with 2 different HPVs. In low-grade lesions, the differentiation markers were expressed normally, showing that differentiation proceeds despite upregulation of cell cycle--associated proteins. Loss of involucrin (3 of 33) and cytokeratin 13 (8 of 33) expression occurred only in the high-grade lesions and was therefore related to lesion grade. Loss of cytokeratin 14 expression was also significantly more frequent in high-grade than in low-grade lesions (19 of 33 v 12 of 51; P < .01). In addition, cytokeratin 14 expression was significantly less frequent in the intermediate and superficial layers of low-grade SILs infected with high-risk HPVs than in those infected with low-risk HPVs (3 of 27 v 14 of 24; P < .001). These findings are consistent with in vitro data and suggest that abnormalities of both cell cycle control and squamous differentiation are important in HPV-associated neoplastic transformation.     

Therapeutic HPV DNA vaccines

It is now well established that most cervical cancers are causally associated with HPV infection. This realization has led to efforts to control HPV-associated malignancy through prevention or treatment of HPV infection. Currently, commercially available HPV vaccines are not designed to control established HPV infection and associated premalignant and malignant lesions. To treat and eradicate pre-existing HPV infections and associated lesions which remain prevalent in the U.S. and worldwide, effective therapeutic HPV vaccines are needed. DNA vaccination has emerged as a particularly promising form of therapeutic HPV vaccines due to its safety, stability and ability to induce antigen-specific immunity. This review focuses on improving the potency of therapeutic HPV vaccines through modification of dendritic cells (DCs) by [1] increasing the number of antigen-expressing/antigen-loaded DCs, [2] improving HPV antigen expression, processing and presentation in DCs, and [3] enhancing DC and T cell interaction. Continued improvement in therapeutic HPV DNA vaccines may ultimately lead to an effective DNA vaccine for the treatment of HPV-associated malignancies.     

Detection of antibodies to L1, L2, and E4 proteins of human papillomavirus types 6, 11, and 16 by ELISA using synthetic peptides

Antibodies against eight synthetic peptides spanning different epitopes located on L1, L2, and E4 proteins of human papillomavirus (HPV) types 16, 6, and 11 were examined in sera from 73 women infected by HPV and from 139 healthy controls. Only three of these peptides were reactive. Two located on proteins L2 and E4 of HPV 16 seem type specific since antibodies to these peptides were detected, respectively, in 21% and 15% of the HPV 16 infected patients and in 2.5% and none of women infected by other HPVs. The third peptide located on the L1 protein of HPV 6 bears a common epitope since antibodies to this peptide were detected not only in 85% of women infected by HPV 6 or 11, but also in 82% of women infected by other HPVs, and in 74% and 71% of the control groups (10–12-year-old children and adults, respectively). In conclusion, none of the peptides investigated seems useful to develop ELISAs for serological diagnosis of HPV infection. © 1995 Wiley-Liss, Inc.     

Immunotherapy for Cervical Cancer

The high-risk types of human papillomavirus (HPV) have been found to be associated with most cervical cancers and play an essential role in the pathogenesis of the disease. Despite recent advances in preventive HPV vaccine development, such preventive vaccines are unlikely to reduce the prevalence of HPV infections within the next few years, due to their cost and limited availability in developing countries. Furthermore, preventive HPV vaccines may not be capable of treating established HPV infections and HPV-associated lesions, which account for high morbidity and mortality worldwide. Thus, it is important to develop therapeutic HPV vaccines for the control of existing HPV infection and associated malignancies. Therapeutic vaccines are quite different from preventive vaccines in that they require the generation of cell-mediated immunity, particularly T cell-mediated immunity, instead of the generation of neutralizing antibodies. The HPV-encoded early proteins, the E6 and E7 oncoproteins, form ideal targets for therapeutic HPV vaccines, since they are consistently expressed in HPV-associated cervical cancer and its precursor lesions and thus play crucial roles in the generation and maintenance of HPV-associated disease. Our review covers the various therapeutic HPV vaccines for cervical cancer, including live vector-based, peptide or protein-based, nucleic acid-based, and cell-based vaccines targeting the HPV E6 and/or E7 antigens. Furthermore, we review the studies using therapeutic HPV vaccines in combination with other therapeutic modalities and review the latest clinical trials on therapeutic HPV vaccines.