Type-specific L1 virus-like particle-mediated protection of horses from experimental bovine papillomavirus 1-induced pseudo-sarcoid formation is long-lasting.

Equine sarcoids are common therapy-resistant skin tumours induced by bovine papillomavirus type 1 or 2 (BPV1, BPV2) infection. We have previously shown that prophylactic vaccination with BPV1 L1 virus-like particles (VLPs) efficiently protects horses from experimental BPV1-induced pseudo-sarcoid development. Here, we assessed BPV1 L1 VLP vaccine-mediated long-term protection from experimental tumour formation in seven horses 5 years after immunization with three different doses of BPV1 L1 VLPs, and three unvaccinated control animals. Horses were challenged by intradermal inoculation with infectious BPV1 virions at 10 sites on the neck (106 virions per injection). In vaccinated horses, BPV1 challenge did not result in any apparent lesions irrespective of vaccine dosage and BPV1-neutralizing antibody titres that had dropped considerably over time and below the detection limit in one individual. Control horses developed pseudo-sarcoids at all inoculation sites. We conclude that immunization of horses with BPV1 L1 VLPs induces long-lasting protection against experimental BPV1 virion-induced disease.

Potential of a BPV1 L1 VLP vaccine to prevent BPV1- or BPV2-induced pseudo-sarcoid formation and safety and immunogenicity of EcPV2 L1 VLPs in horse.

We have previously shown that immunization of horses with bovine papillomavirus type 1 (BPV1) L1 virus-like particles (VLPs) is safe and highly immunogenic and that BPV1 and bovine papillomavirus type 2 (BPV2) are closely related serotypes. Here we evaluated the protective potential of a BPV1 L1 VLP vaccine against experimental BPV1 and BPV2 challenge and studied the safety and immunogenicity of a bivalent equine papillomavirus type 2 (EcPV2)/BPV1 L1 VLP vaccine. Fourteen healthy horses were immunized with BPV1 L1 VLPs (100 µg per injection) plus adjuvant on days 0 and 28, while seven remained unvaccinated. On day 42, all 21 horses were challenged intradermally at 10 sites of the neck with 107 BPV1 virions per injection. In analogy, 14 horses immunized twice with EcPV2 plus BPV1 L1 VLPs (50 µg each) and seven control animals were challenged with 107 BPV2 virions per injection. Immunization with BPV1 L1 VLPs alone induced a robust antibody response (day 42 median titre: 12 800), and BPV1-inoculated skin remained unchanged in 13/14 vaccinated horses. Immunization with the bivalent vaccine was safe, resulted in lower median day 42 antibody titres of 400 for BPV1 and 1600 for EcPV2 and conferred significant yet incomplete cross-protection from BPV2-induced tumour formation, with 11/14 horses developing small, short-lived papules. Control horses developed pseudo-sarcoids at all inoculation sites. The monovalent BPV1 L1 VLP vaccine proved highly effective in protecting horses from BPV1-induced pseudo-sarcoid formation. Incomplete protection from BPV2-induced tumour development conferred by the bivalent vaccine is due to the poorer immune response by immune interference or lower cross-neutralization titres to heterologous BPV2 virions.

In planta production of a candidate vaccine against bovine papillomavirus type 1.

Bovine papillomavirus type 1 (BPV-1) is an economically important virus that induces tumourigenic pathologies in horses and cows. Given that the BPV-1 L1 major coat protein can self-assemble into highly immunogenic higher-order structures, we transiently expressed it in Nicotiana benthamiana as a prelude to producing a candidate vaccine. It was found that plant codon optimization of L1 gave higher levels of expression than its non-optimized counterpart. Following protein extraction, we obtained high yields (183 mg/kg fresh weight leaf tissue) of relatively pure L1, which had self-assembled into virus-like particles (VLPs). We found that these VLPs elicited a highly specific and strong immune response, and therefore they may have utility as a potential vaccine. This is the first report demonstrating the viable production of a candidate BPV vaccine protein in plants.

Smurf2 alters BPV1 trafficking and decreases infection.

Papillomavirus capsid proteins L1 and L2 mediate virion attachment, internalization and trafficking. In our studies of the capsid proteins, we identified an interaction of L2 with the E3 ligase Smad ubiquitin regulatory factor 2 (Smurf2). Smurf2 expression alters BPV1 virion trafficking and L2 protein levels. Using BPV1 pseudovirions (PSVs) containing a GFP or DSRed transgene encapsidated by L1 and L2 proteins, our data showed that although only BPV1 L2 interacts with Smurf2, both L1 and L2 levels decrease in a Smurf2- and ubiquitin-dependent manner. The decrease in L2 protein levels corresponded to a decrease in infection (i.e., loss of GFP or DSRed expression). We propose that Smurf2 regulates L2 protein cellular localization and therefore alters L2 protein levels. This change in trafficking and protein level decreases nuclear delivery and transcription of encapsidated pseudoviral transgenes and thus decreases BPV1 infection levels.

IFN-gamma promotes generation of IL-10 secreting CD4+ T cells that suppress generation of CD8 responses in an antigen-experienced host.

Ags characterizing tumors or chronic viral infection are generally presented to the host immune system before specific immunotherapy is initiated, and consequent generation of regulatory CD4(+) T cells can inhibit induction of desired effector CD8 T cell responses. IL-10 produced in response to ongoing Ag exposure inhibits generation of CD8 T cells in an Ag-experienced host. We now show that this IL-10 is produced by Ag experienced CD4(+) glucocorticoid-induced tumor necrosis factor receptor(+) T cells that also secrete IFN-gamma upon antigenic stimulation, that IL-10 secretion by these cells is enhanced through IFN-gamma signaling, and, unexpectedly, that IFN-gamma signaling is required for inhibition of generation of Ag-specific CD8 T cell responses in an Ag-experienced host. Systemic inhibition of both IL-10 and IFN-gamma at the time of immunization may therefore facilitate induction of effective immunotherapeutic responses against tumor specific and viral Ags.

Influenza virus vector iNS1 expressing bovine papillomavirus 1 (BPV1) antigens efficiently induces tumour regression in equine sarcoid patients.

Bovine papillomaviruses types 1 and 2 (BPV1, BPV2) commonly induce skin tumours termed sarcoids in horses and other equids. Sarcoids seriously compromise the health and welfare of affected individuals due to their propensity to resist treatment and reoccur in a more severe form. We have developed influenza (Flu) A and B virus vectors that harbour a truncated NS1 gene (iNS) assuring interferon induction and co-express shuffled BPV1 E6 and E7 antigens for sarcoid immunotherapy. In a safety trial involving 12 healthy horses, intradermal administration of iNSA/E6E7equ and iNSB/E6E7equ was well tolerated, with the only transient side effect being mild fever in four horses. Repeated screening of secretions and faeces by RT-PCR and plaque assay revealed no virus shedding, thus also confirming biological safety. In a patient trial involving 29 horses bearing BPV1-induced single or multiple sarcoids, at least one lesion per horse was intratumourally injected and then boosted with iNSA/E6E7equ and/or iNSB/E6E7equ. The treatment induced a systemic antitumour response as reflected by the synchronous regression of injected and non-injected lesions. Irrespective of vaccination schemes, complete tumour regression was achieved in 10/29 horses. In 10/29 horses, regression is still ongoing (May 2021). Intriguingly, scrapings collected from former tumour sites in two patients tested negative by BPV1 PCR. Nine severely affected individuals with a history of unsuccessful therapeutic attempts did not (6/29) or only transiently (3/29) respond to the treatment. INSA/E6E7equ and iNSB/E6E7equ proved safe and effective in significantly reducing the tumour burden even in severe cases.

Vaccination with prion peptide-displaying papillomavirus-like particles induces autoantibodies to normal prion protein that interfere with pathologic prion protein production in infected cells.

Prion diseases are fatal neurodegenerative disorders caused by proteinaceous infectious pathogens termed prions (PrP(Sc)). To date, there is no prophylaxis or therapy available for these transmissible encephalopathies. Passive immunization with monclonal antibodies recognizing the normal host-encoded prion protein (PrP(C)) has been reported to abolish PrP(Sc) infectivity and to delay onset of disease. Because of established immunologic tolerance against the widely expressed PrP(C), active immunization appears to be difficult to achieve. To overcome this limitation, papillomavirus-like particles were generated that display a nine amino acid B-cell epitope, DWEDRYYRE, of the murine/rat prion protein in an immunogenic capsid surface loop, by insertion into the L1 major capsid protein of bovine papillomavirus type 1. The PrP peptide was selected on the basis of its previously suggested central role in prion pathogenesis. Immunization with PrP-virus-like particles induced high-titer antibodies to PrP in rabbit and in rat, without inducing overt adverse effects. As determined by peptide-specific ELISA, rabbit immune sera recognized the inserted murine/rat epitope and also cross-reacted with the homologous rabbit/human epitope differing in one amino acid residue. In contrast, rat immune sera recognized the murine/rat peptide only. Sera of both species reacted with PrP(C) in its native conformation in mouse brain and on rat pheochromocytoma cells, as determined by immunoprecipitation and fluorescence-activated cell sorting analysis. Importantly, rabbit anti-PrP serum contained high-affinity antibody that inhibited de novo synthesis of PrP(Sc) in prion-infected cells. If also effective in vivo, PrP-virus-like particle vaccination opens a unique possibility for immunologic prevention of currently fatal and incurable prion-mediated diseases.

Induction of mucosal and systemic immune responses against human carcinoembryonic antigen by an oral vaccine.

Carcinoembryonic antigen (CEA) is a tumor-associated antigen targeted for the development of colorectal tumor vaccines. In this study, we developed papillomavirus pseudoviruses encoding the truncated CEA without NH2-terminal signal peptide (PV-CEA) as an oral vaccine to induce CEA-specific CTL responses. In CEA transgenic (CEA-Tg) mice orally immunized with PV-CEA, the immunologic tolerance to CEA as a "self-antigen" was overcome and both mucosal and systemic CEA-specific cytolytic activities were detected by in vitro 51Cr release assays. In a tumor prevention model, the growth rate of CEA+ tumors was significantly delayed in CEA-Tg mice orally immunized with PV-CEA when compared with the control vaccine. Further, the IFN-gamma enzyme-linked ImmunoSPOT and in vitro 51Cr release assay results showed that HLA-A2-restricted, CEA-specific CTL responses were induced in both mucosal and systemic lymphoid tissues in A2 transgenic mice after oral immunization with PV-CEA. Finally, we showed that coadministration of papillomavirus pseudoviruses encoding interleukin-2 with PV-CEA enhanced the generation of A2-restricted, CEA-specific CTLs in aged CEA/A2 double transgenic mice, which were more clinically relevant. Our data suggest that PV-CEA pseudovirus vaccine is a promising oral CEA vaccine for humans to induce CEA-specific CTLs at the site of colorectal tumors (i.e., intestinal mucosa), which might efficiently eliminate CEA+ colorectal tumor cells in the mucosa.

Integrated analysis of recombinant BPV-1 L1 protein for the production of a bovine papillomavirus VLP vaccine.

Bovine papillomatosis is an infectious disease that is caused by bovine papillomavirus (BPV), which results in important economic losses. However, no BPV vaccines or effective treatment methods are commercially available to date. Moreover, the absence of papillomavirus replication in vitro makes the use of recombinant protein a promising candidate for vaccine formulations. Hence, we developed an integrated study on the L1 capsid protein of BPV-1, obtained from a bacterial expression system, regarding its purification, biosafety, thermostability and immunogenicity. The results indicated an absence of genotoxicity of the purified recombinant L1 protein, ß-sheet prevalence of secondary structure folding, protein stability under high temperatures as well as the presence of capsomeres and VLPs. In addition, preliminary experimental vaccination of calves showed the production of specific antibodies against BPV-1 L1.

Recent advances in preclinical model systems for papillomaviruses.

Preclinical model systems to study multiple features of the papillomavirus life cycle have greatly aided our understanding of Human Papillomavirus (HPV) biology, disease progression and treatments. The challenge to studying HPV in hosts is that HPV along with most PVs are both species and tissue restricted. Thus, fundamental properties of HPV viral proteins can be assessed in specialized cell culture systems but host responses that involve innate immunity and host restriction factors requires preclinical surrogate models. Fortunately, there are several well-characterized and new animal models of papillomavirus infections that are available to the PV research community. Old models that continue to have value include canine, bovine and rabbit PV models and new rodent models are in place to better assess host-virus interactions. Questions arise as to the strengths and weaknesses of animal PV models for HPV disease and how accurately these preclinical models predict malignant progression, vaccine efficacy and therapeutic control of HPV-associated disease. In this review, we examine current preclinical models and highlight the strengths and weaknesses of the various models as well as provide an update on new opportunities to study the numerous unknowns that persist in the HPV research field.

Seroepidemiologic studies of bovine papillomavirus infections.

Bovine and human sera were analyzed for the presence of antibodies against bovine papillomavirus types 1 and 2 (BPV 1 and 2) and human papillomavirus type 1 (HPV 1) in a solid-phase radioimmunoassay. Human sera did not react with BPV antigens, and bovine sera showed no evidence of antibodies against HPV 1. In contrast, 19% of all bovine sera tested reacted with BPV 1 and 2 antigens, and 35% of human sera revealed antibodies against HPV 1. No serologic evidence was obtained for heterologous infections of persons exposed preferentially to cattle (farmers, butchers, and patients with Q-fever).

Association of serum immunoglobulin G antibodies against human papillomavirus type 16 capsids with anal epidermoid carcinoma.

BACKGROUND: Anal epidermoid carcinoma is a relatively rare tumor, but its incidence has been increasing rapidly during the past few years. Genetic material from the major oncogenic types of human papillomavirus (HPV), types 16 and 18, has regularly been demonstrated in a substantial proportion of anal cancers, suggesting an etiologic role of HPV infection. Recently, serum antibodies against HPV type 16 capsids were shown to be a serologic measure of HPV16 infection. PURPOSE: We investigated whether serum antibodies against HPV16 capsids are associated with an increased risk of developing anal cancer. METHODS: Serum samples from 64 patients (48 women and 16 men) with untreated anal epidermoid cancer and from 79 age- and sex-matched healthy blood donors were analyzed for the levels of serum immunoglobulin G (IgG) against capsids of HPV16 by the enzyme-linked immunosorbent assay. The levels of serum IgG against HPV type 6 and bovine papillomavirus (BPV) capsids, as well as against HPV16 peptide antigens, were also measured. RESULTS: Whereas antibodies against HPV6 or BPV capsids were not significantly associated with anal cancer, the presence of IgG against HPV16 capsids exceeding the anti-BPV antibody levels was demonstrated among 55% (35 of 64) of the case patients but only among 4% (three of 79) of the control subjects (odds ratio [OR] = 30.4; 95% confidence interval [CI] = 8.4-161.5). Antibodies against HPV16 E2 and E7 peptides were also more common among case patients (OR = 12.8 and 95% CI = 5.4-31.5 for E2; OR = 3.0 and 95% CI = 1.4-6.7 for E7). CONCLUSION: The results suggest that HPV16 capsid antibodies are serologic markers for anal cancer. IMPLICATION: Exposure to HPV16 or related viruses appears to be a major risk factor in the majority of anal cancers.

Bovine papillomavirus type 1 monoclonal antibodies.

Bovine papillomavirus type 1 (BPV-1) and type 2 (BPV-2) are the etiologic agents of fibropapillomas in cattle. Polyclonal antisera produced against BPV-1 structural antigens are cross-reactive with BPV-2. In this study BPV-1 type-specific monoclonal antibodies were produced that were not reactive with BPV-2. These monoclonal antibodies could be used for identification of BPV-1 structural antigens in acetone-fixed, frozen sections by immunofluorescence and Formalin-fixed, paraffin-embedded sections by immunoperoxidase techniques. In addition, these antibodies could be used for identification and purification of BPV-1 virions by immune electron microscopy and immunoadsorption techniques, respectively.

Topographical and conformational epitopes of bovine papillomavirus type 1 defined by monoclonal antibodies.

Monoclonal antibodies (MAbs) were generated against sodium dodecyl sulfate-disrupted bovine papillomavirus type 1 (BPV-1). When screened by enzyme-linked immunosorbent assay (ELISA) on intact and disrupted BPV-1, -2, and deer papillomavirus, three patterns of reactivity were defined: reactivity only with intact virus, with both intact and disrupted virus, and only with disrupted virus. On the basis of ELISA results, the topographical location and requirement for conformation for immunoreactivity of epitopes was defined as external conformational, external linear, and internal linear. Cross-reactivity of MAbs with other papillomavirus types was analyzed by immunofluorescence on warts from different species. Type-specific, BPV-1 and/or -2 cross-reactive, broadly cross-reactive, and genus-specific MAbs were identified. MAb reactivity with structural polypeptides of BPV-1 was analyzed by Western blot. MAbs reactive with epitopes defined as conformational by ELISA did not react in Western blot. All MAbs reactive in Western blot reacted with the major capsid protein (MCP) [55 kilodalton (kDa)], demonstrating that the MCP carries both type-specific and cross-reactive epitopes. Most MAbs reactive with the MCP were cross-reactive with structural polypeptides of 48 and 96 kDa, demonstrating the immunologic relatedness of these three polypeptides.

HPV vaccination to prevent cervical cancer and other HPV-associated disease: from basic science to effective interventions.

Identification of HPV infection as the etiologic agent of virtually all cases of cervical cancer, as well as a proportion of other epithelial cancers, has led to development of three FDA-approved multivalent prophylactic HPV vaccines composed of virus-like particles (VLPs). This essay describes the research and development that led to the VLP vaccines; discusses their safety, efficacy, and short-term effect on HPV-associated disease; and speculates that even a single dose of these vaccines, when given to adolescents, might be able to confer long-term protection. The HPV field exemplifies how long-term funding for basic research has lead to clinical interventions with the long-term potential to eradicate most cancers attributable to HPV infection. Although this essay is the result of my receiving the 2015 Harrington Prize for Innovation in Medicine from the Harrington Discovery Institute and the American Society for Clinical Investigation, this clinical advance has depended on the research of many investigators, development of commercial vaccines by the pharmaceutical companies, and participation of many patient volunteers in the clinical trials.

Infection by bovine papillomavirus and prospects for vaccination.

Infection with bovine papillomavirus (BPV) results in the onset of benign proliferative lesions that usually regress spontaneously through a cell-mediated immune response. Occasionally, warts persist as benign tumours or progress to squamous-cell carcinomas. Vaccines that prevent or cure BPV infection provide a model for the formulation of vaccines against human papillomavirus.

Two antibodies that neutralize papillomavirus by different mechanisms show distinct binding patterns at 13 A resolution.

Complexes between bovine papillomavirus type 1 (BPV1) and examples of two sets of neutralizing, monoclonal antibodies (mAb) to the major capsid protein (L1) were analyzed by low-dose cryo-electron microscopy and three-dimensional (3D) image reconstruction to 13 A resolution. mAb #9 is representative of a set of neutralizing antibodies that can inhibit viral binding to the cell surface, while mAb 5B6 is representative of a second set that efficiently neutralizes papillomaviruses without significantly inhibiting viral binding to the cell surface. The 3D reconstructions reveal that mAb #9 binds to L1 molecules of both pentavalent and hexavalent capsomeres. In contrast, 5B6 binds only to hexavalent capsomeres, reflecting the significant structural or environmental differences for the 5B6 epitope in the 12 pentavalent capsomeres. Epitope localization shows that mAb #9 binds monovalently to the tips of capsomeres whereas 5B6 binds both monovalently and bivalently to the sides of hexavalent capsomeres approximately two-thirds of the way down from the outer tips, very close to the putative stabilizing intercapsomere connections. The absence of mAb 5B6 from the pentavalent capsomeres and its inability to prevent viral binding to the cell surface suggest that receptor binding may occur at one or more of the 12 virion vertices.

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.

Monoclonal antibody neutralization of BPV-1.

Mouse monoclonal antibodies were generated against intact infectious BPV-1 virions by methods previously described (Christensen et al., 1990). ELISA was used to screen for reactivities to intact and/or disrupted BPV-1, CRPV and HPV-11 virions. Several hybridomas were initially selected that showed antibody reactivity by ELISA to both intact and disrupted BPV-1, to disrupted BPV-1 only, or to intact BPV-1 virions. One monoclonal antibody, designated B1.A1, which reacted only to intact BPV-1 was selected for virus neutralization analyses. ELISA demonstrated that this monoclonal antibody bound to intact BPV-1 virions, but not to intact CRPV, HPV-11 or to disrupted papillomavirus (PV) antigens. Strong neutralization of BPV-1-induced focus formation of mouse C127 cells by monoclonal B1.A1 was observed. The neutralization titer was equivalent to the neutralization titer obtained with a polyclonal rabbit anti-BPV-1 virion antisera, and directly correlated with antibody concentration as determined by ELISA. These results extend our previous analyses on the epitopes of infectious papillomaviruses as defined by monoclonal antibodies that identify neutralizing epitopes. The nature of these epitopes is such that maintenance of the quaternary structure of the infectious virions is necessary for preservation of the antigenicity of the neutralizing epitope.

Overcoming antigen masking of anti-amyloidbeta antibodies reveals breaking of B cell tolerance by virus-like particles in amyloidbeta immunized amyloid precursor protein transgenic mice.

BACKGROUND: In prior work we detected reduced anti-Abeta antibody titers in Abeta-vaccinated transgenic mice expressing the human amyloid precursor protein (APP) compared to nontransgenic littermates. We investigated this observation further by vaccinating APP and nontransgenic mice with either the wild-type human Abeta peptide, an Abeta peptide containing the "Dutch Mutation", E22Q, or a wild-type Abeta peptide conjugated to papillomavirus virus-like particles (VLPs). RESULTS: Anti-Abeta antibody titers were lower in vaccinated APP than nontransgenic mice even when vaccinated with the highly immunogenic Abeta E22Q. One concern was that human Abeta derived from the APP transgene might mask anti-Abeta antibodies in APP mice. To test this possibility, we dissociated antigen-antibody complexes by incubation at low pH. The low pH incubation increased the anti-Abeta antibody titers 20-40 fold in APP mice but had no effect in sera from nontransgenic mice. However, even after dissociation, the anti-Abeta titers were still lower in transgenic mice vaccinated with wild-type Abeta or E22Q Abeta relative to non-transgenic mice. Importantly, the dissociated anti-Abeta titers were equivalent in nontransgenic and APP mice after VLP-based vaccination. Control experiments demonstrated that after acid-dissociation, the increased antibody titer did not cross react with bovine serum albumin nor alpha-synuclein, and addition of Abeta back to the dissociated serum blocked the increase in antibody titers. CONCLUSIONS: Circulating human Abeta can interfere with ELISA assay measurements of anti-Abeta titers. The E22Q Abeta peptide vaccine is more immunogenic than the wild-type peptide. Unlike peptide vaccines, VLP-based vaccines against Abeta abrogate the effects of Abeta self-tolerance.