Several wild-type poxviruses have emerged as potential oncolytic viruses (OVs), including orf virus (OrfV), and vaccinia virus (VV). Oncolytic VVs have been modified to include attenuating mutations that enhance their tumour selective nature, but these mutations also reduce overall viral fitness in cancer cells. Previous studies have shown that a VV (Western Reserve) with its E3L gene replaced with the E3L homologue from, OrfV (designated VV-E3LOrfV), maintained its ability to infect cells in vitro, but was attenuated compared to its parental VV in vivo. Our goal was to determine the safety and oncolytic potential VV-E3LOrfV, compared to wild type VV and other attenuated recombinants. VV-E3LOrfV, was unable to replicate to the same titers and was sensitive to IFN compared to its parental virus and other attenuated VVs in normal human fibroblast cells. The virus was also less pathogenic when administered in vivo. Viral replication, spread and cell killing, as measures of oncolytic potential in vitro, along with in vivo efficacy, were also observed.. The Parapoxvirus, OrfV has been shown to have a unique immune-stimulation profile, inducing a number of pro-inflammatory cytokines, as well as potently recruiting and activating a number of immune cells. Despite this unique profile, OrfV is limited in its ability to replicate and spread in human cancer cells. Various strategies were employed to enhance the oncolytic efficacy of wild-type OrfV. A transient transfection/infection screen was created to determine if any of the VV host-range genes (C7L, K1L, E3L or K3L) would augment OrfV oncolysis. Combination therapy, including the use of microtubule targeting agents, Viral Sensitizer (VSe) compounds and the addition of soluble VV B18R gene product were employed to see if they also enhance OrfV efficacy. Unfortunately, none of the strategies mentioned were able to enhance OrfV.

Several wild-type poxviruses have emerged as potential oncolytic viruses (OVs), including orf virus (OrfV), and vaccinia virus (VV). Oncolytic VVs have been modified to include attenuating mutations that enhance their tumour selective nature, but these mutations also reduce overall viral fitness in cancer cells. Previous studies have shown that a VV (Western Reserve) with its E3L gene replaced with the E3L homologue from, OrfV (designated VV-E3LOrfV), maintained its ability to infect cells in vitro, but was attenuated compared to its parental VV in vivo. Our goal was to determine the safety and oncolytic potential VV-E3LOrfV, compared to wild type VV and other attenuated recombinants. VV-E3LOrfV, was unable to replicate to the same titers and was sensitive to IFN compared to its parental virus and other attenuated VVs in normal human fibroblast cells. The virus was also less pathogenic when administered in vivo. Viral replication, spread and cell killing, as measures of oncolytic potential in vitro, along with in vivo efficacy, were also observed.. The Parapoxvirus, OrfV has been shown to have a unique immune-stimulation profile, inducing a number of pro-inflammatory cytokines, as well as potently recruiting and activating a number of immune cells. Despite this unique profile, OrfV is limited in its ability to replicate and spread in human cancer cells. Various strategies were employed to enhance the oncolytic efficacy of wild-type OrfV. A transient transfection/infection screen was created to determine if any of the VV host-range genes (C7L, K1L, E3L or K3L) would augment OrfV oncolysis. Combination therapy, including the use of microtubule targeting agents, Viral Sensitizer (VSe) compounds and the addition of soluble VV B18R gene product were employed to see if they also enhance OrfV efficacy. Unfortunately, none of the strategies mentioned were able to enhance OrfV.

abstract: The concept of vaccination dates back further than Edward Jenner's first vaccine using cowpox pustules to confer immunity against smallpox in 1796. Nevertheless, it was Jenner's success that gave vaccines their name and made vaccinia virus (VACV) of particular interest. More than 200 years later there is still the need to understand vaccination from vaccine design to prediction of vaccine efficacy using mathematical models. Post-exposure vaccination with VACV has been suggested to be effective if administered within four days of smallpox exposure although this has not been definitively studied in humans. The first and second chapters analyze post-exposure prophylaxis of VACV in an animal model using v50ΔB13RMγ, a recombinant VACV expressing murine interferon gamma (IFN-γ) also known as type II IFN. While untreated animals infected with wild type VACV die by 10 days post-infection (dpi), animals treated with v50ΔB13RMγ 1 dpi had decreased morbidity and 100% survival. Despite these differences, the viral load was similar in both groups suggesting that v50ΔB13RMγ acts as an immunoregulator rather than as an antiviral. One of the main characteristics of VACV is its resistance to type I IFN, an effect primarily mediated by the E3L protein, which has a Z-DNA binding domain and a double-stranded RNA (dsRNA) binding domain. In the third chapter a VACV that independently expresses both domains of E3L was engineered and compared to wild type in cells in culture. The dual expression virus was unable to replicate in the JC murine cell line where both domains are needed together for replication. Moreover, phosphorylation of the dsRNA dependent protein kinase (PKR) was observed at late times post-infection which indicates that both domains need to be linked together in order to block the IFN response. Because smallpox has already been eradicated, the utility of mathematical modeling as a tool for predicting disease spread and vaccine efficacy was explored in the last chapter using dengue as a disease model. Current modeling approaches were reviewed and the 2000-2001 dengue outbreak in a Peruvian region was analyzed. This last section highlights the importance of interdisciplinary collaboration and how it benefits research on infectious diseases. / Dissertation/Thesis / Ph.D. Molecular and Cellular Biology 2011

Several wild-type poxviruses have emerged as potential oncolytic viruses (OVs), including orf virus (OrfV), and vaccinia virus (VV). Oncolytic VVs have been modified to include attenuating mutations that enhance their tumour selective nature, but these mutations also reduce overall viral fitness in cancer cells. Previous studies have shown that a VV (Western Reserve) with its E3L gene replaced with the E3L homologue from, OrfV (designated VV-E3LOrfV), maintained its ability to infect cells in vitro, but was attenuated compared to its parental VV in vivo. Our goal was to determine the safety and oncolytic potential VV-E3LOrfV, compared to wild type VV and other attenuated recombinants. VV-E3LOrfV, was unable to replicate to the same titers and was sensitive to IFN compared to its parental virus and other attenuated VVs in normal human fibroblast cells. The virus was also less pathogenic when administered in vivo. Viral replication, spread and cell killing, as measures of oncolytic potential in vitro, along with in vivo efficacy, were also observed.. The Parapoxvirus, OrfV has been shown to have a unique immune-stimulation profile, inducing a number of pro-inflammatory cytokines, as well as potently recruiting and activating a number of immune cells. Despite this unique profile, OrfV is limited in its ability to replicate and spread in human cancer cells. Various strategies were employed to enhance the oncolytic efficacy of wild-type OrfV. A transient transfection/infection screen was created to determine if any of the VV host-range genes (C7L, K1L, E3L or K3L) would augment OrfV oncolysis. Combination therapy, including the use of microtubule targeting agents, Viral Sensitizer (VSe) compounds and the addition of soluble VV B18R gene product were employed to see if they also enhance OrfV efficacy. Unfortunately, none of the strategies mentioned were able to enhance OrfV.

abstract: Since the molecular biology revolution in the 1980s, ease of gene editing had led to the resurgence of Oncolytic Virotherapy. Countless viruses have been engineered yet only three are approved for clinical use worldwide, with only one being approved by the U.S Food and Drug Administration (FDA). Vaccinia virus (VACV) has a large genome, contains many immune evasion genes and has been thoroughly studied, making it a popular candidate for an oncolytic platform. VACV mutants with deletions in the E3 immune evasion protein have been shown to have oncolytic efficacy but the mechanism of tumor selectivity has not been fully elucidated. These mutants have been shown to be regulated by the necroptosis pathway, a pathway that has been shown to be deficient in certain cancers. Using a pan-cancer screening method that combines dye exclusion assays, western blot analysis, and viral growth curve, the role of necroptosis in regulating VACV replication and oncolytic efficacy in cancer was further characterized. Results demonstrate a preliminary correlation between necroptosis, viral replication, and oncolytic efficacy. This correlation is clearest in breast cancer and melanomas yet may apply to other cancer subgroups. This data was also used to guide the development of a receptor-interacting protein kinase 3 (RIP3) matched pair mouse model in the E0771 mouse breast cancer line which can be used to further study the role of necroptosis and oncolytic efficacy in vivo. Understanding the contribution necroptosis plays in oncolytic efficacy can guide to design enhance the design of clinical trials to test VACV E3L mutants and may lead to better efficacy in humans and an improvement in clinical oncology. / Dissertation/Thesis / Masters Thesis Biology 2020

The innate immune system is the first line of defence against pathogen invasion. The range of diseases that are caused by deficiencies in or deregulation of the innate immune system illustrates the importance of maintaining an effective balance between clearance of infectious agents and minimisation of inflammatory mediated tissue damage. This thesis explores the role of two proteins in the regulation of the innate immune system. Primarily, this work investigates the effect of human β-defensin 3 (hBD3) on the response to self-DNA and pathogenic DNA. HBD3 is an antimicrobial peptide (AMP), which has been shown to have a role in regulating the immune response; increased copy number of the region containing the gene for hBD3, DEFB103, is linked to an increased risk of psoriasis. Additionally, a similar cationic AMP, LL37, has been shown to exacerbate the pathogenesis of psoriasis by forming an immunogenic complex with self-DNA. This lead to the hypothesis that hBD3 may also affect the innate immune response to DNA. Therefore this project investigates what effect hBD3 has on the response of the innate immune system to self and pathogenic DNA. Flt-3 dendritic cells were used to show that whilst hBD3 increased cellular uptake of self-DNA, it did not convert self-DNA into an immune stimulus. However, hBD3 significantly exacerbated the response to bacterial DNA in a TLR9-dependent manner, also by increasing cellular uptake into FLDCs. The finding that hBD3 increased cellular uptake of both self- and pathogenic DNA suggests that at sites of infection or increased cell death, where DNA would be found in the extracellular environment, hBD3 may increase uptake into immune cells and could induce an increased immune response. Since increased hBD3 expression is induced by inflammatory stimuli, this process would cause a positive feedback loop of inflammation during bacterial infections. In conclusion, hBD3’s role in regulating the innate immune response to DNA is at the ligand-receptor level rather than affecting signalling pathways. Furthermore, hBD3 promotes the innate immune response to bacterial DNA by increasing the efficiency of cellular uptake possibly by inducing DNA aggregation. These results implicate a possible role for hBD3 in the earliest stages of psoriatic plaque development, which is often initiated or exacerbated by an infection, and this could be investigated further. Secondly, I investigated the innate immune function of an E3 ubiquitin ligase (E3L) not previously associated with human disease. Mutations in E3L have been identified in three microcephalic primordial dwarfism families; these patients also presented with recurrent respiratory illnesses. E3L has been implicated in the regulation of the innate immune system via interactions with signalling pathways downstream of the receptor, though its role is not clear. We hypothesised that E3L had a dual role both in regulating growth and cell division and in regulating the immune system. Primary patient fibroblasts did not demonstrate an altered cytokine response to bacterial or viral ligands, implying that E3L may have a specific function in immune cells. To investigate this further, and to provide a system to study E3L in vivo, two transgenic mouse lines were designed and engineered, firstly a conditional ‘knock-out’ designed to replicate some of the alternative isoforms of E3L seen in RT-PCRs, and secondly a ‘knock-in’ line to recapitulate the human mutation in exon 7 of E3L, R185X. These mouse lines should offer an insight into the developmental role for E3L, and contribute to establishing a potential role for E3L in the innate immune system. This thesis exemplifies the complexity of the innate immune system and the regulatory pathways that interact to maintain a delicate homeostasis preventing pathogenic inflammation. Understanding these regulatory mechanisms may shed light on the pathogenicity of diseases and identification of potential targets for therapeutics.