Cancer is one of the leading causes of death worldwide, with currently assessed chances to develop at least one cancer in a lifetime for about 20%. High cases rates and mortality require the development of new anticancer therapies and treatment strategies. Another important concern is toxicity normally associated with conventional therapy methods, such as chemo- and radiotherapy. Among many proposed antitumoral agents, oncolytic viruses are still one of the promising and fast-developing fields of research with almost a hundred studies published data on over 3000 patients since the beginning of the new millennia. Among all oncolytic viruses, the Vaccinia virus is arguably one of the safest, with an extremely long and prominent history of use, since it was the one and only vaccine used in the Smallpox Eradication Program in the 1970s. Interestingly enough, it was the first oncolytic virus proven to have tumor tropism in vitro and in vivo in laboratory settings, and this year we can celebrate an unofficial 100th anniversary since the publication of the fact. While being highly immunogenic, Vaccinia virus DNA replication takes place in the cytoplasm of the infected cell, and virus genes never integrate into the host genome. Another advantage of using Vaccinia as an oncolytic agent is its high genome capacity, which allows inserting up to 25 kbps of exogenous genes, thus allowing to additionally arm the virus against the tumor. Oncolytic virus action consists of two major parts: direct oncolysis and immune activation against the tumor, with the latter being the key to successful treatment. To this moment, preclinical research data are mostly generated in immunocompromised xenograft models, which have hurdles to be properly translated for clinical use. In the first part of the current study, fourteen different recombinant Vaccinia virus strains were tested in two different murine tumor cell lines and corresponding immunocompetent animal models. We found, that Copenhagen backbone Vaccinia viruses while being extremely effective in cell culture, do not show significant oncolytic efficacy in animals. In contrast, several of the LIVP backbone viruses tested (specifically, IL-2 expressing ones) have little replication ability when compared to the Copenhagen strain, but are able to significantly delay tumor growth and prolong survival of the treated animals. We have also noted cytokine related toxicity of the animals to be mouse strain specific. We have also tested the virus with the highest therapeutic benefit in combination with romidepsin and cyclophosphamide. While the combination with histone deacetylase inhibitor romidepsin did not result in therapeutic benefit in our settings, the addition of cyclophosphamide significantly improved the efficacy of the treatment, at the same time reducing cytokine-associated toxicity of the IL-2 expressing virus. In the second part of the work, we analyzed the ability of adipose-derived mesenchymal stem cells to serve as a carrier for the oncolytic Vaccinia virus. We showed for the first time that the cells can be infected with the virus and can generate virus progeny. They are also able to survive for a substantially long time and, when injected into the bloodstream of tumor-bearing animals, produce the virus that is colonizing the tumor. Analysis of the systemic distribution of the cells after injection revealed that infected and uninfected cells are not distributed in the same manner, possibly suggesting that infected cells are getting recognized and cleared by an impaired immune system of athymic mice faster than non-infected cells. Despite this, injection of virus-loaded adipose-derived mesenchymal stem cells to human A549 tumor-bearing xenograft mice resulted in rapid tumor regression and reduced virus-related side effects of the treatment when compared to injection of the naked virus. In conclusion, we have tested two different approaches to augmenting oncolytic Vaccinia virus therapy. First, the combination of recombinant Vaccinia virus expressing IL-2 and cyclophosphamide showed promising results in a syngeneic mouse model, despite the low permissivity of murine cells to the virus. Second, we loaded the oncolytic Vaccinia virus into mesenchymal stem cells and have proven that they can potentially serve as a vehicle for the virus. / Krebs ist eine der häufigsten Todesursachen weltweit, wobei die Wahrscheinlichkeit, im Laufe des Lebens an mindestens einer Krebsart zu erkranken, derzeit auf etwa 20 % geschätzt wird. Die hohen Fallzahlen und die hohe Sterblichkeit erfordern die Entwicklung neuer Krebstherapien und Behandlungsstrategien. Ein weiteres wichtiges Problem ist die Toxizität, die normalerweise mit konventionellen Behandlungsmethoden, wie Chemo- und Strahlentherapie, einhergeht. Unter den vielen vorgeschlagenen antitumoralen Wirkstoffen sind onkolytische Viren nach wie vor eines der vielversprechendsten und sich schnell entwickelnden Forschungsgebiete mit fast hundert veröffentlichten Studien an über 3000 Patienten seit Beginn des neuen Jahrtausends. Unter allen onkolytischen Viren ist das Vaccinia Virus wohl eines der Sichersten und hat eine extrem lange und prominente Anwendungsgeschichte, da es der einzige Impfstoff war, der im Pockenausrottungsprogramm in den 1970er Jahren verwendet wurde. Interessanterweise war es das erste onkolytische Virus, dessen Tumortropismus in vitro und in vivo im Labor nachgewiesen wurde. In diesem Jahr (2022) können wir das inoffizielle 100-jährige Jubiläum seit der Veröffentlichung dieser Tatsache feiern. Obwohl Vaccinia hoch immunogen ist, findet die Replikation im Zytoplasma der infizierten Zelle statt, und die Virusgene werden niemals in das menschliche Genom integriert. Ein weiterer Vorteil der Verwendung von Vaccinia als onkolytisches Agens ist seine hohe Genomkapazität, die es ermöglicht, bis zu 25 kbit/s an exogenen Genen einzufügen, wodurch das Virus zusätzlich gegen den Tumor aufgerüstet werden kann. Die Wirkung des onkolytischen Virus besteht aus zwei Hauptbestandteilen: der direkten Onkolyse und die Aktivierung des Immunsystems gegen den Tumor, wobei letztere der Schlüssel zum Behandlungserfolg ist. Bislang wurden präklinische Forschungsdaten meist in immungeschwächten Xenotransplantationsmodellen gewonnen, die sich nur schwer für den klinischen Einsatz eignen. Im ersten Teil der aktuellen Studie wurden vierzehn verschiedene rekombinante Vaccinia-Virusstämme in zwei verschiedenen murinen Tumorzelllinien und in entsprechenden immunkompetenten Tiermodellen getestet. Wir fanden heraus, dass Kopenhagener Backbone-Vaccinia-Viren zwar in der Zellkultur äußerst wirksam sind, im Tiermodell jedoch keine signifikante onkolytische Wirksamkeit zeigen. Im Gegensatz dazu haben mehrere der getesteten LIVP-Backbone-Viren (insbesondere die IL-2 exprimierenden) im Vergleich zum Kopenhagener Stamm nur eine geringe Replikationsfähigkeit, sind aber in der Lage, das Tumorwachstum deutlich zu verzögern und das Überleben der behandelten Tiere zu verlängern. Wir haben auch festgestellt, dass die Zytokin-bedingte Toxizität der Tiere mausstammspezifisch ist. Wir haben auch das Virus mit dem höchsten therapeutischen Nutzen in Kombination mit Romidepsin und Cyclophosphamid getestet. Während die Kombination mit dem Histon-Deacetylase-Inhibitor Romidepsin in unseren Versuchsreihen keinen therapeutischen Nutzen erbrachte, verbesserte die Zugabe von Cyclophosphamid die Wirksamkeit der Behandlung erheblich und verringerte gleichzeitig die zytokinbedingte Toxizität des IL-2-exprimierenden Virus. Im zweiten Teil der Arbeit analysierten wir die Fähigkeit von aus Fettgewebe gewonnenen mesenchymalen Stammzellen, als Träger für das onkolytische Vaccinia-Virus zu dienen. Wir konnten zum ersten Mal zeigen, dass die Zellen mit dem Virus infiziert werden können und Virusnachkommen erzeugen können. Sie sind auch in der Lage, sehr lange zu überleben und, wenn sie in den Blutkreislauf von Tieren mit Tumoren injiziert werden, das Virus zu produzieren, das den Tumor besiedelt. Die Analyse der systemischen Verteilung der Zellen nach der Injektion ergab, dass infizierte und nicht infizierte Zellen nicht auf die gleiche Weise verteilt werden, was möglicherweise darauf hindeutet, dass infizierte Zellen von einem beeinträchtigten Immunsystem der athymischen Mäuse schneller erkannt und beseitigt werden, als nicht infizierte Zellen. Trotzdem führte die Injektion von virusbeladenen mesenchymalen Stammzellen aus Fettgewebe in A549-Tumor-tragende Xenograft-Mäuse zu einer schnellen Tumorregression und zu geringeren virusbedingten Nebenwirkungen der Behandlung, als bei der Injektion des nackten Virus. Zusammenfassend lässt sich sagen, dass wir zwei verschiedene Ansätze zur Verstärkung der onkolytischen Vaccinia-Virus-Therapie getestet haben. Erstens zeigte die Kombination aus rekombinantem Vaccinia-Virus, das IL-2 exprimiert, und Cyclophosphamid in einem syngenen Mausmodell vielversprechende Ergebnisse, trotz der geringen Permissivität der Mäusezellen für das Virus. Zweitens haben wir onkolytische Vaccinia-Viren in mesenchymale Stammzellen eingebracht und nachgewiesen, dass diese als Vehikel für das Virus dienen können.

Indiana University-Purdue University Indianapolis (IUPUI) / Vaccinia virus (VV) has been used as a vaccine, yet safety concerns remain due to its viral immunoevasive properties. Among these, VV infection of antigen presentation cells (APC) perturbs MHC class II-mediated antigen (Ag) presentation. The goals of this project include: 1) to define mechanisms by which VV disrupts class II presentation; and 2) to examine whether disruption of the class II pathway by VV alters T cell responses in vitro and in vivo. A significant reduction in the expression of the class II chaperone, invariant chain (Ii), was observed during the late stage of VV infection. Yet surface expression of MHC class II molecules was maintained along with cell viability. To examine whether VV acts solely to disrupt host protein synthesis, B cells were treated with an inhibitor of translation-cycloheximide (CHX). Like VV, CHX negatively regulated Ii protein expression and class II presentation. Ii proteolysis also contributed in part to reduce Ii expression in VV infected and CHX treated APC. Yet only VV infection altered lysosomal protease expression, potentially influencing Ii degradation. Over-expression or ectopic-expression of Ii partially protected cells from VV-induced class II dysfunction. These studies suggest VV destabilizes class II molecules by disrupting Ii expression. To examine the presentation of viral Ags by class II, CD4 T cells from VV-primed mice were used. Viral proteins were presented by class II shortly after APC exposure to low concentrations of VV. The presentation of VV Ags correlated temporally with reductions in exogenous peptide presentation. At higher MOI (≥ 1), class II presentation of VV Ags was reduced. To examine the in vivo effects of VV on Ag presentation, a mouse model of ovalbumin-induced airway hypersensitivity was used. Th2 cytokine production was reduced, while a novel inflammatory cytokine Interleukin-17 (IL-17) production was enhanced in asthmatic VV-infected mice. In health mice, repeated VV infections lead to enhanced CD8 T cell production of Interferon-γ (IFN-γ) and IL-17. Finally, antibodies to a viral protein H3 were generated and shown to preserve class II presentation. Together these studies suggest VV disruption of the class II pathway may blunt T cell responses to VV.

Viral appropriation of chemokine receptors is an effective way to prevent a host immune response against the invading virus. Many viruses, including poxviruses, subvert the host immune response by encoding several chemokine receptor homologues, capable of binding to and thereby precluding chemokines from activating their cognate cell surface receptors. All poxviruses employ strategies to modulate chemokine activity, including virus-encoded chemokine-binding proteins, receptor homologues and ligand mimics. The potential for the involvement of certain chemokine receptors in poxviral infection was suggested in studies utilizing the rabbit poxvirus, myxoma. Specifically, CCR5 was implicated in mediating cell target susceptibility to infection. Our data suggest virus-CCR5 interactions may lead to the selective activation of distinct signaling pathways that are advantageous for the virus. VACV, a member of the poxvirus family, produces two structurally distinct forms of virions, the intracellular mature virus (IMV) and the extracellular enveloped virus (EEV), for which the immediate events following cell entry are ill-defined. Using confocal microscopy, we provided evidence that IMV and EEV enter both permissive and non-permissive cells, and that introduction of CCR5 into non-permissive cells – mouse fibroblasts and human PM1 T cells - renders them permissive for VACV replication. We showed that virus activation of CCR5 leads to the selective activation of distinct signaling pathways that are advantageous for the virus. We demonstrated that VACV infection in permissive cells is inhibited by siRNA knockdown of cell surface CCR5 expression and by the CCR5 antagonist, TAK-779. The importance of tyrosine phosphorylation of CCR5 was suggested by the observation that introduction of a CCR5 mutant, in which all the intracellular tyrosines are replaced by phenylalanines, effectively reduces VACV infection in permissive cells. Moreover, tyrosine-339 was implicated in CCR5 as the critical residue for mediating viral infection, since cells expressing CCR5.Y339F do not support viral replication. The cascade of events that leads to permissive phenotype of these cells includes phosphorylation activation of multiple signaling effectors: Jak-2, IRS-2, ERK1/ 2 and Grb2. These data were supported by findings that viral replication in permissive CCR5 expressing cells is blocked by Herbimycin A, and the Jak2 inhibitor, tyrophostin AG490, but not pertussis toxin. Viewed altogether, a critical role of post-entry events, specifically intracellular tyrosine phosphorylation events, was established in determining permissiveness of cells to VACV replication. Furthermore, evidence was provided that introduction of CCR5 in primary human T cells renders them permissive to VACV replication. Since permissive infection of T cells might represent a mechanism for VACV dissemination throughout the lymphatic system, we hypothesized that the absence of CCR5 may be protective against VACV infection in vivo. To test this hypothesis, wild-type and CCR5 null mice were challenged with VACV by intranasal inoculation. In time course studies we identified aggressive viral replication in the lungs and spleens of CCR5+/+ mice, with no evidence of infection in the CCR5-/- mice. Moreover, associated with VACV infection, we provided evidence for CD4+ and CD8+ T as well as CD11c+ and F4/80+ cell infiltration into the lungs of CCR5+/+ but not CCR5-/- mice, and showed that CCR5-expressing T cells harbor replicating virus. We showed that this CCR5-dependence is VACV-specific, since CCR5-/- mice were as susceptible to intranasal influenza (A/WSN/33) infection as CCR5+/+ mice. In a final series of experiments we provided evidence that adoptive transfer of CCR5+/+ bone marrow into CCR5-/- mice restored VACV permissiveness, with evidence of lung and spleen infection. Taken together, our data showed a critical and novel role for CCR5 in VACV infection and dissemination in vivo. Moreover, our confocal studies suggested a possible physical interaction between cellular proteins and the VACV in cytosole. Using mass spectrometry-based proteomics, glomulin was identified as a host cell protein that interacts with VACV. Knockdown of glomulin expression in human PM1.CCR5 T cells reduced VACV infection. We demonstrate that treatment of PM1.CCR5 T cells with a c-Met phosphorylation inhibitor led to a significant reduction in VACV infectivity. The data indicated that inhibition of c-Met phosphorylation, reduces the cytosolic availability of activated glomulin, thus leading to a decrease in VACV infectivity. These data identify glomulin as a permissivity factor for VACV infection, and as a potential therapeutic target for VACV.

<p>Natural killer (NK) cells are lymphocytes of the innate immune response with well-demonstrated activities against viral infections and tumors. Because of these abilities, we sought to glean insights into the mechanisms of NK cell activation so that they may be applied toward the design of new therapies.</p><p>NK cells are particularly critical for the control of poxviral infections. Vaccinia virus (VV) is the most-studied member of the poxviral family. It is robustly immunogenic and functions as the live vaccine responsible for the successful elimination of smallpox. VV infection provides a useful model for studying NK cell activation: NK cells play an important role in its clearance and the virus efficiently activates NK cells and recruits them to the site of infection. We had previously used this model to identify Toll-like receptor (TLR)-dependent and -independent mechanisms of NK cell activation to VV. One method of TLR-independent activation to VV requires the activation receptor NKG2D, which recognizes host ligands expressed upon viral infection by accessory cells such as dendritic cells (DCs) and macrophages.</p><p>In the first aim of this thesis, we sought to determine how the ligands for the NKG2D activation receptor become upregulated in the context of VV infection. Specifically, we asked whether interleukin-18 (IL-18), known to play a role in the innate immune response, could boost the expression of NKG2D ligands on DCs in response to viral infection. Using an in vivo infection model with IL-18R-deficient mice, our results confirmed an important role for IL-18 in NK cell activation to VV and viral control. We then made use of an NK-DC co-culture to show that IL-18 signaling on DCs, in addition to NK cells, is necessary to achieve efficient NK cell activation to viral infection. We further demonstrated in a cell-transfer experiment that cell-extrinsic IL-18 signaling is critical for NK cell activation in vivo. DC ablation via a mouse model designed to specifically ablate CD11c+ cells showed that DCs are also required for NK cell activation to VV in vivo. We finally showed how IL-18 can act on DCs in vivo and in vitro to boost the expression of Rae-1, an NKG2D ligand. Collectively, our data uncover a novel mechanism whereby NK cells become activated by IL-18 control of NKG2D ligand expression on DCs.</p><p>In the second aim of this project, we detailed how IL-18 signaling results in the upregulation of the NKG2D ligand Rae-1. Using an in vitro macrophage model, we showed how recombinant IL-18 was sufficient to upregulate Rae-1 expression. We compared IL-18 control of Rae-1 expression to LPS, a TLR ligand that also signals through the common adaptor MyD88 to govern Rae-1 expression. Using chemical inhibitors to cell signaling molecules, we then identified the importance of MyD88 signaling through PI3K. We then revealed that glycogen synthase kinase 3 (GSK-3) can act as a negative regulator of Rae-1 expression downstream of IL-18/TLR signaling. Specifically, we have shown that during inflammatory signaling, PI3K (acting downstream of MyD88) can inhibit GSK-3 to relieve its tonic suppression of Rae-1 expression and upregulate the NKG2D ligand. Finally, we showed that PI3K and GSK-3 signaling are also important to Rae-1 expression on DCs - the accessory cell where IL-18 signals to control Rae-1 expression to boost NK cell activation against VV.</p><p>In its entirety, this work seeks to address how NK cells become activated in the context of VV infection in order to identify new ways NK cells may be harnessed therapeutically.</p> / Dissertation

Vaccinia virus (VACV) is a large double-stranded DNA virus with a cytoplasmic site of replication. It has a complex life cycle that produces two distinct infectious virion forms, Intracellular Mature Virions (IMVs) and Extracellular Enveloped Virions (EEVs). The host cell microtubule trafficking machinery is hijacked by the virus at three distinct positions of the viral life cycle. After virus entry, the virus cores are transported to pre-nuclear sites where they form viral factories that ultimately produce fully functional and infectious IMVs. A small proportion of IMVs are further transported to sites of wrapping, where they are enveloped by a host-derived double membrane to form Intracellular Enveloped Virions (IEVs). The IEVs are then transported to the cell periphery to facilitate efficient viral spread. The viral proteins A36, F12 and E2 together with the kinesin-1 microtubule motor protein are thought to be involved in IEV egress from the site of wrapping to the cell periphery, although the exact mechanism of movement is unclear. Until recently, A36 was the only known protein to interact with the kinesin-1 motor through kinesin light chain (KLC), but F12 has also been shown to interact with KLC through E2. The precise mechanism of how the IEV interacts with and activates the kinesin-1 motor protein is unclear, and this study explores the interactions of IEV proteins with KLCs in detail, mapping interactions between KLC and A36 or F12/E2. A36, F12 and E2 also show no sequence or predicted structural homology to any other known proteins, and structural studies were performed in an attempt solve their 3D structure. The CRISPR-Cas9 targeted genome editing tool was also utilised to knockout different KLC isoforms in multiple cell lines to assess their contribution to IEV egress as well as cellular trafficking. These studies will provide insight into the mechanisms behind the spatial and temporal control of kinesin motor activity in the cell.

Oncolytic viral therapies have shown great promise pre-clinically and in human clinical trials for the treatment of various cancers. Oncolytic viruses selectively infect and replicate in cancer cells, destroying tumor tissue via cell lysis, while leaving noncancerous tissues unharmed. Vaccinia virus (VACV) is arguably one of the safest viruses, which has been intensively studied in molecular biology and pathogenesis as a vaccine for the eradication of smallpox in more than 200 million people. It has fast and efficient replication, and cytoplasmic replication of the virus lessens the chance of recombination or integration of viral DNA into the genome of host cells. Anti-tumor therapeutic efficacy of VACV has been demonstrated for human cancers in xenograft models with a variety of tumor types. In addition recombinant oncolytic VACVs carrying imaging genes represent an advance in treatment strategy that combines tumor-specific therapeutics as well as diagnostics. As for other targeted therapies, a number of challenges remain for the clinical translation of oncolytic virotherapy. These challenges include the potential safety risk of replication of oncolytic virus in non-tumor tissue, the relatively poor virus spread throughout solid tumor tissue and the disadvantageous ratio between anti-viral and anti-tumoral immunity. However, manipulation of components of the tumor microenvironment may help oncolytic virus infection in killing the tumor tissue and thereby increasing the anti-tumor efficacy. Furthermore, dogs with natural cancer are considered as one of the best animal models to develop new drugs for cancer therapy. Traditionally, rodent cancer models have been used for development of cancer therapeutics. However, they do not adequately represent several features that define cancer in humans, including biology of initiation of tumor, the complexity of cancer recurrence and metastasis and outcomes to novel therapies. However, the tumor microenvironment, histopathology, molecular and genomics data from dog tumors has significant similarities with corresponding human tumors. These advantages of pet dog cancers provide a unique opportunity to integrate canine cancer patients in the studies designed for the development of new cancer drugs targeted against both human and canine cancers. This dissertation centers on the use of VACV strains in canine cancer xenografts with the aim of understanding the effects of modulation of tumor microenvironment on VACV-mediated tumor therapy. In the first studies, wild-type VACV strain LIVP6.1.1 was tested for its oncolytic efficiency in canine soft tissue sarcoma (STSA-1) and canine prostate carcinoma (DT08/40) cells in culture and xenografts models. LIVP6.1.1 infected, replicated within, and killed both STSA-1 and DT08/40 cells in cell culture. The replication of virus was more efficient in STSA-1 cells compared to DT08/40 cells. In xenograft mouse models, LIVP6.1.1 was safe and effective in regressing both STSA-1 and DT08/40 xenografts. However, tumor regression was faster in STSA-1 xenografts compared to DT08/40 xenografts presumably due to more efficient replication of virus in STSA-1 cells. Biodistribution profiles revealed persistence of virus in tumors 5 and 7 weeks post virus injection in STSA-1 and DT08/40 xenografts, respectively, with the virus mainly cleared from all other major organs. Immunofluorescence staining detected successful colonization of VACV in the tumor. Consequently, LIVP6.1.1 colonization in the tumor showed infiltration of innate immune cells mainly granulocytes and macrophages in STSA-1 tumor xenografts. These findings suggest that virotherapy-mediated anti-tumor mechanism in xenografts could be a combination of direct viral oncolysis of tumor cells and virus-dependent infiltration of tumor-associated host immune cells. In further studies, the effects of modulation of tumor angiogenesis of VACV therapy were analyzed in canine cancer xenografts. GLV-1h109 VACV strain (derived from prototype virus GLV-1h68) encoding the anti-VEGF single chain antibody GLAF-1 was characterized for its oncolytic efficacy in STSA-1 and DT08/40 cancer cells in culture and tumor xenografts. Concomitantly, the effects of locally expressed GLAF- 1 in tumors on virus replication, host immune infiltration, tumor vascularization and tumor growth were also evaluated. GLV-1h109 was shown to be similar to the parental virus GLV-1h68 in expression of the two marker genes that both virus strains have in common (Ruc-GFP and gusA) in cell cultures. Additionally, the anti-VEGF single-chain antibody GLAF-1 was expressed by GLV-1h109 in both cell cultures and tumor xenografts. The insertion of GLAF-1 did not significantly affect the replication and cytotoxicity of GLV-1h109 in the STSA-1 and DT08/40 cell lines, although at early time points (24-48 hpi), the replication of GLV-1h109 was higher in STSA-1 cells compared to DT08/40 cells. In addition, STSA-1 cells were more susceptible to lysis with GLV-1h109 than DT08/40 cells. GLV-1h109 achieved a significant inhibition of tumor growth in both STSA-1 and DT08/40 canine xenografts models. Consequently, the significant regression of tumor growth was initiated earlier in STSA-1 tumor xenografts compared to regression in DT08/40 xenografts. The reason for the higher efficacy of GLV-1h109 in STSA-1 xenografts than DT08/40 xenografts was attributed to more efficient replication of virus in STSA-1 cells. In addition, tumor-specific virus infection led to a continued presence of GLAF-1 in peripheral blood, which could be useful as a pharmacokinetic marker to monitor virus colonization and persistence in GLV-1h109- injected xenograft mice. GLAF-1 is a single-chain antibody targeting human and murine VEGF. It was demonstrated that GLAF-1 was functional and recognized both canine and human VEGF with equal efficiency. Histological analysis of tumor sections 7 days after GLV-1h109 injection confirmed that colonization of VACV and intratumoral expression of GLAF-1 translated into a significant decrease in blood vessel number compared to GLV-1h68 or PBS-treated control tumors. Subsequently, reduction in blood vessel density significantly improved the spread and replication of VACV as observed by FACS analysis and standard plaque assay, respectively. Inhibition of tumor angiogenesis and increased replication of virus further improved the infiltration of innate immune cells mainly granulocytes and macrophages in STSA-1 tumor xenografts. Both the results, i.e. improved virus spread and increased infiltration of innate immune cells in tumor, were explained by a phenomenon called “vascular normalization”, where anti-VEGF therapy normalizes the heterogeneous tumor vasculature thereby improving delivery and spread of VACV. In summary, the effects of inhibition of tumor angiogenesis on virus spread and replication were demonstrated using a vaccinia virus caring an anti- angiogenic payload targeting vascular endothelial growth factor (VEGF) in canine cancer xenografts. In the final studies, the effects of VACV therapy on modulation of the immune system were analyzed in canine cancer patients enrolled in a phase I clinical trial. V-VET1 (clinical grade LIVP6.1.1 VACV) injection significantly increased the percentages of CD3+CD8+ T lymphocytes at 21 days after initiation of treatment. CD3+CD8+ T lymphocytes are mainly cytotoxic T lymphocytes that have potential to lyse cancer cells. Subsequently, the frequency of immune suppressor cells, mainly MDSCs and Treg was also analyzed in peripheral blood of canine cancer patients. Increase in the MDSC population and decreased CD8/Treg ratio is known to have inhibitory effects on the functions of cytotoxic T cells. We demonstrated that injection of V-VET1 in canine cancer patients significantly reduced the percentages of MDSCs at 21 days post initiation of treatment. Additionally, CD8/Treg ratio was increased 21 days after initiation of V-VET1 treatment. We also showed that changes in the frequency of immune cells neither depends on dose of virus nor depends on tumor type according to the data observed from this clinical trial with eleven analyzed patients. This preclinical and clinical data have important clinical implications of how VACV therapy can be used for the treatment of canine cancers. Moreover, dogs with natural cancers can be used as an ideal animal model to improve the oncolytic virotherapy for human cancers. Furthermore, modulation of tumor microenvironment mainly tumor angiogenesis and tumor immunity has significant impact on the success of oncolytic virotherapy. / Therapien für verschiedenste Krebsarten mittels onkolytischer Viren zeigten sowohl in präklinischen- als auch in humanen klinischen Studien ein erfolgversprechendes Potenzial. Onkolytische Viren infizieren selektiv Krebszellen und replizieren ausschließlich in diesen. In der Folge zerstören sie Tumorgewebe durch Zelllyse, während gesundes Gewebe unbeeinträchtigt bleibt. Das Vaccinia-Virus besitzt ein äußerst geringes Risikopotential, und wurde intensiv auf molekularbiologischer Ebene und in Bezug auf seine Pathogenese untersucht. All das qualifizierte es als Vakzin zur Ausrottung der Pocken und seit Markteinführung mehr als 200 Millionen Menschen injiziert. Das Vaccinia-Virus zeigt eine schnelle und effiziente Replikation, welche im Zytoplasma der Zelle stattfindet. Dies verringert die Möglichkeit der Rekombination oder Integration der viralen DNA in das Wirtsgenom. Die therapeutische Wirksamkeit onkolytischer Vaccinia-Viren (VACVs) wurde in humanen Xenograft-Mausmodellen mit unterschiedlichen Tumorarten gezeigt. Rekombinante onkolytische VACVs, welche mit fluoreszierenden Genen ausgestattet sind, kombinieren die Vorteile tumorspezifischer Therapeutika und dienen gleichzeitig als Diagnostika. Wie auch andere spezifische Therapien, steht auch die onkolytische Virustherapie vor einer Reihe von Herausforderungen. Dazu gehören die Replikation onkolytischer Viren in nicht-kanzerogenem Gewebe, relativ schlechte Virusverbreitung durch solides Tumorgewebe und ein unvorteilhaftes Verhältnis zwischen antiviraler und antitumoraler Immunität. Die gezielte Manipulation einzelner Komponenten des Tumormikromilieus kann jedoch zu einer verbesserten Virusinfektion und Lyse des Tumorgewebes führen und somit die Effizienz der antitumoralen Therapie verstärken. ...

Die Familie der Pockenviren zeichnet sich durch ein komplexes DNA Genom aus und hat großes medizinisches Potential. Am eindrucksvollsten ist dies für das Vaccinia-Virus (VACV) belegt, welches nicht nur als Pocken-Impfstoff eingesetzt wird, sondern auch als onkolytisches Virus in der Tumorbiologie. VACV hat einen außergewöhnlichen Replikationszyklus, welcher ausschließlich im Zytoplasma der Wirtszelle stattfindet. Somit ist die gesamte virale Genexpressionsmaschinerie völlig unabhängig von kernvermittelten Reaktionen des Wirts und somit auch aus Sicht der Grundlagenforschung von größtem Interesse. Die Schlüsselkomponente der viralen Genexpression ist die makromolekulare DNA-abhängige RNA Polymerase (vvRPO), deren Untereinheiten allesamt Virus-kodiert sind. Zwar wurden in den letzten Jahren Protokolle zur biochemischen und funktionellen Charakterisierung der vvRPO etabliert, ein detailliertes Wissen über deren Zusammenlagerung in vivo und die räumlichen und zeitlichen Interaktionen mit den Transkriptions- bzw. Prozessierungsfaktoren sind aber weitgehend unbekannt. Diese Arbeit umfasst Untersuchungen zur strukturellen und funktionellen Charakterisierung der vvRPO und seiner assoziierten Faktoren. Grundlage hierfür war die Etablierung eines Reinigungsprotokolls mithilfe eines neu konstruierten rekombinanten VACV (GLV-1h439). Diese Strategie erlaubte es hoch-molekulare native vvRPO Komplexe zu isolieren. Ein transkriptions-inaktiver Komplex (Komplex I) mit einer kalkulierten Masse von 575 kDa bestand aus den acht Untereinheiten des vvRPO Holoenzyms und den Polymerase-assoziierten Faktoren RAP94 und D6. Ein zweiter, transkriptionell aktiver Komplex (Komplex II) mit einer Masse von 803 kDa enthielt, neben dem Holoenzym der vvRPO, noch weitere Faktoren, die primär die Erkennung der DNA-Matrize und die Prozessierung der naszierenden RNA vermitteln. Hierbei handelt es sich um RAP94, das virale Capping Enzym bestehend aus den zwei Untereinheiten D1 und D12, A7 und dem Terminationsfaktor NPH I. Interessanterweise enthielt dieser Komplex zusätzlich mit E11 eine bislang unbekannte weitere Protein-Komponente, sowie tRNAGln und tRNAArg. Der isolierte Kompelx II ist daher ein Ribonukleoprotein (RNP). Die Verfügbarkeit von hoch-reinen vvRPO Komplexen erlaubte es erstmals deren strukturelle Architektur zu untersuchen. Hierfür wurden drei experimentelle Ansätze, die klassische Röntgenstrukturanalyse, die Kryo-Elektronenmikroskopie (Kryo-EM) und Quervernetzungssstudien miteinander kombiniert. Die Strukturen der Komplexe I und II haben eine Auflösung von 11-12 Å, wobei auffällig war, dass beide eine markante strukturelle Ähnlichkeit zur eukaryotischen RNA Polymerase II aufwiesen. Darüber hinaus gelang es zusätzliche Bereiche im Komplex II zu definieren, welche die Polymerase-assoziierten Prozessierungsfaktoren beherbergen. Zudem konnte die atomare Struktur von E11, mittels Röntgenstrukturanalyse bei einer Auflösung von 1,9 Å, gelöst werden. Das E11 Protein besitzt ein neuartiges Faltungsmuster und weist einen intensiven Dimerisierungskontakt auf, welcher sich über vier ß-Faltblätter ausbildet. Die im Rahmen dieser Arbeit erhaltenen Daten legen die Grundlage für ein detailliertes Verständnis der räumlichen Organisation der viralen Transkriptonsmaschinerie. Darüber hinaus werden sie funktionelle Studien ermöglichen, welche die Rolle der einzelnen Proteine, sowie der tRNAs bei der mRNA Synthese klären helfen. / Poxviruses comprise a diverse family of complex DNA-genome viruses with great medical potenial. This is exemplified by vaccinia virus, which not only served as a vaccine against smallpox but is also used as a promising tool in viral anti-cancer therapies. A key feature that distinguishes the poxvirus family from other DNA viruses is their replication cycle, which is confined to the cytoplasm. This results in a high level of independence from the host cell, which supports transcription and replication events only in the nucleus. Accordingly, virus specific, rather than host cell enzymes mediate most processes including DNA replication and mRNA synthesis. The key component of viral gene expression is the DNA-dependent RNA polymerase (vvRPO), which constitutes the virus-encoded macromolecular machine ensuring viral mRNA synthesis. Although this enzyme has been studied in some details in the past years, neither its mode of assembly in vivo nor its spatio-temporal association with transcription and processing factors has been understood in detail. In this thesis I present work that focuses on the structural and functional characterization of vvRPO and its associated factors. To gain insights into the structure and the assembly of the VACV transcription system we established an efficient purification protocol by generating recombinant virus strains expressing tagged subunits of vvRPO (GLV-1h439). These recombinant virus strains enabled the isolation of high molecular weight vvRPO complexes. Complex I, which was transcriptionally inactive in vitro displayed a calculated mass of about 575 kDa, consisted of eight subunits of the vvRPO holoenzym and two additional polymerase-associated factors termed RAP94 and D6. A second, transcriptionally active complex (complex II) with a mass of 803 kDa, was related to the first one. It consisted apart from the factors of the holoenzyme already found in complex I additional factors that mediate primarily binding of the polymerase to its DNA template and the processing of nascent RNA. These factors comprise the viral capping enzyme (D1, D12), A7 and the termination factor NPH I. Interestingly, complex II contained in addition the viral protein E11, thus far not connected to viral transcription als well as tRNAGln, tRNAArg). Complex II is hence a ribonucleoprotein (RNP). The availability of highly pure vvRPO complexes allowed for the first time to investigate their structure. To this end, three experimental approaches, the classic X-ray crystallography, cryo-electron microscopy (cryo-EM) and chemical crosslinking were combined. Structures of both polymerase complexes were obtained at a resolution of 11-12 Å and revealed a striking structural similarity to eukaryotic RNA polymerase II. Moreover, it was possible to allocate positions in the structure of complex II that are likely to harbour the polymerase-associated processing factors. In addition we were able to solve atomic structure of E11 by X-ray crystallography at a resolution of 1.9 Å. Interestingly, the structure of E11 showed a novel folding pattern that forms a dimer, which is mostly composed of four ß-sheets. These studies provide the basis for a detailed investigation of the architecture of the viral transcriptional machinery. Furthermore, the pave the way for functional studies aimed at elucidating the function of individual proteins and tRNA in the generation of viral mRNA.

The classical Nuclear Factor kappa B (NF-B) signaling pathway is an important regulator of inflammation and innate immune responses. Poxviruses, including vaccinia virus, encode multiple immune evasion proteins, including a growing number of NF-B inhibitors. To determine if additional vaccinia virus gene products disrupted NF-B signaling, we utilized VV811, a mutant virus missing 55 open reading frames and devoid of the known inhibitors of TNF-induced NF-B activation. NF-B nuclear translocation was inhibited in VV811 infected cells stimulated with TNF. Furthermore, VV811 infection suppressed IB degradation and resulted in accumulation of phosphorylated IB in cells stimulated with TNF. Coimmunoprecipitation assays demonstrated that the inhibitory IB-p65-p50 complex was intact in VV811 infected cells, and, significantly, treatment with AraC revealed the involvement of late protein synthesis in stabilization of IB. This work indicates that unidentified inhibitors of NF-B exist in vaccinia virus and illustrates the importance of NF-B activation in the antiviral response. / Virology

Thesis (Ph.D.)--University of Alberta, 2010. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Virology, Medical Microbiology and Immunology. Title from pdf file main screen (viewed on January 10, 2010). Includes bibliographical references.

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