Replicating viruses for the treatment of cancer have a number of advantages over traditional therapeutic modalities. They are highly targeted, self-amplifying, and have the added potential to act as both gene-therapy delivery vehicles and oncolytic agents. ORFV, (Parapoxvirus ovis, or Orf virus) is the prototypic species of the Parapoxvirus genus, causing a benign disease in its natural ungulate host. ORFV possesses a number of unique properties that make it an ideal viral backbone for the development of a cancer therapeutic: it is safe in humans, has the ability to cause repeat infections even in the presence of antibody, and it induces a potent Th-1 dominated immune response. Here I show for the first time that live replicating ORFV induces an anti-tumour immune response in multiple syngeneic mouse models of cancer that is mediated largely by the potent activation of both cytokine-secreting, and tumouricidal natural killer (NK) cells. I have also highlighted the clinical potential of the virus by demonstration of human cancer cell oncolysis including efficacy in an A549 xenograft model of cancer. The mechanism of ORFV-mediated activation of NK cells has been explored, where I have demonstrated activation via direct ex vivo infection of NK cells. I have also highlighted ORFV-mediated activation of dendritic cells (DCs), both in vivo and by direct infection ex vivo. An in vivo DC depletion study demonstrated an indirect mechanism for ORFV NK cell activation, where in the absence of DCs, NK cell activation was diminished, as was the ability of ORFV to clear lung metastases. The ORFV innate immune stimulatory profile has been harnessed for therapeutic application in an experimental surgery model of cancer, where ORFV therapy at the time of surgery reduces the number of cancer metastases. These data highlight the clinical potential of a live, immune stimulating Parapoxvirus therapeutic.

Replicating viruses for the treatment of cancer have a number of advantages over traditional therapeutic modalities. They are highly targeted, self-amplifying, and have the added potential to act as both gene-therapy delivery vehicles and oncolytic agents. ORFV, (Parapoxvirus ovis, or Orf virus) is the prototypic species of the Parapoxvirus genus, causing a benign disease in its natural ungulate host. ORFV possesses a number of unique properties that make it an ideal viral backbone for the development of a cancer therapeutic: it is safe in humans, has the ability to cause repeat infections even in the presence of antibody, and it induces a potent Th-1 dominated immune response. Here I show for the first time that live replicating ORFV induces an anti-tumour immune response in multiple syngeneic mouse models of cancer that is mediated largely by the potent activation of both cytokine-secreting, and tumouricidal natural killer (NK) cells. I have also highlighted the clinical potential of the virus by demonstration of human cancer cell oncolysis including efficacy in an A549 xenograft model of cancer. The mechanism of ORFV-mediated activation of NK cells has been explored, where I have demonstrated activation via direct ex vivo infection of NK cells. I have also highlighted ORFV-mediated activation of dendritic cells (DCs), both in vivo and by direct infection ex vivo. An in vivo DC depletion study demonstrated an indirect mechanism for ORFV NK cell activation, where in the absence of DCs, NK cell activation was diminished, as was the ability of ORFV to clear lung metastases. The ORFV innate immune stimulatory profile has been harnessed for therapeutic application in an experimental surgery model of cancer, where ORFV therapy at the time of surgery reduces the number of cancer metastases. These data highlight the clinical potential of a live, immune stimulating Parapoxvirus therapeutic.

Replicating viruses for the treatment of cancer have a number of advantages over traditional therapeutic modalities. They are highly targeted, self-amplifying, and have the added potential to act as both gene-therapy delivery vehicles and oncolytic agents. ORFV, (Parapoxvirus ovis, or Orf virus) is the prototypic species of the Parapoxvirus genus, causing a benign disease in its natural ungulate host. ORFV possesses a number of unique properties that make it an ideal viral backbone for the development of a cancer therapeutic: it is safe in humans, has the ability to cause repeat infections even in the presence of antibody, and it induces a potent Th-1 dominated immune response. Here I show for the first time that live replicating ORFV induces an anti-tumour immune response in multiple syngeneic mouse models of cancer that is mediated largely by the potent activation of both cytokine-secreting, and tumouricidal natural killer (NK) cells. I have also highlighted the clinical potential of the virus by demonstration of human cancer cell oncolysis including efficacy in an A549 xenograft model of cancer. The mechanism of ORFV-mediated activation of NK cells has been explored, where I have demonstrated activation via direct ex vivo infection of NK cells. I have also highlighted ORFV-mediated activation of dendritic cells (DCs), both in vivo and by direct infection ex vivo. An in vivo DC depletion study demonstrated an indirect mechanism for ORFV NK cell activation, where in the absence of DCs, NK cell activation was diminished, as was the ability of ORFV to clear lung metastases. The ORFV innate immune stimulatory profile has been harnessed for therapeutic application in an experimental surgery model of cancer, where ORFV therapy at the time of surgery reduces the number of cancer metastases. These data highlight the clinical potential of a live, immune stimulating Parapoxvirus therapeutic.

Parapoxvirus ovis (PPVO) is known for its immunostimulatory capacities and has been successfully used to generate vector vaccines effective especially in non-permissive host species. Murine conventional and plasmacytoid dendritic cells (cDC and pDC) are able to recognize PPVO. The PPVO-sensing receptor on pDC is hitherto unknown. In this study we aimed to define the pattern recognition receptor responsible for the activation of murine pDC by inactivated and replication-competent PPVO. We show that PPVO-induced expression of type I and type III interferons, pro-inflammatory cytokines, and costimulatory CD86 by bone marrow-derived pDC but not cDC is blocked by chloroquine, an inhibitor of endosomal maturation. The activation of pDC is independent of viral replication and depends mainly on TLR9. Moreover, the use of phosphatidylinositol 3-kinase inhibitor wortmannin or C-Jun-N-terminal kinase inhibitor SP600125 results in significant reduction of PPVO-induced pDC activation. Taken together, our data identify endosomal TLR9 as PPVO-sensing receptor in pDC.

Parapoxvirus ovis (PPVO) is known for its immunostimulatory capacities and has been successfully used to generate vector vaccines effective especially in non-permissive host species. Murine conventional and plasmacytoid dendritic cells (cDC and pDC) are able to recognize PPVO. The PPVO-sensing receptor on pDC is hitherto unknown. In this study we aimed to define the pattern recognition receptor responsible for the activation of murine pDC by inactivated and replication-competent PPVO. We show that PPVO-induced expression of type I and type III interferons, pro-inflammatory cytokines, and costimulatory CD86 by bone marrow-derived pDC but not cDC is blocked by chloroquine, an inhibitor of endosomal maturation. The activation of pDC is independent of viral replication and depends mainly on TLR9. Moreover, the use of phosphatidylinositol 3-kinase inhibitor wortmannin or C-Jun-N-terminal kinase inhibitor SP600125 results in significant reduction of PPVO-induced pDC activation. Taken together, our data identify endosomal TLR9 as PPVO-sensing receptor in pDC.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Orf virus (ORFV), the type member of the genus Parapoxvirus of the family Poxviridae, is the etiologic agent of orf or contagious ecthyma, a contagious and ubiquitous disease of sheep and goats. ORFV genome consists of a double stranded DNA molecule with approximately 138 Kb, and contains 131 putative genes. Among those, 15 are novel genes, unique to parapoxviruses, which lack homology to other known viral or cellular genes. In the present study we describe the functional characterization of three of these genes, ORFV024, ORFV002, and ORFV121. Results presented here demonstrate that the proteins encoded by these genes inhibit the activation of the nuclear factor-kappa B (NF-κB) signaling pathway. ORFV-encoded ORFV024 inhibits activation of the NF-κB signaling pathway in the cell cytoplasm by inhibiting phosphorylation of the IκB kinases, IKKα and IKKβ, consequently inhibiting the activation of the IKK complex. Deletion of ORFV024 from the ORFV genome had no significant effect on disease severity, progression or time to resolution in sheep, indicating that ORFV024 does not contribute to ORFV virulence. ORFV-encoded ORFV002 functions in the cell nucleus, where it interacts with the NF-κB subunit NF-κB-p65, inhibiting its acetylation, a p300-mediated modification of NF-κB-p65 which modulates its transcriptional activity. Similarly to ORFV024, deletion of ORFV002 from the ORFV genome had no significant effect on ORFV virulence and disease pathogenesis in sheep. ORFV-encoded ORFV121 functions in the cell cytoplasm, where it binds to and inhibits phosphorylation and nuclear translocation of NF-κB-p65. Deletion of ORFV121 from the ORFV genome resulted in a marked attenuated disease phenotype in sheep, indicating that ORFV121 is a determinant of virulence of ORFV in the natural host. These results indicate that ORFV, like other poxviruses, has evolved multiple strategies to modulate NF-κB, targeting different steps of the signaling pathway. Results obtained in the pathogenesis studies performed here suggest that multiple NF-κB inhibitors encoded by ORFV may exert complementary and/or redundant functions to effectively block host cell responses regulated by the NF-κB signaling pathway. Additionally, it is possible that ORFV-encoded NF-κB inhibitors modulate distinct cellular processes regulated by NF-κB in vivo. A better understanding of ORFV-host interactions may provide valuable insights for the development of improved vaccines against orf, or yet for the development of novel ORFV-based therapeutic agents and vaccine vectors with enhanced safety and efficacy, and a broader applicability. / O vírus da orf (ORFV), protótipo do gênero Parapoxvirus da família Poxviridae, é o agente etiológico da orf ou ectima contagioso, uma enfermidade contagiosa de distribuição mundial que afeta primariamente ovinos e caprinos. O genoma do ORFV consiste de uma molécula de DNA de fita dupla com aproximadamente 138 Kb, que contém presumidamente 131 genes. Dentre estes, 15 são genes novos, identificados apenas nos parapoxvírus e que não possuem homologia com outros genes de origem viral ou celular. O presente estudo descreve a caracterização funcional de três destes genes, ORFV024, ORFV002 e ORFV121. Os resultados apresentados no presente estudo demonstram que as proteínas codificadas pelos genes ORFV024, ORFV002 e ORFV121 inibem a ativação da via de sinalização do fator de transcrição nuclear-kappa B (NF-κB). O produto da ORFV024 bloqueia a ativação da via do NF-κB no citoplasma celular, inibindo a fosforilação das quinases IκB (IKK), IKKα e IKKβ e, consequentemente inibindo a ativação do complexo IKK. A deleção do gene ORFV024 do genoma do ORFV não alterou a severidade, a progressão, ou o tempo de resolução das lesões produzidas pelo ORFV em ovinos, indicando que o produto deste gene não contribui para a virulência do vírus. O gene ORFV002 codifica um inibidor do NF-κB que atua no núcleo das células. O produto do ORFV002 interage com a subunidade NF-κB-p65 do NF-κB, inibindo a sua acetilação, uma modificação pós-traducional do NF-κB-p65 mediada pela acetiltransferase p300 que regula a sua atividade transcripcional. Semelhante ao ORFV024, a deleção do gene ORFV002 do genoma do ORFV não afetou a virulência do vírus nem alterou a patogenia da enfermidade em ovinos. O produto do gene ORFV121 atua no citoplasma das células, onde esta proteína viral interage com o NF-κB-p65 inibindo sua fosforilação e translocação nuclear. A deleção do gene ORFV121 do genoma do ORFV reduziu significativamente a severidade, a progressão e o tempo de resolução da doença em ovinos, indicando que este produto viral constitui-se em um fator de virulência para o ORFV em seu hospedeiro natural. Estes resultados demonstram que, assim como outros poxvírus, o ORFV também desenvolveu múltiplas estratégias para modular a via de sinalização do NF-κB, codificando proteínas que atuam em diferentes eventos desta complexa via de sinalização intracelular. Os resultados obtidos nos estudos de patogenia sugerem que os inibidores do NF-κB codificados pelo ORFV desempenham funções complementares e/ou redundantes, provavelmente, para promover um bloqueio efficiente dos processos biológicos regulados pelo NF-κB. Além disso, estes produtos virais podem modular diferentes processos biológicos controlados pelo NF-κB in vivo. Um melhor entendimento das interações do ORFV com o seu hospedeiro pode favorecer o desenvolvimento de vacinas mais eficazes para o ectima contagioso, ou ainda, promover o desenvolvimento de vacinas vetoriais ou imunoterápicos, baseados no ORFV, mais eficazes e com uma maior espectro de aplicações.

Submitted by (edna.saturno@ufrpe.br) on 2016-11-04T13:11:29Z No. of bitstreams: 1 Rosana Leo de Santana.pdf: 693786 bytes, checksum: b9d2b7d60669ce82e4b87ed63463daad (MD5) / Made available in DSpace on 2016-11-04T13:11:29Z (GMT). No. of bitstreams: 1 Rosana Leo de Santana.pdf: 693786 bytes, checksum: b9d2b7d60669ce82e4b87ed63463daad (MD5) Previous issue date: 2008-02-28 / Contagious ectima is a severe and proliferartive virus among ovine and caprine caused by the contagious ectima virus (ECV) of the Parapoxvirus genus. The control of the infection in endemic regions is done with vaccines, however there are limitations in the vaccine production due to the difficulties in replicating the virus in cell cultures. The purpose of this paper is to evaluate the behavior of ECV samples in primary cultures of fetal caprine cornea cells, a system of cultures yet untested for the replication of ECV. Crust samples from nine sheep and two goats from the states of Bahia, Sergipe and Paraiba and presenting the clinical symptoms of EC were inoculated in monolayers of the cells, during seven consecutive passages at weekly intervals. During all the occasions we observed, after 24 hours of infection, the cytopathic effect (CE) characterized by cell rounding, fusion with the formation of small sincicia, cytoplasmatic inclusion and vacuolation. The intensity of detachment from the cell layers ranged from 25% to 100% which varied according to the sample. We concluded that the primary cell cultures of the fetal caprine cornea appeared highly permissible to replication of ECV and the isolated samples of ECV appeared to adapt to the utilized culture with slight variation among samples. / Ectima contagioso (EC) é uma virose aguda e proliferativa de ovinos e caprinos, causada pelo vírus do ectima contagioso (ECV), do gênero Parapoxvirus. O controle da infecção em regiões endêmicas é realizado através de vacinação, porém existem limitações nas técnicas de produção de vacinas, que é a dificuldade de replicação do vírus em cultivo celular. Este trabalho foi conduzido com o objetivo de isolar e avaliar o comportamento das amostras de ECV em cultivo primário de células de córnea fetal caprina (CFC), sistema de cultivo ainda não testado para replicação de ECV. Amostras de crostas de nove ovinos e de dois caprinos que apresentavam sintomatologia clínica de EC, originários dos Estados da Bahia, Sergipe e Paraíba, foram inoculadas em monocamadas de células epiteliais de córnea de feto caprino, durante sete passagens consecutivas, a intervalos semanais. Observou-se em todas passagens, a partir de 24 horas pós infecção, efeito citopático (ECP) caracterizado pelo arredondamento celular, fusão com formação de pequenos sincícios, vacuolização e corpúsculos de inclusão citoplasmático, com intensidade de 25% a 100% de desprendimento da camada celular, que variou de acordo com a amostra. Conclui-se que as culturas de células primárias de córnea fetal caprina mostraram-se altamente permissíveis à replicação do ECV e que as amostras de ECV isoladas mostraram-seadaptadas ao cultivo utilizado, com pequena variação entre as amostras.

Fusionspeptid-, Redox-, Viruscore- und sonstige Proteine. Alle analysierten Single ORF (SO)-VVOV Rekombinanten vermittelten einen signifikanten Schutz vor einer tödlichen Belastung mit Aujeszky-Virus. Zwei der Rekombinanten (SO 93-, SO 94-VVOV) enthalten ORFs, die für ATI/Fusionspeptid-Proteine kodieren. In SO 19- und SO 70-VVOV sind dagegen für Redoxproteine kodierende ORFs integriert. Weiterführende Untersuchungen zeigten, dass SO 94- und SO 19-VVOV in zwei weiteren Modellsystemen immunstimulatorisch aktiv sind. Im Baculo-Virussystem exprimierte Proteine waren nur in Kombination mit Vaccinia Lister-Virus (VV) wirksam. Dabei zeigten jeweils Virus-Protein-Gemische mit dem geringsten Proteinanteil den stärksten immunstimulatorischen Effekt. Proben in denen VV durch bovines Herpes-Virus-1 ersetzt wurde, sind dagegen nicht wirksam. Dies lässt auf eine Beteiligung VV-spezifischer Faktoren schließen. Übereinstimmend mit diesen Ergebnissen führte eine Frameshift-Mutation in ORF 94r von SO 94mut-VVOV nur zur Abschwächung und nicht zum vollständigen Verlust der immunstimulatorischen Wirkung. Beide in Schizosaccharomyces pombe exprimierten Proteine, sp-ORF19 und sp-ORF94r, induzierten keinen signifikanten Schutz im Aujeszky Maus Modell. Mit der Identifikation einzelner immunstimulatorisch aktiver PPVO-Komponenten ist es erstmals gelungen, den paramunisierenden Effekt von Parapox-Viren einzelnen viralen Genen zu zuordnen. Insbesondere stellen SO 94- und SO 19-VVOV viel versprechende Kandidaten für die prophylaktische bzw. therapeutische Anwendung in verschiedenen Indikationen als auch für weitere Untersuchungen des Wirkmechanismus dar.

Fusionspeptid-, Redox-, Viruscore- und sonstige Proteine. Alle analysierten Single ORF (SO)-VVOV Rekombinanten vermittelten einen signifikanten Schutz vor einer tödlichen Belastung mit Aujeszky-Virus. Zwei der Rekombinanten (SO 93-, SO 94-VVOV) enthalten ORFs, die für ATI/Fusionspeptid-Proteine kodieren. In SO 19- und SO 70-VVOV sind dagegen für Redoxproteine kodierende ORFs integriert. Weiterführende Untersuchungen zeigten, dass SO 94- und SO 19-VVOV in zwei weiteren Modellsystemen immunstimulatorisch aktiv sind. Im Baculo-Virussystem exprimierte Proteine waren nur in Kombination mit Vaccinia Lister-Virus (VV) wirksam. Dabei zeigten jeweils Virus-Protein-Gemische mit dem geringsten Proteinanteil den stärksten immunstimulatorischen Effekt. Proben in denen VV durch bovines Herpes-Virus-1 ersetzt wurde, sind dagegen nicht wirksam. Dies lässt auf eine Beteiligung VV-spezifischer Faktoren schließen. Übereinstimmend mit diesen Ergebnissen führte eine Frameshift-Mutation in ORF 94r von SO 94mut-VVOV nur zur Abschwächung und nicht zum vollständigen Verlust der immunstimulatorischen Wirkung. Beide in Schizosaccharomyces pombe exprimierten Proteine, sp-ORF19 und sp-ORF94r, induzierten keinen signifikanten Schutz im Aujeszky Maus Modell. Mit der Identifikation einzelner immunstimulatorisch aktiver PPVO-Komponenten ist es erstmals gelungen, den paramunisierenden Effekt von Parapox-Viren einzelnen viralen Genen zu zuordnen. Insbesondere stellen SO 94- und SO 19-VVOV viel versprechende Kandidaten für die prophylaktische bzw. therapeutische Anwendung in verschiedenen Indikationen als auch für weitere Untersuchungen des Wirkmechanismus dar.