Expression of the bovine papillomavirus type 1 E5B gene reveals a protein-protein interaction of the E5A and E5B gene products.

The bovine papillomavirus type 1 (BPV-1) genome has been shown to contain a small open-reading frame designated E5B (nucleotides 4013-4167) which is predicted to encode a hydrophobic, 52 amino acid protein. In order to detect and characterize the E5B protein, an 18 nucleotide sequence encoding a 6 amino acid epitope was added to the 3' end of the E5B open-reading frame which was then expressed in COS-1 cells using a SV40 vector. Immunoprecipitation, immunofluorescence, and cell fractionation studies identified the E5B protein as a 4-kDa protein and localized it primarily to membranes of the endoplasmic reticulum and nucleus. Unlike the E5A protein of BPV-1, E5B did not form dimers (despite containing a cysteine residue) or form complexes with growth factor receptors such as the PDGF receptor or erb B-2 receptor. Interestingly, the E5B protein formed physical complexes with the hydrophobic E5A oncoprotein, apparently via transmembrane interactions. Additionally, expression of E5B inhibited the transforming capability of BPV-1 E5A. These observations suggest that the expression of this viral protein may play a significant role in BPV/host cell interactions.

The pathogenesis of advanced cervical cancer provides the basis for an empirical therapeutic vaccine.

The pathogenesis of carcinogenic human papillomavirus (HPV) infections of the cervix includes early induction of peripheral tolerance of tissue-infiltrating lymphocytes and an imbalanced Th2 response to HPV early virus proteins. As lesions become progressively dysplastic, major histocompatibility complex (MHC)-1 molecules are down-regulated on the surface of abnormal keratinocytes. When the target of MHC-1 class-restricted cytotoxic lymphocytes disappears, immune deviation to a Th2 response becomes more dominant. After severely dysplastic lesions become invasive, cervical cancer cells die and release HPV E6 and E7 oncoproteins that react with anti-E6 and anti-E7 antibodies to form insoluble immune complexes in antibody excess under the continuing influence of immune deviation. On the basis of this knowledge of the pathogenesis of advanced cervical cancer, we believe that successful immunotherapeutic treatments of these patients will use a vaccine formulation that will break peripheral tolerance in association with biological response modifiers that will enable the patient's immune system to switch classes from Th2 to Th1 while up-regulating MHC-1 molecules on cancer cells. Like prophylactic vaccines against HPV, successful therapeutic vaccine against cervical cancer may have to be universal rather than individualized to be efficacious.

Human papillomavirus and skin cancer.

Human papillomavirus (HPV) appears to be the most ubiquitous of the human viruses. Over 100 HPV types have been identified. A minority of HPV cause cutaneous warts and mucosal condylomata. The HPV that cause mucosal condylomata put the patient at various degrees of risk for developing cancers, particularly cervical cancer. The majority of HPV infect the skin of normal and immunocompromised individuals. In normal people, most of these HPV appear to establish a latent infection of the skin, most likely as normal flora residing in hair follicles; however, in patients with various systemic and localized depressions of cell-mediated immunity, some HPV infections appear to be involved in the development of nonmelanotic skin cancer and its precursor lesions in skin, usually in sunlight-exposed areas. Circumstantial evidence suggests that these HPV may have a role in promoting proliferative lesions of the skin, although their sites of active infection and mode of transmission to susceptible individuals remain unknown.

Spontaneously regressing oral papillomas induce systemic antibodies that neutralize canine oral papillomavirus.

Canine oral papillomavirus (COPV) infection of naive beagle dogs causes oral papillomas, most of which spontaneously regress. Regressor beagles do not develop new oral papillomas because of COPV type-specific, cell-mediated immunity, COPV neutralizing antibodies, or both. Formalin-fixed native and recombinant COPV vaccines that target the systemic immune system induce neutralizing antibodies that prevent development of oral papillomas. This study was designed to determine whether spontaneously regressing mucosal papillomas also targeted the systemic immune system to induce circulating, neutralizing IgG antibodies that protect against infection by COPV. To accomplish this goal, IgG was fractionated from sera collected from weanling beagles and regressor beagles and tested for conferring protection by passive immunization. Serum was tested by ELISA for antibodies against intact virions and then pooled for passive transfer to naive beagles. Preimmune sera were neither reactive by ELISA nor protective by passive transfer. On the other hand, IgG antibodies from regressor beagles were reactive by ELISA and passive transfer conferred protection against COPV challenge. Circulating IgG antibodies induced by spontaneous regression of canine oral papillomas protect beagles against intraoral infection by COPV, a model for mucosotropic HPV.

Feline papillomas and papillomaviruses.

Papillomaviruses (PVs) are highly species- and site-specific pathogens of stratified squamous epithelium. Although PV infections in the various Felidae are rarely reported, we identified productive infections in six cat species. PV-induced proliferative skin or mucous membrane lesions were confirmed by immunohistochemical screening for papillomavirus-specific capsid antigens. Seven monoclonal antibodies, each of which reacts with an immunodominant antigenic determinant of the bovine papillomavirus L1 gene product, revealed that feline PV capsid epitopes were conserved to various degrees. This battery of monoclonal antibodies established differential expression patterns among cutaneous and oral PVs of snow leopards and domestic cats, suggesting that they represent distinct viruses. Clinically, the lesions in all species and anatomic sites were locally extensive and frequently multiple. Histologically, the areas of epidermal hyperplasia were flat with a similarity to benign tumors induced by cutaneotropic, carcinogenic PVs in immunosuppressed human patients. Limited restriction endonuclease analyses of viral genomic DNA confirmed the variability among three viral genomes recovered from available frozen tissue. Because most previous PV isolates have been species specific, these studies suggest that at least eight different cat papillomaviruses infect the oral cavity (tentative designations: Asian lion, Panthera leo, P1PV; snow leopard, Panthera uncia, PuPV-1; bobcat, Felis rufus, FrPV; Florida panther, Felis concolor, FcPV; clouded leopard, Neofelis nebulosa, NnPV; and domestic cat, Felis domesticus, FdPV-2) or skin (domestic cat, F. domesticus, FdPV-1; and snow leopard, P. uncia, PuPV-2).

Characterization of a major neutralizing epitope on human papillomavirus type 16 L1.

Persistent infection with human papillomavirus type 16 (HPV-16) is strongly associated with the development of cervical cancer. Neutralizing epitopes present on the major coat protein, L1, have not been well characterized, although three neutralizing monoclonal antibodies (MAbs) had been identified by using HPV-16 pseudovirions (R. B. Roden et al., J. Virol. 71:6247-6252, 1997). Here, two of these MAbs (H16.V5 and H16.E70) were demonstrated to neutralize authentic HPV-16 in vitro, while the third (H16.U4) did not. Binding studies were conducted with the three MAbs and virus-like particles (VLPs) composed of the reference L1 sequence (114K) and three variant L1 sequences: Rochester-1k (derived from viral stock DNA), GU-1 (derived from cervical biopsy DNA), and GU-2 (derived from biopsy DNA, but containing some sequence changes likely to be artifactual). While all three MAbs bound to 114K and Rochester-1k VLPs, GU-1 VLPs were not recognized by H16.E70, and both H16.E70 and H16.V5 failed to bind to GU-2 VLPs. Site-directed mutagenesis was used to replace disparate amino acids in the GU-2 L1 with those found in the 114K L1. Alteration of the amino acid at position 50, from L to F, completely restored H16.V5 binding and partially restored H16.E70 binding, while complete restoration of H16.E70 binding occurred with GU-2 VLPs containing both L50F and T266A alterations. Immunization of mice with L1 variant VLPs revealed that GU-2 VLPs were poorly immunogenic. The L50F mutant of GU-2 L1, in which the H16.V5 epitope was restored, elicited HPV-16 antibody responses comparable to those obtained with 114K VLPs. These results demonstrate the importance of the H16.V5 epitope in the generation of potent HPV-16 neutralizing antibody responses.

Prevalence of antibodies against virus-like particles of Epidermodysplasia verruciformis-associated HPV8 in patients at risk of skin cancer.

There is increasing evidence for widespread occurrences of infection with Epidermodysplasia verruciformis-related human papillomaviruses, both in the general population and in immunosuppressed patients. In order to test for the prevalence of antibodies directed against the native L1 epitopes exposed on the surface of the virions, we have established an IgG-specific enzyme-linked immunosorbent assay with L1 virus-like particles of the Epidermodysplasia verruciformis-specific human papillomavirus 8 as antigen to screen 567 representative serum samples from the general population and immunosuppressed/dermatologic patients. Among healthy European donors (n = 210), 7.6% were found to be seropositive. In a group of renal transplant recipients (n = 185) the antibody prevalence was elevated to 21.1%, irrespective of the presence or absence of skin cancer. High positivity rates could be detected among (i) immunocompetent patients with nonmelanoma skin tumors (45.6%, n = 79) and (ii) Psoralene/UVA treated psoriasis patients (42.9%, n = 42). In contrast, anti-human papillomavirus 8-virus-like particle antibodies were found in only 6.8% of Hodgkin lymphoma patients (n = 44).

Mutant canine oral papillomavirus L1 capsid proteins which form virus-like particles but lack native conformational epitopes.

Recently, the L1 capsid protein of canine oral papillomavirus (COPV) has been used as an effective systemic vaccine that prevents viral infections of the oral mucosa. The efficacy of this vaccine is critically dependent upon native L1 conformation and, when purified from Sf9 insect cells, the L1 protein not only displays type-specific, conformation-dependent epitopes but it also assembles spontaneously into virus-like particles (VLPs). To determine whether VLP formation was coupled to the expression of conformation-dependent epitopes, we generated a series of N- and C-terminal L1 deletion mutants and evaluated their ability to form VLPs (by electron microscopy) and to react with conformation-dependent antibodies (by immunofluorescence microscopy). We found that (a) deletion of the 26 C-terminal residues generated a mutant protein which formed VLPs efficiently and folded correctly both in the cytoplasm and in the nucleus; (b) further truncation of the L1 C terminus (67 amino acids) resulted in a capsid protein which formed VLPs but which failed to express conformational epitopes; (c) deletion of the first 25 N-terminal amino acids also abolished expression of conformational epitopes (without altering VLP formation) but the native conformation of this deletion mutant could be restored by the addition of the human papillomavirus type 11 N terminus. These results demonstrate that VLP formation and conformational epitope expression can be dissociated and that the L1 N terminus has a critical role in protein folding. In addition, it appears that correct L1 protein folding is not dependent upon the nucleoplasmic environment.

Prospects for human papillomavirus vaccine development: emerging HPV vaccines.

This review concentrates on recent advances in human papillomavirus vaccine development. Strategies for prophylactic HPV subunit vaccines utilizing recombinantly synthesized, immunogenic virus-like particles are discussed. Therapeutic strategies focusing on the induction of cell-mediated immunity and gene manipulation for the treatment of established HPV-associated disease are also reviewed.

Prospects for human papillomavirus vaccine development: emerging HPV vaccines.

This review concentrates on recent advances in human papillomavirus vaccine development. Strategies for prophylactic HPV subunit vaccines utilizing recombinantly synthesized, immunogenic virus-like particles are discussed. Therapeutic strategies focusing on the induction of cell-mediated immunity and gene manipulation for the treatment of established HPV-associated disease are also reviewed.

Multiplicity of uses of monoclonal antibodies that define papillomavirus linear immunodominant epitopes.

During the last 10 yr, we have derived monoclonal antibodies from animals immunized with denatured bovine papillomaviruses type 1 major capsid (L1) protein, mapped their corresponding immunodominant epitopes to within a single amino acid (aa), and compared the reactivity of authentic L1 proteins to the predicted response by collinear analysis of the aa sequences of the same and other papillomaviruses (PVs). The data obtained from this approach has provided us with new insights into the sensitivity and specificity of the antibody response to viral proteins. We have included here some observations and conclusions that appear to be generic for the immune response, some of which might have applications for working with linear epitopes in other experimental systems.

Development and progression of psoriasiform dermatitis and systemic lesions in the flaky skin (fsn) mouse mutant.

Flaky skin (fsn) mutant mice were originally described as a mouse model for psoriasis accompanied by hematological abnormalities. However, homozygous (fsn/fsn) mice develop a number of other pathological changes. Systematic evaluation of over 300 fsn/fsn and normal littermate control (+/+ or +/fsn) mice was carried out to characterize these changes. Psoriasiform skin lesions were first evident as focal epidermal hyperplasia and inflammation at 2 weeks of age. These lesions became confluent and diffuse by 3-4 weeks of age and were associated with marked dermal infiltration of lymphocytes and small numbers of neutrophils and macrophages. Mast cell numbers increased significantly in the dermis from 2 weeks of age onward. Diffuse dermal neovascularization accompanied these cutaneous changes. Systemic lesions included progressive and massive papillomatosis of the stratified squamous epithelium of the forestomach, hyperplasia and dysplasia of the glandular stomach, increased apoptosis of cecal enterocytes, renal glomerulopathy associated with immune complex and complement deposition, testicular degeneration, mixed inflammatory cell infiltrates and fibrosis around portal triads in the liver, splenomegaly due to massive erythropoiesis, and granulomatous lymphadenitis. This spontaneous mouse mutation provides a useful model for modulating neovascularization and keratinocyte hyperproliferation, especially since the cutaneous changes resemble some forms of psoriasis in humans.

Tissue effects and host response. The key to the rational triage of cervical neoplasia.

Genital HPV infections are associated with a spectrum of lesions ranging from benign condylomata to invasive cancer and its precursor lesions. The transformation zone of the cervix is the most frequent target of the high-risk HPV types. Depending on the nomenclature used, cancer precursors are subdivided on the basis of their morphologic presentation into dysplasias (mild, moderate, and severe); cervical intraepithelial neoplasias (CIN I, II, and III); or low-grade and high-grade squamous intraepithelial lesions (LGSILs and HGSILs). The HGSILs (i.e., moderate and severe dysplasias, CIN II and III lesions) are recognized universally as cancer precursors. The LGSILs (i.e., very mild dysplasia and mild dysplasias, condylomata and CIN I lesions), have shown that one of the most important denominators of their cancer potential is the presence of intermediate and particularly high-risk HPV types. HPV typing provides the most rational basis for selecting women with LGSILs to be colposcoped and treated or given follow-up treatment with Pap smears. Until the clinical significance of HPV typing is known, management decisions may be based on an individual's risk factors such as age, compliance, past history of abnormal Pap smears, sexual habits, and access to adequate cytologic diagnosis.

Antigenicity of bovine papillomavirus type 1 (BPV-1) L1 virus-like particles compared with that of intact BPV-1 virions.

Virus-like-particles (VLPs) of various papillomavirus (PV) types have been produced by expressing recombinant L1 proteins in eukaryotic cells. Although VLPs have the same ultrastructural appearance as native virions and their immunogenicity appears to be similar, their antigenicity has not been carefully evaluated. For this reason, the antigenicity of intact bovine PV type 1 (BPV-1) virions was compared with that of BPV-1 recombinant L1 VLPs by ELISA using a well-characterized panel of polyclonal and monoclonal antibodies generated against intact and denatured BPV-1 particles. The structural integrity of the authentic virions and recombinant VLPs was verified by electron microscopy. The specificity of antibodies raised against intact BPV-1 virions and their reactivity with VLPs revealed that the immunodominant, type-specific, conformational epitopes of intact virions were reproduced on VLPs. However, many monoclonal antibodies that define cross-reactive, non-conformational (linear) epitopes cryptic to the authentic BPV-1 virion tested positively when reacted with intact VLPs. One monoclonal antibody, which recognizes a BPV-1 and deer PV surface conformational epitope, did not react with VLPs. Therefore, although VLPs can be used to immunize animals against infection, the external exposure of broadly cross-reactive epitopes of intact L1 VLPs suggests that the use of L1 VLPs in antigenicity studies such as serological screening should be done with caution.

Systemic immunization with papillomavirus L1 protein completely prevents the development of viral mucosal papillomas.

Infection of mucosal epithelium by papillomaviruses is responsible for the induction of genital and oral warts and plays a critical role in the development of human cervical and oropharyngeal cancer. We have employed a canine model to develop a systemic vaccine that completely protects against experimentally induced oral mucosal papillomas. The major capsid protein, L1, of canine oral papillomavirus (COPV) was expressed in Sf9 insect cells in native conformation. L1 protein, which self-assembled into virus-like particles, was purified on CsCl gradients and injected intradermally into the foot pad of beagles. Vaccinated animals developed circulating antibodies against COPV and became completely resistant to experimental challenge with COPV. Successful immunization was strictly dependent upon native L1 protein conformation and L1 type. Partial protection was achieved with as little as 0.125 ng of L1 protein, and adjuvants appeared useful for prolonging the host immune response. Serum immunoglobulins passively transferred from COPV L1-immunized beagles to naive beagles conferred protection from experimental infection with COPV. Our results indicate the feasibility of developing a human vaccine to prevent mucosal papillomas, which can progress to malignancy.

Prospects for a vaccine against human papillomavirus.

OBJECTIVE: To summarize existing data regarding the feasibility of developing strategies for prophylactic and therapeutic vaccination against human papillomavirus (HPV) infection. DATA SOURCES: We used the Medline data base and reference lists of articles to identify English-language papers that evaluate strategies for prophylactic and therapeutic vaccination against HPV infection. METHODS OF STUDY SELECTION: Our search uncovered several reports of systems that produce recombinant HPV major capsid proteins as antigens for biochemical, molecular, and immunologic studies and investigations that evaluate cell-mediated immune responses to HPV-induced, tumor-associated peptides. DATA EXTRACTION AND SYNTHESIS: Recombinant HPV major capsid proteins, which self-assemble into virus-like particles, are produced in quantity, mimic the conformation of native virions, react with neutralizing antibodies, and are type-specific. Human papillomavirus early viral peptides induce cytotoxic T lymphocyte responses that retard tumor progression and protect against tumor development after challenge in animal models. CONCLUSIONS: Recombinant papillomavirus virus-like particles are highly antigenic, protective in animal models, lack potentially carcinogenic viral DNA, and are, therefore, ideal candidates for a prophylactic vaccine against HPV infection. Immunization with HPV tumor peptides may be beneficial in tumor prevention, regression, and rejection. Vaccines against HPV infection can be important in reducing the incidence of cervical dysplasia and carcinoma worldwide, particularly in developing countries.

Human papillomaviruses: their clinical significance in the management of cervical carcinoma.

Studies have shown a strong association between certain human papillomaviruses and the development of cervical carcinoma and its precursor lesions. The oncogenic potential of papillomaviruses has been clearly demonstrated in both laboratory animals and cultured cells. Recent advances in our understanding of viral pathogenesis have provided insights into the natural history of papillomavirus infection and subsequent development of neoplasia. A more thorough understanding of the molecular mechanisms responsible for viral oncogenesis will facilitate the development of novel preventive and therapeutic strategies to prevent and treat papillomavirus-associated cervical neoplasias. Strategies under current investigation are focusing on the induction of effective humoral and cell-mediated immunity, the expression of HPV gene products, and cofactors that interact with HPV gene products to affect cell transformation. As a result of these investigative efforts, prophylactic HPV capsid vaccines and other gene therapies may soon become clinically available.