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.

abstract: Host organisms have evolved multiple mechanisms to defend against a viral infection and likewise viruses have evolved multiple methods to subvert the host's anti-viral immune response. Vaccinia virus (VACV) is known to contain numerous proteins involved in blocking the cellular anti-viral immune response. The VACV E3L protein is important for inhibiting the anti-viral immune response and deletions within this gene lead to a severe attenuation. In particular, VACV containing N-terminal truncations in E3L are attenuated in animal models and fail to replicate in murine JC cells. Monkeypox virus (MPXV) F3L protein is a homologue of the VACV E3L protein, however it is predicted to contain a 37 amino acid N-terminal truncation. Despite containing an N-terminal truncation in the E3L homologue, MPXV is able to inhibit the anti-viral immune response similar to wild-type VACV and able to replicate in JC cells. This suggests that MPXV has evolved another mechanism(s) to counteract host defenses and promote replication in JC cells. MPXV produces less dsRNA than VACV during the course of an infection, which may explain why MPXV posses a phenotype similar to VACV, despite containing a truncated E3L homologue. The development of oncolytic viruses as a therapy for cancer has gained interest in recent years. Oncolytic viruses selectively replicate in and destroy cancerous cells and leave normal cells unharmed. Many tumors possess dysregulated anti-viral signaling pathways, since these pathways can also regulate cell growth. Creating a mutation in the N-terminus of the VACV-E3L protein generates an oncolytic VACV that depends on dysregulated anti-viral signaling pathways for replication allowing for direct targeting of the cancerous cells. VACV-E3Ldel54N selectively replicates in numerous cancer cells lines and not in the normal cell lines. Additionally, VACV-E3Ldel54N is safe and effective in causing tumor regression in a xenograph mouse model. Lastly, VACV-E3Ldel54N was capable of spreading from the treated tumors to the untreated tumors in both a xenograph and syngeneic mouse model. These data suggest that VACV-E3Ldel54N could be an effective oncolytic virus for the treatment of cancer. / Dissertation/Thesis / Ph.D. Microbiology 2010

Poxviruses are double stranded (ds) DNA viruses with large brick-shaped virions (~200x300nm) that can be seen by light microscopy. The Chordopoxvirus (ChPV) subfamily demonstrates a vast genetic diversity in poxvirus virulence and evolution, and infects a wide range of vertebrate hosts including human/primates, rodents, birds, squirrels, and many economically important ruminants. There are at least 14 distinct ChPV genera, whose members have genomes that range between 127-360 kbp, and can be either GC-rich (33-38% A+T base composition) or AT-rich (up to 76% A+T). My work in the assembly and annotation of novel poxviruses serves to enrich the poxvirus sequence repository and further virulence characterization, comparative analysis, and phylogenetic studies. Using a variety of programs, as well as tools developed by the Virus Bioinformatics Research Centre, a protocol is created, refined, and applied to the assembly and annotation of novel poxviruses: Pteropox virus (PTPV) from a south Australian megabat Pteropus scapulatus, Eptesipox virus (EPTV) from a north American microbat Eptesicus fuscus, sea otter poxvirus (SOPV) from the north American Enhydra lutris, and two Kangaroopox viruses western and eastern Kangaroopox viruses (WKPV, EKPV) from the Australian Macropus fuliginosus and Macropus giganteus. This is the first time poxviruses from these vertebrate hosts are assembled in full, and the result supports the establishment of 4 new ChPV genera. The two bat-isolated poxviruses, PTPV and EPTV, likely did not co-speciate with their hosts despite infection of related host species. Instead, EPTV forms a sister clade with the Clade II virus, and together forms a sister group with the orthopoxviruses. On the other hand, PTPV and SOPV are each other’s closest extant relatives despite the distant geographical location from which they were isolated; together they share a novel homolog of TRAIL (Tumor necrosis factor-Related Apoptosis-Inducing Ligand) never before seen in poxviruses. SOPV additionally encodes distinct interleukin (IL)-18 binding protein and tumor necrosis factor (TNF) receptor-like protein that could have novel immune-evasion roles. The KPVs present the first case of a putative viral cullin-like protein, which might be involved in regulating the host ubiquitination pathway. Altogether, these novel proteins can potentially serve as new virokines and viroceptors in the form of viromimicry pathogenesis; they demonstrate the capacity and diversity with which poxviruses modulate host immune responses in their favour, and should be studied further. / Graduate / 2019-04-11

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.

Made available in DSpace on 2016-04-28T20:18:33Z (GMT). No. of bitstreams: 1 2008 - Renata Vitoria Campos Costa.pdf: 1895440 bytes, checksum: a7d7c26a7f184a241826971db909015b (MD5) Previous issue date: 2008-10-29 / In the South of Rio de Janeiro State, Brazil, numerous outbreaks of poxvirus infection in cattle and humans are described. Clinical, epidemiological and pathological aspects are presented and discussed. Serum neutralization, ultramicroscopy, viral isolation and PCR showed poxvirus strains very close to those employed in preparation of the human variola vaccines made in Rio de Janeiro State at 60/70 s. The disease is a zoonosis and causes important economic losses due to decrease of milk production and temporary incapacitation of milkmen. / Neste estudo descrevem-se surtos de infec??o por um v?rus do grupo pox em bovinos e humanos ocorridos entre maio de 2006 a outubro de 2007, nos munic?pios de Pira?, Barra do Pira?, Rio das Flores, Resende, Rio Claro e Valen?a, no Estado do Rio de Janeiro. S?o apresentadas, detalhadamente, as caracter?sticas cl?nicas, epidemiol?gicas e patol?gicas dessa enfermidade. Exames de soroneutraliza??o, visualiza??o pelo microsc?pio eletr?nico, isolamento viral e identifica??o molecular atrav?s da PCR (Rea??o em cadeia de polimerase), demonstraram tratar-se de uma cepa molecularmente muito pr?xima ?s amostras do Vaccinia v?rus (Orthopoxvirus) que foram utilizadas no preparo de vacinas durante as campanhas de erradica??o da var?ola humana, nas d?cadas de 60 e 70. Trata-se de uma importante zoonose adquirida por ordenhadores que t?m contato direto com tetas e ?beres de vacas infectadas durante a ordenha, e que cursa com consider?veis perdas econ?micas, principalmente devidas ? redu??o na produ??o de leite no animal e incapacita??o tempor?ria dos ordenhadores.

Poxviruses are renowned for encoding proteins that modulate virtually every aspect of the host immune system. One effective barrier against virus infection is apoptosis, a form of programmed cell death. Apoptosis is controlled at the mitochondria by pro- and anti-apoptotic members of the highly conserved Bcl-2 family of proteins, and two members in particular, Bak and Bax, are absolutely critical to the induction of cell death. Although poxviruses encode an array of effective inhibitors of apoptosis, only members of the Avipoxvirus genus, of which fowlpox virus is the prototypical member, encode proteins with obvious, albeit limited, sequence identity to cellular Bcl-2 proteins. Fowlpox virus, the prototypical avipoxvirus, encodes FPV039, a protein that possesses two of the four highly conserved Bcl-2 homology (BH) domains that characterise the Bcl-2 family. Here we demonstrate that, like cellular Bcl-2 proteins, FPV039 localised to the mitochondria where it prevented apoptosis induced by a variety of cytotoxic stimuli, including virus infection itself. FPV039 inhibited apoptosis induced by Bak and Bax through an interaction with Bak and activated Bax. FPV039 also interacted with a discrete subset of BH3-only proteins, the upstream activators of Bak and Bax, to prevent Bax activation in the first place. Additionally, we have characterised the function and mechanism of action of a novel deerpox virus protein, DPV022. Intriguingly, DPV022 lacks obvious homology to cellular Bcl-2 proteins but shares limited regions of amino acid identity with two other poxviral inhibitors of apoptosis, vaccinia virus F1L and myxoma virus M11L, which are themselves unrelated. Here we demonstrate that DPV022 localised to the mitochondria where it interacted directly with Bak and Bax to inhibit apoptosis, even in the absence all cellular anti-apoptotic Bcl-2 proteins. We have also embarked on a preliminary analysis of the apical events that initially trigger apoptosis during infection with vaccinia virus, the prototypical poxvirus. Accordingly, we demonstrate that the BH3-only protein Noxa contributed to the vaccinia virus-induced apoptotic response, possibly through an involvement with dsRNA. Together, this study represents a comprehensive analysis of the ways in which poxviruses manipulate the cellular Bcl-2 family of proteins, the arbiters of cell death. / Virology

Vaccines are one of the most effective advances in medical science and continue to be developed for applications against infectious diseases, cancers, and autoimmunity. A common strategy for vaccine construction is the use of viral vectors derived from various virus families, with Adenoviruses (Ad) and Poxviruses (Pox) being extensively used. Studies utilizing viral vectors have shown a broad variety of vaccine-elicited immune response phenotypes. However, innate immune stimulation elicited by viral vectors and its possible role in shaping these vaccine-elicited adaptive immune responses remains unclear. Here we show that Ad and Pox vectors display profound intra- and inter-group differences in innate immune cytokine and chemokine elicitation. The CD46-utilizing vectors Ad35, Ad26, and Ad48 induced greater anti-viral and proinflammatory cytokines and chemokines relative to Ad5 in vaccinated rhesus monkeys and stimulated human PBMC. Ad fiber protein, as well as other capsid components, influenced resultant Ad vector innate stimulatory phenotypes. Analysis of human sera from Ad26-vaccinated volunteers showed similar anti-viral and proinflammatory cytokine and chemokine elicitation. Mechanistic analysis of Ad innate immune stimulation showed greater amounts Ad35 and Ad26, and small amounts of Ad5, traffic to the late endosome following infection. Innate immune stimulation by all three was reduced by inhibition of endosomal acidification, Cathepsin B, and Caspase-1, suggesting a common set of innate immune sensors triggered by Ads between 0-6 hours post-infection, in agreement with trafficking data showing Ad vector colocalization in the late endosome at similar time points. These data suggest a model mechanism explaining differences in observed Ad vector innate immune stimulation phenotypes. Similar to results obtained with Ad vectors, analysis of innate cytokine and chemokine responses elicited by Pox vectors ALVAC, MVA, and NYVAC showed that all three were distinct, with the canarypox-based vector ALVAC eliciting a unique potent proinflammatory response. Together these results reveal surprising and pronounced differences in innate immune stimulatory properties of viral vectors. Furthermore, these results could lead to possible strategies for targeted construction of vaccines for desired innate immune phenotypes, and have profound implications on vaccine design against infectious diseases and cancers, as well as gene therapy.

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