Prophylactic potential of an anti-BPV-1/-2 vaccine in horses

Equine sarcoids are persistent, locally aggressive skin tumours induced by bovine papillomaviruses of types 1 and 2 (BPV-1, BPV-2). Given that no prophylactic vaccine is so far available, this common disease constitutes a significant veterinary problem. Papillomavirus (PV)-like particles (VLPs) have been shown to be well tolerated and effective vaccines against PV infection-mediated tumour formation in several species. A VLP-based vaccine for prevention of human PV (HPV)-induced cervical cancer in women has been recently introduced on the market. We have shown that immunisation of horses with BPV-1 L1 VLPs has minimal side effects and results in high titres of type-specific neutralising antibodies. We could also demonstrate that BPV-1 and BPV-2 are sero-analogues. We hence postulate that vaccination with BPV-1 VLPs can avert infection by BPV-1 and -2 and thus prevent equine sarcoid development. To be able to address this assumption, we then established a virus challenge model. Four weanling foals were intradermally inoculated with BPV-1 virion, equine fibroblasts harbouring BPV-1 episomes and naked full-length BPV-1 genome. BPV-1 virions reliably induced transient sarcoid-like skin tumours in all horses, whereas injection with DNA-based inocula failed to induce lesions. This result confirmed earlier findings supporting that intact virions are required to produce infection and associated disease in horses, and that intradermal inoculation of horses with virion constitutes a robust infection model. Based on these preliminary findings, we now propose to address the protective potential of BPV-1 L1 VLPs in horses, hypothesising that this vaccine candidate will prevent pseudo-tumour formation or significantly reduce the size and/or number of lesions. To this aim, 42 non-infected foals will be assigned to 2 equivalent groups. Fourteen randomly selected horses from each group will be immunised with BPV-1 L1 VLPs on days 0 and 28. The remaining 7 horses per group will receive adjuvant only and serve as controls. In addition, the study will include 4 naive horses subjected to passive serum protein transfer, and 8 horses immunised in 2007. Then, all horses will be challenged by intradermal inoculation with BPV-1 or BPV-2 virions. Horses will then be clinically monitored for a minimum of 6 month. Tumours expected in the control group and skin biopsies, as well as blood samples collected from all individuals will be subjected to down-stream analyses. Provided that a substantial protection from pseudo- tumour formation can be achieved, BPV-1 L1 VLPs are envisaged to be routinely employed for prevention of BPV-1/-2-associated disease in equids.

Introduction: Bovine papillomavirus type 1 or 2 (BPV1/2)-induced sarcoids and equine papillomavirus type 2 (EcPV2)-associated genital squamous cell carcinoma (gSCC) and precursor lesions are common tumours in horses and other equids. To establish vaccines for future protection of equids from sarcoids and gSCC, we have previously generated empty BPV1 capsids (virus-like particles; VLPs) and shown in horses that they are safe and induce a robust immune response. We also demonstrated in vitro that antibodies against BPV1 also neutralize BPV2. Then we established a reliable BPV1 horse challenge model that we used to assess the protective potential of BPV1 L1 VLPs in vivo. We further addressed the safety and immunogenicity of a bivalent BPV1/EcPV2 L1 VLP vaccine. Material and Methods: In a first trial 14 horses were immunized with BPV1 VLPs and 7 served as unvaccinated control animals. Then all horses were intradermally inoculated with BPV1 virion at 10 sites of the neck. In addition 7 horses vaccinated six years ago and three control horses were challenged likewise. In a second trial 14 horses were immunized with a bivalent vaccine containing BPV1 and EcPV2 VLPs whilst 7 control horses remained unvaccinated. Then, all horses were challenged with BPV2 virion. Finally, we immunized rabbits with EcPV2 or control VLPs, transferred serum to mice and then challenged these mice with EcPV2 pseudo-virions. All animal trials were accompanied by molecular biological and immunological analyses and legally approved. Major Results: Intramuscular administrations of VLPs were safe and induced a robust antibody response. In trial 1, all control horses developed self-resolving pseudo-sarcoids at all inoculation sites, whilst vaccinated horses were completely protected. Importantly, horses vaccinated six years ago were like-wise protected from experimental BPV1 infection and associated pseudo-sarcoid formation. EcPV2/BPV1 L1 VLPs conferred partial protection from BPV2 challenge, with some individuals showing tiny, transient lesions upon challenge. Mice transferred with serum from EcPV2 VLP-immunized rabbits were completely protected from challenge with EcPV2 pseudo-virions. Conclusions: Vaccination with BPV1 VLPs protected horses efficiently from BPV1 infection, also when carried out six years ago. EcPV2/BPV1 VLPs were safe and conferred partial protection from BPV2 infection. This may be due to a BPV2 L1 protein sequence deviation of 7% compared to BPV1 L1. Anti-EcPV2 rabbit antibodies protected mice efficiently from EcPV2 challenge. However, lack of detectable anti-BPV1antibody titres six years after BPV1 VLP immunization in one protected horse suggests that VLP-induced protection from BPV1 infection is not exclusively based antibodies but also on memory B cell immunity.