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Summary Reasons for performing study: Infection with bovine papillomaviruses types 1 and 2 (BPV-1, BPV-2) can lead to the development of therapy-resistant skin tumours termed sarcoids and possibly other skin diseases in equids. Although sarcoids seriously compromise the welfare of affected animals and cause considerable economic losses, no prophylactic vaccine is available to prevent this common disease. In several animal species and man, immunisation with papillomavirus-like particles (VLP) has been shown to protect efficiently from papillomaviral infection. Hypothesis: BPV-1 L1 VLPs may constitute a safe and highly immunogenic vaccine candidate for protection of horses against BPV-1/-2-induced disease. Methods: Three groups of 4 horses each received 50, 100 or 150 µg of BPV-1 L1 VLPs, respectively, on Days 0, 28 and 168. Three control horses received adjuvant only. Horses were monitored on a daily basis for one week after each immunisation and then in 2 week intervals. Sera were collected immediately before, 2 weeks after each vaccination and one and 2 years after the final boost and analysed by pseudovirion neutralisation assay. Results: None of the horses showed adverse reactions upon vaccination apart from mild and transient swelling in 2 individuals. Irrespective of the VLP dose, all VLP-immunised horses had developed a BPV-1-neutralising antibody titre of ≥1600 plaque forming units (pfu)/ml 2 weeks after the third vaccination. Eight of 10 trial horses still available for follow-up had neutralising antibody titres ≥1600 pfu/ml one year and ≥800 pfu/ml 2 years after the last immunisation. Conclusion: Intramuscular BPV-1 L1 VLP vaccination in horses is safe and results in a long-lasting antibody response against BPV-1. Neutralisation titres were induced at levels that correlate with protection in experimental animals and man. Potential relevance: BPV-1 L1 VLPs constitute a promising vaccine candidate for prevention of BPV-1/-2-induced disease in equids. [ABSTRACT FROM AUTHOR]

Cathelicidins have been reported to inhibit human papillomavirus infection in vitro; however, nothing is known about their activity in vivo. In this study, experimental skin infection with Mus musculus papillomavirus 1 resulted in robust development of cutaneous papillomas in cyclosporine A-treated C57BL/6J mice deficient for the murine cathelicidin-related antimicrobial peptide (CRAMP), in contrast to wild-type controls. Analysis of the underlying mechanisms revealed moderate disruption of virion integrity and lack of interference with viral entry and intracellular trafficking by a synthetic CRAMP peptide. Differences in the immune response to Mus musculus papillomavirus 1 infection were observed between CRAMP-deficient and wild-type mice. These included a stronger reduction in CD4+ and CD8+ T-cell numbers in infected skin, and lack of Mus musculus papillomavirus 1-specific neutralizing antibodies in response to cyclosporine A in the absence of endogenous CRAMP. CRAMP has modest direct anti-papillomaviral effects in vitro, but exerts protective functions against Mus musculus papillomavirus 1 skin infection and disease development in vivo, primarily by modulation of cellular and humoral immunity.

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.

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.

Common cutaneous human papillomavirus (HPV) types induce skin warts, whereas species beta HPV are implicated, together with UV-radiation, in the development of non-melanoma skin cancer (NMSC) in immunosuppressed patients. Licensed HPV vaccines contain virus-like particles (VLP) self-assembled from L1 major capsid proteins that provide type-restricted protection against mucosal HPV infections causing cervical and other ano-genital and oro-pharyngeal carcinomas and warts (condylomas), but do not target heterologous HPV. Experimental papillomavirus vaccines have been designed based on L2 minor capsid proteins that contain type-common neutralization epitopes, to broaden protection to heterologous mucosal and cutaneous HPV types. Repetitive display of the HPV16 L2 cross-neutralization epitope RG1 (amino acids (aa) 17-36) on the surface of HPV16 L1 VLP has greatly enhanced immunogenicity of the L2 peptide. To more directly target cutaneous HPV, L1 fusion proteins were designed that incorporate the RG1 homolog of beta HPV17, the beta HPV5 L2 peptide aa53-72, or the common cutaneous HPV4 RG1 homolog, inserted into DE surface loops of HPV1, 5, 16 or 18 L1 VLP scaffolds. Baculovirus expressed chimeric proteins self-assembled into VLP and VLP-raised NZW rabbit immune sera were evaluated by ELISA and L1- and L2-based pseudovirion (PsV) neutralizing assays, including 12 novel beta PsV types. Chimeric VLP displaying the HPV17 RG1 epitope, but not the HPV5L2 aa53-72 epitope, induced cross-neutralizing humoral immune responses to beta HPV. In vivo cross-protection was evaluated by passive serum transfer in a murine PsV challenge model. Immune sera to HPV16L1-17RG1 VLP (cross-) protected against beta HPV5/20/24/38/96/16 (but not type 76), while antisera to HPV5L1-17RG1 VLP cross-protected against HPV20/24/96 only, and sera to HPV1L1-4RG1 VLP cross-protected against HPV4 challenge. In conclusion, RG1-based VLP are promising next generation vaccine candidates to target cutaneous HPV infections., Competing Interests: Reinhard Kirnbauer and Christina Schellenbacher are co-inventors on a patent on RG1-VLP technology: Title: Papillomavirus-like particles (VLP) as broad spectrum human papillomavirus (hpv) vaccines (US 20120093821 A1). The technology has been licensed to Pathovax, Baltimore, a biotech startup company developing this technology. RK and CS also own equity in PathoVax LLC and are member of its scientific advisory board. These arrangements have been reviewed and approved by the Medical University of Vienna and Johns Hopkins University in accordance with their conflict of interest policies. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

The consistent and specific presence of Equus caballus papillomavirus type 2 (EcPV2) DNA and mRNA in equine genital squamous cell carcinoma (gSCC) is suggestive of an etiological role in tumor development. To further validate this concept, EcPV2-neutralizing serum antibody titers were determined by an EcPV2 pseudovirion (PsV) neutralization assay. Furthermore, an EcPV2 L1 virus-like particle (VLP)-based vaccine was generated and its prophylactic efficacy evaluated in vivo. All 6/6 gSCC-affected, but only 3/20 tumor-free age-matched animals revealed EcPV2-neutralizing serum antibody titers by PsV assay. Vaccination of NZW rabbits and BalbC mice with EcPV2 L1 VLP using Freund׳s or alum respectively as adjuvant induced high-titer neutralizing serum antibodies (1600-12,800). Passive transfer with rabbit EcPV2-VLP immune sera completely protected mice from experimental vaginal EcPV2 PsV infection. These findings support the impact of EcPV2 in equine gSCC development and recommend EcPV2 L1 VLP as prophylactic vaccine against EcPV2 infection and associated disease in equids., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Persistent infection with high-risk human papillomavirus (HPV) types, most often HPV16 and HPV18, causes all cervical and most anal cancers, and a subset of vulvar, vaginal, penile and oropharyngeal carcinomas. Two prophylactic virus-like particle (VLPs)-based vaccines, are available that protect against vaccine type-associated persistent infection and associated disease, yet have no therapeutic effect on existing lesions or infections. We have generated recombinant live-attenuated influenza A viruses expressing the HPV16 oncogenes E6 and E7 as experimental immunotherapeutic vaccine candidates. The influenza A virus life cycle lacks DNA intermediates as important safety feature. Different serotypes were generated to ensure efficient prime and boost immunizations. The immune response to vaccination in C57BL/6 mice was characterized by peptide ELISA and IFN-γ ELISpot, demonstrating induction of cell-mediated immunity to HPV16 E6 and E7 oncoproteins. Prophylactic and therapeutic vaccine efficacy was analyzed in the murine HPV16-positive TC-1 tumor challenge model. Subcutaneous (s.c.) prime and boost vaccinations of mice with recombinant influenza A serotypes H1N1 and H3N2, followed by challenge with TC-1 cells resulted in complete protection or significantly reduced tumor growth as compared to control animals. In a therapeutic setting, s.c. vaccination of mice with established TC-1 tumors decelerated tumor growth and significantly prolonged survival. Importantly, intralesional vaccine administration induced complete tumor regression in 25% of animals, and significantly reduced tumor growth in 50% of mice. These results suggest recombinant E6E7 influenza viruses as a promising new approach for the development of a therapeutic vaccine against HPV-induced disease.

Persistent infection with oncogenic human papillomaviruses (HPV) types causes all cervical and a subset of other anogenital and oropharyngeal carcinomas. Four high-risk (hr) mucosal types HPV16, 18, 45, or 59 cause almost all cervical adenocarcinomas (AC), a subset of cervical cancer (CxC). Although the incidence of cervical squamous cell carcinoma (SCC) has dramatically decreased following introduction of Papanicolaou (PAP) screening, the proportion of AC has relatively increased. Cervical SCC arise mainly from the ectocervix, whereas AC originate primarily from the endocervical canal, which is less accessible to obtain viable PAP smears. Licensed (bivalent and quadrivalent) HPV vaccines comprise virus-like particles (VLP) of the most important hr HPV16 and 18, self-assembled from the major capsid protein L1. Due to mainly type-restricted efficacy, both vaccines do not target 13 additional hr mucosal types causing 30% of CxC. The papillomavirus genus alpha species 7 (α7) includes a group of hr types of which HPV18, 45, 59 are proportionally overrepresented in cervical AC and only partially (HPV18) targeted by current vaccines. To target these types, we generated a chimeric vaccine antigen that consists of a cross-neutralizing epitope (homologue of HPV16 RG1) of the L2 minor capsid protein of HPV45 genetically inserted into a surface loop of HPV18 L1 VLP (18L1-45RG1). Vaccination of NZW rabbits with 18L1-45RG1 VLP plus alum-MPL adjuvant induced high-titer neutralizing antibodies against homologous HPV18, that cross-neutralized non-cognate hr α7 types HPV39, 45, 68, but not HPV59, and low risk HPV70 in vitro, and induced a robust L1-specific cellular immune response. Passive immunization protected mice against experimental vaginal challenge with pseudovirions of HPV18, 39, 45 and 68, but not HPV59 or the distantly related α9 type HPV16. 18L1-45RG1 VLP might be combined with our previously described 16L1-16RG1 VLP to develop a second generation bivalent vaccine with extended spectrum against hr HPV.

Licensed human papillomavirus (HPV) vaccines, based on virus-like particles (VLPs) self-assembled from major capsid protein L1, afford type-restricted protection against HPV types 16/18/6/11 (or 16/18 for the bivalent vaccine), which cause 70% of cervical cancers (CxCas) and 90% of genital warts. However, they do not protect against less prevalent high-risk (HR) types causing 30% of CxCa, or cutaneous HPV. In contrast, vaccination with the minor capsid protein L2 induces low-level immunity to type-common epitopes. Chimeric RG1-VLP presenting HPV16 L2 amino acids 17-36 (RG1 epitope) within the DE-surface loop of HPV16 L1 induced cross-neutralizing antisera. We hypothesized that RG1-VLP vaccination protects against a large spectrum of mucosal and cutaneous HPV infections in vivo. Immunization with RG1-VLP adjuvanted with human-applicable alum-MPL (aluminum hydroxide plus 3-O-desacyl-4'-monophosphoryl lipid A) induced robust L2 antibodies (ELISA titers 2,500-12,500), which (cross-)neutralized mucosal HR HPV16/18/45/37/33/52/58/35/39/51/59/68/73/26/69/34/70, low-risk HPV6/11/32/40, and cutaneous HPV2/27/3/76 (titers 25-1,000) using native virion- or pseudovirion (PsV)-based assays, and a vigorous cytotoxic T lymphocyte response by enzyme-linked immunospot. In vivo, mice were efficiently protected against experimental vaginal challenge with mucosal HR PsV types HPV16/18/45/31/33/52/58/35/39/51/59/68/56/73/26/53/66/34 and low-risk HPV6/43/44. Enduring protection was demonstrated 1 year after vaccination. RG1-VLP is a promising next-generation vaccine with broad efficacy against all relevant mucosal and also cutaneous HPV types.

Infection with bovine papillomavirus type 1 (BPV1) or BPV2 induces fibropapillomas in cows and skin sarcoids in horses. Prophylactic vaccination targeting BPV1 and BPV2 may reduce the incidence of these economically important diseases. The L1 major capsid proteins of BPV1 and BPV2 were expressed in Sf-9 insect cells and both self-assembled into virus-like particles (VLPs). Using conformation-dependent monoclonal antibodies (mAb) both type-specific and shared epitopes were detected. Antisera were raised against BPV1 or BPV2 VLP using alum adjuvant, and their (cross)neutralization capacity was tested by C127 neutralization assays using native BPV1 and BPV2 virions, or by BPV1 pseudovirion assay. Antisera induced by either VLP vaccine were able to robustly (cross-)neutralize heterologous as well as homologous types, indicating that BPV1 and BPV2 are closely related serotypes. These results suggest that a monovalent BPV1 (or BPV2) VLP vaccine may potentially protect against both BPV1 and BPV2 infections and associated diseases.

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.

Peptides of the papillomavirus L2 minor capsid protein can induce antibodies (Ab) that neutralize a broad range of human papillomavirus (HPV) genotypes. Unfortunately, L2 is antigenically subdominant to L1 in the virus capsid. To induce a strong anti-L2 Ab response with cross-neutralizing activity to other mucosal types, chimeric virus-like particles (VLP) were generated in which HPV16 L2 neutralization epitopes (comprising L2 residues 69-81 or 108-120) are inserted within an immunodominant surface loop (between residues 133 and 134) of the L1 major capsid protein of bovine papillomavirus type 1 (BPV1). These chimeras self-assembled into pentameric capsomers, or complete VLP similar to wild type (wt) L1 protein. Immunization of rabbits with assembled particle preparations induced L2-specific serum Ab with titers 10-fold higher than those induced by cognate synthetic L2 peptides coupled to KLH. Antisera to both chimeric proteins partially neutralized HPV16 pseudovirions, confirming that both HPV16 L2 peptides define neutralization epitopes. When analyzed for the ability to cross-neutralize infection by authentic HPV11 virions, using detection of early viral RNA by RT-PCR-assays as the readout, immune serum to chimeric protein comprising L2 residues 69-81, but not 108-120, was partially neutralizing. In addition, mouse-antiserum induced by vaccinations with synthetic L2 peptide 108-120, but not 69-81, was partially neutralizing in this assay. Induction of cross-neutralization Ab by L2 epitopes displayed on chimeric VLP represents a possible strategy for the generation of broad-spectrum vaccines to protect against relevant mucosal HPV and associated neoplasia.

Immunization with amyloid-beta (Abeta) prevents the deposition of Abeta in the brain and memory deficits in transgenic mouse models of Alzheimer's disease (AD), opening the possibility for immunotherapy of AD in humans. Unfortunately, the first human trial of Abeta vaccination was complicated, in a small number of vaccinees, by cell-mediated meningoencephalitis. To develop an Abeta vaccine that lacks the potential to induce autoimmune encephalitis, we have generated papillomavirus-like particles (VLP) that display 1-9 aa of Abeta protein repetitively on the viral capsid surface (Abeta-VLP). This Abeta peptide was chosen because it contains a functional B cell epitope, but lacks known T cell epitopes. Rabbit and mouse vaccinations with Abeta-VLP were well tolerated and induced high-titer autoAb against Abeta, that inhibited effectively assembly of Abeta(1-42) peptides into neurotoxic fibrils in vitro. Following Abeta-VLP immunizations of APP/presenilin 1 transgenic mice, a model for human AD, we observed trends for reduced Abeta deposits in the brain and increased numbers of activated microglia. Furthermore, Abeta-VLP vaccinated mice also showed increased levels of Abeta in plasma, suggesting efflux from the brain into the vascular compartment. These results indicate that the Abeta-VLP vaccine induces an effective humoral immune response to Abeta and may thus form a basis to develop a safe and efficient immunotherapy for human AD.

Papillomaviruses replicate in stratified epithelia of skin and mucosa. Infection with certain human papillomavirus (HPV) types is the main cause of anogenital neoplasia, in particular cervical cancer. Early events of papillomavirus infectivity are poorly understood. While heparan sulfate proteoglycans (HSPGs) mediate initial binding to the cell surface, the class of proteins carrying heparan sulfates has not been defined. Here we examined two processes of papillomavirus infection, attachment of virus-like particles (VLP) to cells and infection with authentic HPV type 11 (HPV11) virions. Of the HSPGs, syndecan-1 is the major epithelial form and is strongly upregulated in wound edge keratinocytes. We employed K562 cells, which lack HSPGs except minor amounts of endogenous betaglycan, and stable clones that express cDNAs of syndecan-1, syndecan-4, or glypican-1. Binding of VLP correlated with levels of heparan sulfate on the cell surface. Parental K562 bound HPV16 VLP weakly, whereas all three K562 transfectants demonstrated enhanced binding, with the highest binding capacity observed for syndecan-1-transfected cells, which also expressed the most HSPG. For HPV11 infectivity assays, a high virion inoculum was required to infect K562 cells, whereas ectopic expression of syndecan-1 increased permissiveness eightfold and expression of syndecan-4 or glypican-1 fourfold. Infection of keratinocytes was eliminated by treatment with heparitinase, but not phospholipase C, further implicating the syndecan family of integral membrane proteins as receptor proteins. Human keratinocytes with a homozygous deletion of alpha6 integrin are permissive for HPV11 infection. These results indicate that several HSPGs can serve as HPV receptors and support a putative role for syndecan-1, rather than alpha6 integrin, as a primary receptor protein in natural HPV infection of keratinocytes.

Neutralization capsid epitopes are important determinants for antibody-mediated immune protection against papillomavirus (PV) infection and induced disease. Chimeric L1 major capsid proteins of the human PV type 16 (HPV-16) and the bovine PV type 1 (BPV-1) with a foreign peptide incorporated into several capsid surface loops self-assembled into pentamers or virus-like particles (VLP). Binding patterns of neutralizing monoclonal antibodies (MAb) and immunization of mice confirmed (i) that regions around aa 282-286 and 351-355 contribute to neutralization epitopes and identified the latter region as an immunodominant site and (ii) that placing a foreign peptide in the context of an assembled structure markedly enhanced its immunogenicity. Pentamers disassembled from wild-type HPV-16 and BPV-1 VLPs displayed some of the neutralization epitopes that were detected on fully assembled VLPs, but were deficient for binding a subset of neutralizing MAb that inhibit cell attachment.