Influenza epidemics continue to cause morbidity and mortality within the human population despite widespread vaccination efforts. This, along with the ominous threat of an avian influenza pandemic (H5N1), demonstrates the need for a much improved, more sophisticated influenza vaccine. Our group has developed an in vitro model system for producing a membrane-bound Cytokine-bearing Influenza Vaccine (CYT-IVAC). Numerous cytokines are involved in directing both innate and adaptive immunity and it is our goal to utilize the properties of individual cytokines and other immunomodulatory proteins to create a more immunogenic vaccine. Here we report methodologies for the construction of membrane-bound cytokine fusion constructs in which our cytokine of interest (mouse GM-CSF, mouse IL-2, mouse IL-4) was fused to the membrane anchoring regions of viral Hemagglutinin (HA). Progeny virions, produced from influenza infected MDCK cells expressing membrane-bound cytokines, readily incorporated membrane-bound cytokines during budding and these cytokines on the virus particles retained bioactivity following viral inactivation. In vivo vaccination studies in mice showed enhanced antibody titers and improved protection following lethal challenge in those mice vaccinated with IL-2 and IL-4-bearing CYT-IVAC's compared to the conventional wild-type vaccine without membrane-bound cytokines. In addition, the immune response induced by IL-2 and IL-4-bearing CYT-IVACs was skewed toward Th1 (cellular) mediated immunity compared to the Th2 (humoral) dominated response induced with wild-type vaccination. Cellular mediated immunity afforded by IL-2 and IL-4 CYT-IVACs was manifested as enhanced influenza specific T cell proliferation and activation. In conclusion, we have developed a novel methodology to introduce bioactive membrane-bound cytokines directly into virus particles in order to augment the immunogenicity of inactivated, whole virus influenza vaccines. / Ph. D.

Through the years, cancer therapies have progressed rapidly, pouring out novel treatments such as gene therapy, small molecule therapies and immunotherapy. One such immunotherapy, adoptive cell transfer (ACT), augmented through the addition of a chimeric antigen receptor (CAR), has proven success in treatment of hematological malignancies. Additionally, oncolytic viruses (OV) and OV-based (OVV) therapies, have shown promising results in both clinical and pre-clinical studies. In most instances, when applied as a monotherapy, the aforementioned treatment methods are incapable of inducing complete tumour remission. The Wan lab has developed an approach combining ACT with OVV therapies that dramatically increase therapeutic benefit resulting in complete regression of well-established solid tumours. Despite promising results, certain tumours can still escape this combination therapy through antigen loss resulting in antigen negative relapse (ANR). To further augment the therapy, the addition of a secondary receptor (CAR) provides the ACT multiple avenues of attack to prevent ANR. In this dissertation, we define culture conditions that promote strong expression of the CAR alongside confirmation of function in an in vitro setting. Following, it is demonstrated that OVV boosted dual-targeting T cells carry strong T cell activity by measure of cytokine release in vivo. Despite promising T cell activity data, dual-specific T cells are unable to improve tumour control and survival once relapse occurs. The failure to control relapse remains unclear however evidence points towards lack of T cell persistence, poor CAR function in vivo and a lack of endogenous T cell response leading to compounding effects that prevent dual-targeting T cells from preventing ANR. Although dual specific therapies have shown poor efficacy in preventing ANR, further study must be completed to identify areas of improvement – such as persistence, as the potential for success in using dual-targeting T cells coupled with OVVs still lies untapped. / Thesis / Master of Science (MSc)

A major obstacle in the implementation of adoptive cell therapy (ACT) for solid tumors is CD8+ T cell quantity and functional quality. In order to address this issue, the ACT field has directed considerable effort toward the generation of less-differentiated memory T cells (Tm), which demonstrate superior effector function and engraftment over effector T cells. An obstacle in using Tm for ACT is their requirement for in vivo activation before full effector function can be acquired. We sought to determine if a rhabdovirus expressing a defined tumor antigen (i.e. a rhabdoviral oncolytic vaccine) could activate adoptively-transferred Tm in vivo and eliminate established tumors. We used ex vivo cultured DUC18 TCR-transgenic Tm combined with a rhabdoviral oncolytic vaccine to target established CMS5 fibrosarcomas in both balb/c and NRG mice, and we compared the efficacy of the combination treatment versus monotherapies. Our data demonstrate that the rhabdoviral oncolytic vaccine was capable of expanding adoptively-transferred Tm in order to eliminate established tumors. Furthermore, synergy between ACT and oncolytic vaccination was required for optimal therapeutic outcome. Interestingly, we observed a population of endogenous, tumor-primed lymphocytes which appeared to be required for complete tumor elimination and subsequent memory formation. This was in contrast to the current consensus in the ACT field which is that endogenous lymphocytes are detrimental to therapeutic outcome, thus necessitating lymphodepletion prior to the commencement of therapy. Our data suggest that endogenous lymphocytes may be a beneficial cell population which is overlooked by current approaches to ACT. / Thesis / Master of Science (MSc) / Current approaches to the T cell therapy of cancer are hindered by poor cell quality. It is simple to grow higher quality T cells, but it is difficult to grow very large numbers of them. Furthermore, higher quality T cells need a signal in order to “switch on” before they can start killing cancer cells. Here, we use a cancer-targeting virus as a signal for these cells to activate, grow to very large numbers in the patient, and destroy their tumor. Our vaccine also switches on other immune cells in the patient, which help guarantee the destruction of the tumor. The significance of this work is that it will improve T cell therapy for cancer by opening the possibility of using higher-quality T cells which are much better at killing cancer than the currently used type of T cells.