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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.

Bovine papillomavirus types 1 and 2 (BPV-1 and BPV-2) are known to induce common equine skin tumours, termed sarcoids. Recently, it was demonstrated that vaccination with BPV-1 virus-like particles (VLPs) is safe and highly immunogenic in horses. To establish a BPV-1 challenge model for evaluation of the protective potential of BPV-1 VLPs, four foals were injected intradermally with infectious BPV-1 virions and with viral genome-based and control inocula, and monitored daily for tumour development. Blood was taken before inoculation and at weekly intervals. BPV-1-specific serum antibodies were detected by a pseudo-virion neutralization assay. Total nucleic acids extracted from tumours, intact skin and PBMCs were tested for the presence of BPV-1 DNA and mRNA using PCR and RT-PCR, respectively. Intralesional E5 oncoprotein expression was determined by immunofluorescence. Pseudo-sarcoids developed exclusively at sites inoculated with virions. Tumours became palpable 11-32 days after virion challenge, reached a size of ≤20 mm in diameter and then resolved in ≤6 months. No neutralizing anti-BPV-1 serum antibodies were detectable pre- or post-challenge. BPV-1 DNA was present in lesions but not in intact skin. In PBMCs, viral DNA was already detectable before lesions were first palpable, in concentrations correlating directly with tumour growth kinetics. PBMCs from two of two foals also harboured E5 mRNA. Immunofluorescence revealed the presence of the E5 protein in tumour fibroblasts, but not in the apparently normal epidermis overlying the lesions. Together with previous findings obtained in horses and cows, these data suggest that papillomavirus infection may include a viraemic phase.

Bovine papillomaviruses of types 1 and 2 (BPV-1 and -2) chiefly contribute to equine sarcoid pathogenesis. However, the mode of virus transmission and the presence of latent infections are largely unknown. This study established a PCR protocol allowing detection of

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.