Anti-tumor Effects Evaluation And Mechanism Study Of Oncolytic HSV-1 Encoding CⅡTA In Tumor Immunotherapy

Objectives:Cancer remains one of the leading causes of death worldwide,and its development is caused by the activation of oncogenic drivers or mutations in onco-suppressor factors.With the further study of tumor therapy,more and more evidence show that the immune system disorder is closely related to the occurrence,progression and recurrence of tumor,which also stimulates people’s interest in the development of tumor immunotherapy.As a new immunotherapy method,oncolytic virus therapy directly lyses tumor cells in the form of viral infection and exposes tumor antigens in inflammatory environment to promote the formation of anti-tumor immune response.So far,a variety of oncolytic viruses represented by T-VEC have entered the stage of clinical research and achieved good application effects.However,it should not be ignored that the treatment of solid tumors often has poor immune response,which limits the efficacy of immunotherapy.In addition,after entering the host,the virus itself is suppressed until it is cleared,which leads to the weakening of the effect of tumor therapy.Therefore,in the process of tumor therapy,it is the key to correctly grasp the anti-tumor adaptive immune response caused by virus infection to exert the effect of oncolytic virus therapy.In order to solve this problem,this study proposed to construct a new type of immune-response-enhanced oncolytic virus（o HSV-1）using it as a carrier to induce effective local antigen presentation process of tumor,thus fully amplifying the anti-tumor immune response aroused by virus invasion into tumor.To provide a more effective oncolytic immunotherapy strategy for weak immune response tumors.Methods:In this study,we used CRISPR-Cas9 gene editing technique and homologous recombinational repair technique to design and construct immune-response-enhanced oncolytic virus OV-CⅡTA(ICP34.5^-/-ICP47^-CⅡTA⁺)expressing exogenous CⅡTA gene.First,we verified the successful construction of the recombinant strain by Western blot and in vitro functional studies.Subsequently,the ability of OV-CⅡTA to kill tumor lysis and the way of inducing cell death were preliminarily explored by in vitro killing activity detection and cell immune death detection.In addition,the ability of OV-CⅡTA to regulate tumor cells was macroscopically analyzed by whole transcriptome sequencing.Further,special tumor models such as mouse tumor syngeneic transplantation model,bilateral tumor challenge model and rechallenge tumor model were used to objectively evaluate the tumor therapeutic effect of OV-CⅡTA and the systemic immune activation ability of in situ tumor vaccine,and to explore the feasibility of combining OV-CⅡTA with immune checkpoint inhibitor.After achieving good therapeutic effects,we used single-cell sequencing,flow cytometry and multiple immunofluorescences staining techniques to explore the ability of OV-CⅡTA to reshape tumor immune microenvironment and the mechanism of anti-tumor immune response.Subsequently,immune cell elimination experiments in mice were performed to identify the immune cell groups that played a central role in the OV-CⅡTA-mediated antitumor effect.Finally,the ability of OV-CⅡTA-infected tumor cells to induce activation of naive T cells was verified by in vitro co-culture models.Results1.Engineered virus to achieve the success of the exogenous CⅡTA gene can insert and mediated by CⅡTA protein expression tumor cell MHCII molecules increases.2.Engineered virus ensures and improves the oncolytic ability of the strain and can further enhance the process of inducing immune death of tumor cells.3.Engineered virus can mediate the regulation of several classical immune-related pathways such as"Antigen processing and presentation","Th cell polarization" and N-glycosylation.4.Engineered virus has shown excellent anti-cancer effect in mouse tumor models and can act as a"tumor vaccine"-mediating the formation of long-term memory anti-tumor immune response,producing a good synergistic effect when combined with immune checkpoint inhibitors.5.Engineered virus can improve the effective infiltration of immune inflammatory cells into the tumor and drive the formation of a"hot tumor immune microenvironment"conducive to anti-tumor response.6.Engineered virus can effectively activated CD4⁺T cells as the core dominance of systemic antitumor response.7.Engineered virus successfully give infected the tumor cell immune activation of naive T cells.Conclusions:Studies have shown that the immune-response-enhanced OV-CⅡTA can mediate the effective antigen-presenting process within the tumor and induce the enhanced activation ability of infected tumor cells to T cells,showing a strong and lasting antitumor effect in animal tumor models,and forming a highly efficient specific anti-tumor immune response dominated by CD4⁺T cells.As a novel tumor treatment strategy,the proposed OV-CⅡTA with enhanced immune response is helpful to further deepen the understanding of the"virus-organism-tumor"interaction and provide strong support for the treatment of weak immunogenic tumors.