Glioblastoma is the most common primary brain tumor in humans and has a poor prognosis. Current therapies are not curative. Oncolytic viruses (OVs) are being investigated as tools to induce immunogenic cell death (ICD), cell death capable of activating the immune system. Semliki Forest Virus (SFV) strain 4 is an OV being investigated to treat glioblastoma. Previous studies in our lab have shown that SFV4 can induce ICD in human osteosarcoma (HOS) cells and ongoing in vivo studies show that SFV4 infected GL261 cell vaccination providesprotective immunity in mouse models. This study aimed to characterize the ICD induced by SFV4in glioblastoma cell lines, namely GL261, SB28 and CT2A, and to explain some of our in vivoobservations, namely why vaccination with SFV infected GL261 provides protective immunity but vaccination with infected CT2A and SB28 does not. Our in vitro studies found that GL261 is resistant to SFV4 while SB28 and CT2A are susceptible. We show that the virus can replicate in all three cell lines as seen by the presence of dsRNA, but that viral translation is delayed or inhibited in GL261 cells as not all cells positive for dsRNA were positive for SFV4 protein. Additionally, the type I interferon (IFN) pathway, responsible for antiviral defense, was highly upregulated in CT2A and SB28 but not as much in GL261 after infectionas seen by surveying IFIT1, IFITM3 and IFN-beta genes. Interferon stimulated genes (ISG) like CXCL10, a chemoattractant, was highly upregulated in GL261 after infection and might account for the protective immunity seen in vivo after vaccination. PDL1, an interferon stimulated gene responsible for self-tolerance, was highly upregulated in CT2A after infection. The IFN-beta ELISA revealed that both infected and uninfected GL261 cells produce IFN-beta suggesting a constitutively active pathway. Our DC phagocytosis assay showed that SFV4 infection of CT2A and SB28 cells induced a significant increase in DC phagocytosis and SFV4 infection of all three cell lines significantly increased DC maturation. We conclude that SFV4 infection of GL261 cells may induce ICD in vivo through a persistent viral infection and increased expression of CXCL10.

Le premier objectif a été de sélectionner des promoteurs de gènes cellulaires actifs spécifiquement dans les HCC à l’aide d’une recherche bibliographique puis en utilisant la base de donnée UniGene. Leur activité a été vérifiée par RT-qPCR et CHIP dans des lignées modèles HCC et dans des hépatocytes. Ces promoteurs ont été clonés en amont de la luciférase dans la région intergénique 20 du génome HSV-1 afin d’étudier leur force d’activité, 2 types de cinétiques et leur activité différentielle en fonction du type cellulaire et dans le contexte d’une infection virale. Le deuxième objectif a été de construire des virus oncolytiques ciblés pour l’expression de la protéine Us3, une protéine virale impliquée dans le contrôle de la réponse apoptotique induite par HSV-1. L’expression de la protéine Us3 est placée sous contrôle d’un promoteur cellulaire spécifique d’HCC. L’hypothèse est qu’en l’absence d’activité du promoteur cellulaire dans les cellules non HCC, la protéine Us3 ne sera pas synthétisée et, par conséquent, l’apoptose qui ne sera pas réprimée, inhibera le cycle de réplication et par conséquent, la production virale dans les cellules saines. Dans les cellules HCC, le promoteur actif permettra la réplication virale aboutissant à la destruction de lamasse tumorale. Un virus HSV-1 Us3- a été construit en utilisant la technique de recombinaison en plasmide BAC (Bacterial artificial chromosome), puis 2 virus oncolytiques en réintroduisant le gène Us3 sous contrôle du promoteur ANGPTL3 ou du promoteur HRE (hypoxia responsive element). Leur comportement oncolytique a été étudié en réalisant des courbes de croissance sur lignées cellulaires d’HCC et cellules hepatocyte-like. / Our long-term purpose is to develop transcriptionally targeted oncolytic vectors, derived from herpes simplex virus type 1 (HSV-1), designed to eradicate hepatocellular carcinomas (HCC). We have identified several HCC-specific promoters, as well as other cancer-specific promoters, that maintain their specificity when expressed from the virus genome. More precisely, we have demonstrated that these promoters are able to drive reporter gene (luciferase) expression from the virus genome in HCC-derived cells, both in cultured cells and in nude mice, but not in fresh human hepatocytes or in the WRL38 hepatocyte-like cells. HSV-1 infection induces, but then inhibits, a cellular antiviral apoptotic response, and the early virus protein US3 is a key actor in inhibiting apoptosis. We have hypothesized that inhibition of US3 expression in hepatocytes should led to early apoptotic death of these cells, therefore precluding virus multiplication and spread. In contrast, expression of US3 in cancer cells is expected to block apoptosis, leading to the achievement of the virus life cycle, cell lysis, and virus spread within the tumours. We report in this communication the construction and properties of two different potentially oncolytic HSV-1 vectors. One of them expresses US3 protein under the control of the HCC-specific promoter ANGPTL3, while the second promoter contains 9 repeats of the hypoxia responsive elements of vascular-endothelial growth factor (VEGF) (9xHRE promoter). Growth curves of these viruses were performed on different HCC cell lines to show their oncolytic properties.

No / Oncolytic viruses (OV) are promising treatments for cancer, with several currently undergoing testing in randomised clinical trials. Measles virus (MV) has not yet been tested in models of human melanoma. This study demonstrates the efficacy of MV against human melanoma. It is increasingly recognised that an essential component of therapy with OV is the recruitment of host antitumour immune responses, both innate and adaptive. MV-mediated melanoma cell death is an inflammatory process, causing the release of inflammatory cytokines including type-1 interferons and the potent danger signal HMGB1. Here, using human in vitro models, we demonstrate that MV enhances innate antitumour activity, and that MV-mediated melanoma cell death is capable of stimulating a melanoma-specific adaptive immune response.

Pancreatic cancer is characterized by its desmoplastic tumor microenvironment and the infiltration of immunosuppressive cells. It is a devastating disease where most patients are diagnosed at a late stage and the treatment options are few. The development of new treatments is surly needed. One treatment option explored is the use of immunotherapy with the intent to activate the immune system and change the balance from pro-tumor to anti-tumor. This thesis presents the idea of using oncolytic adenoviruses called LOAd-viruses that are armed with immunostimulatory- and microenvironment-modulating transgenes. For effective treatment of pancreatic cancer, the virus needs to be able to be given in addition to standard therapy, the chemotherapy gemcitabine. In paper I, the immunomodulatory effect of gemcitabine was evaluated in blood from pancreatic cancer patients receiving their first 28-day cycle of treatment with infusions day 1, 8 and 15 followed by a resting period. Gemcitabine reduced the level of immunosup-pressive cells and molecules but the effect did not last throughout the resting period. On the other hand, gemcitabine did not affect the level or proliferative function of effector T cells indicating that gemcitabine could be combined with immunotherapy. The LOAd700 virus expresses a novel membrane-bound trimerized form of CD40L (TMZ-CD40L). In paper II, LOAd700 showed to be oncolytic in pancreatic cancer cell lines as well as being immunostimulatory as shown by its capacity to activate dendritic cells (DCs), myeloid cells, endothelium, and to promote expansion of antigen-specific T cells. In paper III, LOAd703 armed with both 4-1BBL and TMZ-CD40L was evaluated. LOAd703 gave a more profound effect than LOAd700 on activation of DCs and the virus was also capable of reducing factors in stellate cells connected to the desmo-plastic and immunosuppressive microenvironment. In paper IV, LOAd713 armed with TMZ-CD40L in combination with a single-chain variable fragment against IL-6R was evaluated. The virus could kill pancreatic cancer cells lines through oncolysis and could also reduce factors involved in desmoplasia in stellate cells. Most interestingly, LOAd713 could reduce the up-regulation of PD-1/PD-L1 in DCs after CD40 activation. Taken together, LOAd703 and LOAd713 seem to have interesting features with their combination of immunostimulation and microenvironment modulation. At present, LOAd703 is evaluated in a clinical trial for pancreatic cancer (NCT02705196).