Immunotherapy of Equine Sarcoids-From Early Approaches to Innovative Vaccines.

Horses and other equid species are frequently affected by bovine papillomavirus type 1 and/or 2 (BPV1, BPV2)-induced skin tumors termed sarcoids. Although sarcoids do not metastasize, they constitute a serious health problem due to their BPV1/2-mediated resistance to treatment and propensity to recrudesce in a more severe, multiple form following accidental or iatrogenic trauma. This review provides an overview on BPV1/2 infection and associated immune escape in the equid host and presents early and recent immunotherapeutic approaches in sarcoid management.

Design, Synthesis, and Evaluation of Peptides Derived from L1 Protein Against Bovine Papillomavirus-1/2 Identified Along Mexico's Cattle Export Route.

Introduction: Bovine papillomatosis affects animal health and represents one of the greatest economic losses in the livestock sector. New control and prevention methods to protect the livestock industry from this disease are necessary. The aim of this study was to evaluate a candidate peptide for antibody production against bovine papillomavirus (BPV). Material and Methods: A total of 64 cattle underwent wart excision among 5,485 cattle distributed over 2 to 4 farms per state and 12 farms in total in the four Mexican states of Tabasco, Chiapas, Veracruz, and Nuevo León. The prevalence of bovine papillomatosis per farm was calculated by wart visualisation. The warts were genotyped by PCR and sequenced, then a phylogenetic tree was built using MEGA X software. A synthetic peptide was designed in the ABCpred, Bepipred 2.0, Bepipred IDBT, Bepitope, LBtope, and MHC II predictor online server software's based on the C-terminal region of the L1 protein. Mice antibody production was induced by subcutaneous immunisation with 50 µg of synthetic peptide and evaluated by indirect ELISA. Results: The prevalence of BPV was higher in Tabasco, Chiapas, and Veracruz. Bovine papillomaviruses 1 and 2 were found in all representative samples. A phylogenetic tree showed that Mexican sequences were located in exclusive clades yet were highly related to international ones. The peptide immunisation induced antibody titres of 1 : 10,000/1 : 1,000,000 against synthetic peptide and whole wart lysate (WWL), respectively. Conclusion: Co-infections of BPV-1 and -2 were found in all four states. Immunisation of BALB/C mice with BPV-1/2-derived synthetic peptide based on the C-terminal region of the major viral capsid protein L1 induced the production of specific antibodies able to recognise BPV-1/2 viral particles from bovine WWL.

Papillomavirus-like Particles in Equine Medicine.

Papillomaviruses (PVs) are a family of small DNA tumor viruses that can induce benign lesions or cancer in vertebrates. The observation that animal PV capsid-proteins spontaneously self-assemble to empty, highly immunogenic virus-like particles (VLPs) has led to the establishment of vaccines that efficiently protect humans from specific PV infections and associated diseases. We provide an overview of PV-induced tumors in horses and other equids, discuss possible routes of PV transmission in equid species, and present recent developments aiming at introducing the PV VLP-based vaccine technology into equine medicine.

Bovine Papillomavirus Type 1 or 2 Virion-Infected Primary Fibroblasts Constitute a Near-Natural Equine Sarcoid Model.

Equine sarcoids are common, locally aggressive skin tumors induced by bovine papillomavirus types 1, 2, and possibly 13 (BPV1, BPV2, BPV13). Current in vitro models do not mimic de novo infection. We established primary fibroblasts from horse skin and succeeded in infecting these cells with native BPV1 and BPV2 virions. Subsequent cell characterization was carried out by cell culture, immunological, and molecular biological techniques. Infection of fibroblasts with serial 10-fold virion dilutions (2 × 106-20 virions) uniformly led to DNA loads settling at around 150 copies/cell after four passages. Infected cells displayed typical features of equine sarcoid cells, including hyperproliferation, and loss of contact inhibition. Neither multiple passaging nor storage negatively affected cell hyperproliferation, viral DNA replication, and gene transcription, suggestive for infection-mediated cell immortalization. Intriguingly, extracellular vesicles released by BPV1-infected fibroblasts contained viral DNA that was most abundant in the fractions enriched for apoptotic bodies and exosomes. This viral DNA is likely taken up by non-infected fibroblasts. We conclude that equine primary fibroblasts stably infected with BPV1 and BPV2 virions constitute a valuable near-natural model for the study of yet unexplored mechanisms underlying the pathobiology of BPV1/2-induced sarcoids.

Characterization of Episomal Replication of Bovine Papillomavirus Type 1 DNA in Long-Term Virion-Infected Saccharomyces Cerevisiae Culture.

We have previously reported that bovine papillomavirus type 1 (BPV-1) DNA can replicate its genome and produce infectious virus-like particles in short term virion-infected S. cerevisiae (budding yeast) cultures (Zhao and Frazer 2002, Journal of Virology, 76:3359-64 and 76:12265-73). Here, we report the episomal replications of BPV-1 DNA in long term virion-infected S. cerevisiae culture up to 108 days. Episomal replications of the BPV-1 DNA could be divided into three patterns at three stages, early active replication (day 3-16), middle weak replication (day 23-34/45) and late stable replication (day 45-82). Two-dimensional gel electrophoresis analysis and Southern blot hybridization have revealed further that multiple replication intermediates of BPV-1 DNA including linear form, stranded DNA, monomers and higher oligomers were detected in the virion-infected yeast cells over the time course. Higher oligomers shown as covalently closed circular DNAs (cccDNAs) are the most important replication intermediates that serve as the main nuclear transcription template for producing all viral RNAs in the viral life cycle. In this study, the cccDNAs were generated at the early active replication stage with the highest frequencies and then at late stable replication, but they appeared to be suppressed at the middle weak replication. Our data provided a novel insight that BPV-1 genomic DNA could replicate episomally for the long period and produce the key replication intermediates cccDNAs in S. cerevisiae system.

Clinicopathological characteristics and papillomavirus types in cutaneous warts in bovine.

Thirty-one bovine cutaneous warts were submitted to macroscopic and histological analyses and to molecular analyses to partial amplification and sequencing of the L1 gene of bovine papillomavirus (BPV). Viral types detected were BPV1 (52%), BPV2 (29%), BPV6 (16%) and BPV10 (3%). BPV2 had lower frequency in papilloma in comparison to that in fibropapilloma (p = 0.002).

No evidence of bovine papillomavirus type 1 or 2 infection in healthy equids.

BACKGROUND: There is a large body of evidence supporting bovine papillomavirus types 1 and 2 (BPV1; BPV2) as aetiological agents of equine sarcoids. However, there is conflicting data regarding BPV1/2 infection in sarcoid-free equids. OBJECTIVES: Data obtained between 2007 and 2017 by BPV1/2 screening of sarcoids and nonsarcoid tumours vs. samples from healthy equids are presented to help clarify this issue. STUDY DESIGN: Cross-sectional study. METHODS: Tumour material obtained from horses, donkeys and mules with confirmed sarcoids (n = 130), suspected sarcoids (n = 120), or nonsarcoid lesions (n = 70), skin biopsies from 102 tumour-free horses and dandruff/hair roots from 35 tumour-free donkeys and mules were screened for BPV1/2 infection. Sample DNA was extracted and validated by equine ß-actin PCR. BPV1/2 screening was performed by BPV1/2 E5-specific PCR allowing for the detection of less than 10 viral DNA molecules. Twenty-six amplicons were bidirectionally sequenced and compared to known E5 variants using BLAST program. RESULTS: BPV1/2 E5 PCR scored positive for 130/130 diagnosed sarcoids, 63/120 suspected sarcoids and 13/70 nonsarcoid lesions, whereas 137/137 DNA aliquots derived from tumour-free equids tested negative. On predicted E5 protein level, six different BPV1 E5 variants were identified. MAIN LIMITATIONS: The diagnosis of equine sarcoid was not confirmed in 120 lesions. CONCLUSIONS: Lack of BPV1/2 E5 DNA in tumour-free equids and the prevalence of sarcoid disease in young adult individuals suggest that the time span between initial infection and sarcoid development is short. This contrasts with the long phase of virus latency characterising infection of humans by carcinogenic papillomaviruses. Presence of BPV1/2 DNA in several cases of poor wound healing/hypergranulation and dermatitis points to these skin disorders being possibly co-induced by BPV1/2. PCR screening of tumour tissue/scrapings for BPV1/2 DNA represents a reliable tool for the rapid validation of a clinical diagnosis of equine sarcoid.

Caracterização molecular de DNA de Delta papillomavirus bovino (BPV1, 2 e 13) em sarcoides equinos / Molecular characterization of bovine Deltapapillomavirus (BPV1, 2, and 13) DNA in equine sarcoids

Sarcoides são tumores fibroblásticos, considerados os tumores de pele mais comuns em pele de equinos e que raramente apresentam regressão espontânea. Papilomavírus bovino (BPV) tipos 1 e 2 são relacionados com a patogenia do sarcoide e, provavelmente, o BPV tipo 13 (BPV13), recentemente descrito, também pode estar associado com a formação dessa lesão. Neste estudo, 20 amostras de lesões cutâneas, sendo 12 constituídas por tecidos frescos e 8 amostras de tecido fixado em formalina e embebido em parafina, provenientes de 15 cavalos foram utilizadas para a identificação do DNA de BPV. A análise histopatológica (HE) confirmou todas as lesões como sarcoide. Para a amplificação do DNA de papilomavírus (PV) foram realizadas três reações de PCR. Como triagem, os primers IFNR2/IDNT2 foram utilizados para amplificar um fragmento da ORF L1 do PV. O segundo par de primersutilizado é complementar a sequência dos genes E5 e L2 de BPVs 1, 2 e 13. O terceiro par de primers(FAP59/FAP64) utilizado tem o gene L1 como alvo. A primeira e a segunda PCRs permitiram amplificar produtos em todas as amostras avaliadas. Entretanto, na terceira reação, na qual foram utilizados os primers FAP, foi possível amplificar produtos com tamanho molecular esperado somente nas amostras constituídas por tecidos frescos. O sequenciamento de nucleotídeos e as análises filogenéticas realizadas nos fragmentos E5L2 resultaram na identificação de BPV1, 2 e 13 em 14 (70%), 2 (10%) e em 4 (20%) amostras de sarcoides, respectivamente. As amostras de sarcoides de um dos animais continha somente o DNA de BPV1. Entretanto, nas amostras provenientes do segundo cavalo foi possível identificar o DNA de três tipos de Deltapapillomavirus bovino (BPV1, 2 e 13) em lesões distintas. Este estudo ratifica a presença do DNA de BPV1, 2 e 13 em lesões de sarcoides em equinos, além de identificar três tipos de BPVs em um mesmo animal e descrever pela primeira vez no Brasil a presença de BPV1 e 2 nesse tipo de lesão.

Sarcoids are fibroblastic lesions, which are considered as the most common skin tumors of horses; spontaneous regression rarely occurs. The bovine papillomavirus (BPV) types 1 and 2 may be involved in the pathogenesis of sarcoids, and probably the recently described BPV type (BPV13) might be associated with the pathogenesis of this lesion. This study characterized the DNA of BPVs in sarcoids from 15 horses from Brazil by analyzing 20 cutaneous lesions (12 recently collected; 8 from formalin-fixed paraffin-embedded (FFPE) tissues). Histopathology confirmed the proliferative lesions as sarcoids. Three PCRs were performed to amplify papillomavirus (PV) DNA. For screening, the primers IFNR2/IDNT2 were used to amplify a fragment of the PV L1 ORF. The second primer set was complementary to a common sequence of the E5L2 genomic region of BPV1, 2, and 13. The third primer pair (FAP59/FAP64) targeted a fragment of the PVs L1 ORF. The screening and E5L2 PCRs yielded amplicons in all samples evaluated. The FAP amplicons identified BPV1, 2, and 13 only from fresh tissue samples. The phylogenetic analyses of E5L2 resulted in the identification of BPV1, 2, and 13 in 14 (70%), 2 (10%), and 4 (20%) sarcoids, respectively. Two horses demonstrated multiple lesions: the sarcoids of one of these contained only BPV1 DNA and those of the other contained three types of bovine Deltapapillomavirus (BPV1, 2, and 13). This study confirmed the presence of BPV1, 2, and 13 DNA in equine sarcoids. Moreover, these findings represent the first description of three types of BPV diagnosed in the same horse, as well as the first confirmation of BPV1 and 2 in horses from Brazil.

Sarcoid-derived fibroblasts: links between genomic instability, energy metabolism and senescence.

Bovine papillomavirus 1 (BPV-1) is a well recognized etiopathogenetic factor in a cancer-like state in horses, namely equine sarcoid disease. Nevertheless, little is known about BPV-1-mediated cell transforming effects. It was shown that BPV-1 triggers genomic instability through DNA hypomethylation and oxidative stress. In the present study, we further characterized BPV-1-positive fibroblasts derived from sarcoid tumors. The focus was on cancer-like features of sarcoid-derived fibroblasts, including cell cycle perturbation, comprehensive DNA damage analysis, end-replication problem, energy metabolism and oncogene-induced premature senescence. The S phase of the cell cycle, polyploidy events, DNA double strand breaks (DSBs) and DNA single strand breaks (SSBs) were increased in BPV-1-positive cells compared to control fibroblasts. BPV-1-mediated oxidative stress may contribute to telomere dysfunction in sarcoid-derived fibroblasts. Loss of mitochondrial membrane potential and concurrent elevation in intracellular ATP production may be a consequence of changes in energy-supplying pathways in BPV-1-positive cells which is also typical for cancer cells. Shifts in energy metabolism may support rapid proliferation in cells infected by BPV-1. Nevertheless, sarcoid-derived fibroblasts representing a heterogeneous cell fraction vary in some aspects of metabolic phenotype due to a dual role of BPV-1 in cell transformation and oncogene-induced premature senescence. This was shown with increased senescence-associated ß-galactosidase (SA-ß-gal) activity. Taken together, metabolic phenotypes in sarcoid-derived fibroblasts are plastic, which are similar to greater plasticity of cancer tissues than normal tissues.

Enhanced transgene expression in mammalian cells by recombinant baculovirus vector containing bovine papillomavirus type 1 replication elements.

BACKGROUND: The baculovirus Autographa californica multiple nucleopolyhedrovirus has been widely explored as a transgene expression vector. Further improvement of the expression of the transgene is important for its application. METHODS: Bovine papillomavirus type 1 (BPV-1) cis-element upstream regulatory region (URR) and trans-elements E1, E2, were inserted into the baculovirus genome. The expression of reporter gene, enhanced green fluorescent protein gene (EGFP), and the persistence of viral genome was compared in several mammalian cell lines after virus transduction. The cytotoxicity of the recombinant viruses was also evaluated. RESULTS: The recombinant baculovirus containing URR and E1, E2 genes showed significantly increased expression of EGFP in all cell lines tested, including HEK293, HeLa, BHK-21, CNE, CHO and MDCK cells. In HEK293 cells, the total production of EGFP was approximately five-fold higher than the control. The genome of virus with BPV-1 elements also persisted better than the control virus during the first few days post transduction. No obvious cytotoxicity was observed. CONCLUSIONS: The coexistence of BPV-1 URR and E1, E2 was essential and sufficient to improve the performance of baculovirus with respect to mediating gene expression in various mammalian cells without major cytotoxicity. The results obtained in the present study facilitate the application of baculovirus as an efficient transgene vehicle for protein production and gene delivery.