ANTICANCER IMMUNOGENIC POTENTIAL OF ONCOLYTIC PEPTIDES: RECENT ADVANCES AND NEW PROSPECTS.

Oncolytic peptides are derived from natural host defense peptides/antimicrobial peptides produced in a wide variety of life forms. Over the past two decades, they have attracted much attention in both basic research and clinical applications. Oncolytic peptides were expected to act primarily on tumor cells and also trigger the immunogenic cell death. Their ability in the tumor microenvironment remodeling and potentiating the anticancer immunity has long been ignored. Despite the promising results, clinical application of oncolytic peptides is still hindered by their unsatisfactory bioactivity and toxicity to normal cells. To ensure safer therapy, various approaches are being developed. The idea of the Ukrainian research group was to equip peptide molecules with a "molecular photoswitch" - a diarylethene fragment capable of photoisomerization, allowing for the localized photoactivation of peptides within tumors reducing side effects. Such oncolytic peptides that may induce the membrane lysis-mediated cancer cell death and subsequent anticancer immune responses in combination with the low toxicity to normal cells have provided a new paradigm for cancer therapy. This review gives an overview of the broad effects and perspectives of oncolytic peptides in anticancer immunity highlighting the potential issues related to the use of oncolytic peptides in cancer immunotherapy. We summarize the current status of research on peptide-based tumor immunotherapy in combination with other therapies including immune checkpoint inhibitors, chemotherapy, and targeted therapy.

Synergistic antitumor immune response mediated by paclitaxel-conjugated nanohybrid oncolytic adenovirus with dendritic cell therapy.

Dendritic cell (DC)-based vaccines have emerged as a promising strategy in cancer immunotherapy due to low toxicity. However, the therapeutic efficacy of DC as a monotherapy is insufficient due to highly immunosuppressive tumor environment. To address these limitations of DC as immunotherapeutic agent, we have developed a polymeric nanocomplex incorporating (1) oncolytic adenovirus (oAd) co-expressing interleukin (IL)-12 and granulocyte-macrophage colony-stimulating factor (GM-CSF) and (2) arginine-grafted bioreducible polymer with PEGylated paclitaxel (APP) to restore antitumor immune surveillance function in tumor milieu and potentiate immunostimulatory attributes of DC vaccine. Nanohybrid complex (oAd/APP) in combination with DC (oAd/APP+DC) induced superior expression level of antitumor cytokines (IL-12, GM-CSF, and interferon gamma) than either oAd/APP or DC monotherapy in tumor tissues, thus resulting in superior intratumoral infiltration of both endogenous and exogenous DCs. Furthermore, oAd/APP+DC treatment led superior migration of DC to secondary lymphoid organs, such as draining lymph nodes and spleen, in comparison with either monotherapy. Superior migration profile of DCs in oAd/APP+DC treatment group resulted in more prolific activation of tumor-specific T cells in these lymphoid organs and greater intratumoral infiltration of T cells. Additionally, oAd/APP+DC treatment led to lower subset of tumor infiltrating lymphocytes and splenocytes being immunosuppressive regulatory T cells than any other treatment groups. Collectively, oAd/APP+DC led to superior induction of antitumor immune response and amelioration of immunosuppressive tumor microenvironment to elicit potent tumor growth inhibition than either monotherapy.

Oncolytic Newcastle disease virus carrying the IL24 gene exerts antitumor effects by inhibiting tumor growth and vascular sprouting.

The second-leading cause of death, cancer, poses a significant threat to human life. Innovations in cancer therapies are crucial due to limitations in traditional approaches. Newcastle disease virus (NDV), a nonpathogenic oncolytic virus, exhibits multifunctional anticancer properties by selectively infecting, replicating, and eliminating tumor cells. To enhance NDV's antitumor activity, four oncolytic NDV viruses were developed, incorporating IL24 and/or GM-CSF genes at different gene loci using reverse genetics. In vitro experiments revealed that oncolytic NDV virus augmented the antitumor efficacy of the parental virus rClone30, inhibiting tumor cell proliferation, inducing tumor cell fusion, and promoting apoptosis. Moreover, NDV carrying the IL24 gene inhibited microvessel formation in CAM experiments. Evaluation in a mouse model of liver cancer confirmed the therapeutic efficacy of oncolytic NDV viral therapy. Tumors in mice treated with oncolytic NDV virus significantly decreased in size, accompanied by tumor cell detachment and apoptosis evident in pathological sections. Furthermore, oncolytic NDV virus enhanced T cell and dendritic cell production and substantially improved the survival rate of mice with hepatocellular carcinoma, with rClone30-IL24(P/M) demonstrating significant therapeutic effects. This study establishes a basis for utilizing oncolytic NDV virus as an antitumor agent in clinical practice.

PD-L1 promotes oncolytic virus infection via a metabolic shift that inhibits the type I IFN pathway.

While conventional wisdom initially postulated that PD-L1 serves as the inert ligand for PD-1, an emerging body of literature suggests that PD-L1 has cell-intrinsic functions in immune and cancer cells. In line with these studies, here we show that engagement of PD-L1 via cellular ligands or agonistic antibodies, including those used in the clinic, potently inhibits the type I interferon pathway in cancer cells. Hampered type I interferon responses in PD-L1-expressing cancer cells resulted in enhanced efficacy of oncolytic viruses in vitro and in vivo. Consistently, PD-L1 expression marked tumor explants from cancer patients that were best infected by oncolytic viruses. Mechanistically, PD-L1 promoted a metabolic shift characterized by enhanced glycolysis rate that resulted in increased lactate production. In turn, lactate inhibited type I IFN responses. In addition to adding mechanistic insight into PD-L1 intrinsic function, our results will also help guide the numerous ongoing efforts to combine PD-L1 antibodies with oncolytic virotherapy in clinical trials.

Enhancement of Therapeutic Potential of Oncolytic Virus with Homologous Tumor Cell Membranes for Pancreatic Cancer.

Pancreatic cancer is a leading cause of cancer-related deaths worldwide. Conventional therapies often provide limited success, necessitating the need for novel therapeutic strategies. Oncolytic viruses (OVs) are a class of viruses that specifically target and kill cancer cells while leaving normal cells unharmed. These viruses have shown promise in the treatment of various cancers, including pancreatic cancer. However, their use in clinical settings has been limited by several factors. Their inability to efficiently infect and kill tumor cells. To overcome this limitation, a cell membrane-coated oncolytic virus was developed. However, the necessity of homologous and nonhomologous tumor cell membranes for their function has not yet been proven. This novel virus displayed increased infectivity and killing activity against tumor cells compared to nonhomologous tumor cell membranes and noncoated viruses. We believe that the homologous tumor cell membranes-coated OVs can enhance the therapeutic potential for pancreatic cancer therapy.

Oncolytic adenovirus encoding LHPP exerts potent antitumor effect in lung cancer.

LHPP has been shown to be a new tumor suppressor, and has a tendency to be under-expressed in a variety of cancers. Oncolytic virotheray is a promising therapeutics for lung cancer in recent decade years. Here we successfully constructed a new recombinant oncolytic adenovirus GD55-LHPP and investigated the effect of GD55-LHPP on the growth of lung cancer cells in vitro and in vivo. The results showed that LHPP had lower expression in either lung cancer cells or clinical lung cancer tissues compared with normal cells or tissues, and GD55-LHPP effectively mediated LHPP expression in lung cancer cells. GD55-LHPP could effectively inhibit the proliferation of lung cancer cell lines and rarely affected normal cell growth. Mechanically, the oncolytic adenovirus GD55-LHPP was able to induce stronger apoptosis of lung cancer cells compared with GD55 through the activation of caspase signal pathway. Notably, GD55-LHPP also activated autophagy-related signal pathway. Further, GD55-LHPP efficiently inhibited tumor growth in lung cancer xenograft in mice and prolonged animal survival rate compared with the control GD55 or PBS. In conclusion, the novel construct GD55-LHPP provides a valuable strategy for lung cancer-targeted therapy and develop the role of tumor suppress gene LHPP in lung cancer gene therapy.

The role of immune cells in resistance to oncolytic viral therapy.

Resistance to treatment poses a major challenge for cancer therapy, and oncoviral treatment encounters the issue of viral resistance as well. In this investigation, we introduce deterministic differential equation models to explore the effect of resistance on oncolytic viral therapy. Specifically, we classify tumor cells into resistant, sensitive, or infected with respect to oncolytic viruses for our analysis. Immune cells can eliminate both tumor cells and viruses. Our research shows that the introduction of immune cells into the tumor-virus interaction prevents all tumor cells from becoming resistant in the absence of conversion from resistance to sensitivity, given that the proliferation rate of immune cells exceeds their death rate. The inclusion of immune cells leads to an additional virus-free equilibrium when the immune cell recruitment rate is sufficiently high. The total tumor burden at this virus-free equilibrium is smaller than that at the virus-free and immune-free equilibrium. Therefore, immune cells are capable of reducing the tumor load under the condition of sufficient immune strength. Numerical investigations reveal that the virus transmission rate and parameters related to the immune response significantly impact treatment outcomes. However, monotherapy alone is insufficient for eradicating tumor cells, necessitating the implementation of additional therapies. Further numerical simulation shows that combination therapy with chimeric antigen receptor (CAR T-cell) therapy can enhance the success of treatment.

Frontiers and future of immunotherapy for pancreatic cancer: from molecular mechanisms to clinical application.

Pancreatic cancer is a highly aggressive malignant tumor, that is becoming increasingly common in recent years. Despite advances in intensive treatment modalities including surgery, radiotherapy, biological therapy, and targeted therapy, the overall survival rate has not significantly improved in patients with pancreatic cancer. This may be attributed to the insidious onset, unknown pathophysiology, and poor prognosis of the disease. It is therefore essential to identify and develop more effective and safer treatments for pancreatic cancer. Tumor immunotherapy is the new and fourth pillar of anti-tumor therapy after surgery, radiotherapy, and chemotherapy. Significant progress has made in the use of immunotherapy for a wide variety of malignant tumors in recent years; a breakthrough has also been made in the treatment of pancreatic cancer. This review describes the advances in immune checkpoint inhibitors, cancer vaccines, adoptive cell therapy, oncolytic virus, and matrix-depletion therapies for the treatment of pancreatic cancer. At the same time, some new potential biomarkers and potential immunotherapy combinations for pancreatic cancer are discussed. The molecular mechanisms of various immunotherapies have also been elucidated, and their clinical applications have been highlighted. The current challenges associated with immunotherapy and proposed strategies that hold promise in overcoming these limitations have also been discussed, with the aim of offering new insights into immunotherapy for pancreatic cancer.

Application of Oncolytic Poxviruses: An Emerging Paradigm in Cancer Therapy.

Despite the significant advancement of new tools and technology in the field of medical biology and molecular biology, the challenges in the treatment of most cancer types remain constant with the problem of developing resistance toward drugs and no substantial enhancement in the overall survival rate of cancer patients. Immunotherapy has shown the most promising results in different clinical and preclinical trials in the treatment of various cancer due to its higher efficacy and minimum collateral damage in many cancer patients as compared to conventional chemotherapy and radiotherapy. An oncolytic virus is a new class of immunotherapy that can selectively replicate in tumor cells and destroy them by the process of cell lysis while exerting minimum or no effect on a normal cell. Besides this, it can also activate the host's innate immune system, which generates an anti-tumor immune response to eliminate the tumor cells. Several wild types and genetically modified viruses have been investigated to show oncolytic behavior. Vaccinia virus has been studied extensively and tested for its promising oncolytic nature on various model systems and clinical trials. Recently, several engineered vaccinia viruses have been developed that express the desired genes encoded for selective penetration in tumor cells and enhanced activation of the immune system for generating anti-tumor immunity. However, further investigation is required to prove their potential and enhance their therapeutic efficacy.

Local intralesional talimogene laherparepvec therapy with complete local response in oral palatine mucosal melanoma: a case report.

BACKGROUND: Mucosal melanoma, an aggressive type of malignancy different from the cutaneous melanomas commonly seen in the head and neck region, represents < 1% of all malignant melanomas. The pathogenesis of mucosal melanoma is unknown. Targetable mutations commonly seen in cutaneous melanoma, such as in the BRAF and NRAS genes, have a lower incidence in mucosal melanoma. Mucosal melanoma carries a distinct mutational pattern from cutaneous melanoma. Surgery with negative margins is the first-line treatment for mucosal melanoma, and systemic therapy is not well defined. Talimogene laherparepvec, an oncolytic viral immunotherapy, is United States Food and Drug Administration approved for the treatment of advanced malignant cutaneous melanoma, with local therapeutic benefits. Mucosal melanoma was initially excluded from talimogene laherparepvec's initial phase III clinical trial. CASE PRESENTATION: We present the case of a white female patient in her 40s with past medical history of systemic lupus erythematous, scleroderma, and estrogen-receptor-positive invasive ductal breast carcinoma. Following a bilateral mastectomy, the patient was found to have BRAF-negative mucosal melanoma of her hard palate with a soft palate skip lesion. Owing to the presence of a skip mucosal lesion as well as the anticipated defect and need for free-flap reconstructive surgery, nonsurgical management was considered. The patient was referred to medical oncology, where-based on the patient's complicated medical history and the risk of immunotherapy possibly worsening her prior autoimmune diseases-local talimogene laherparepvec injections were chosen as the primary therapy for her mucosal lesions. Though talimogene laherparepvec is approved for the treatment of cutaneous melanoma, there are limited data available on the use of talimogene laherparepvec in mucosal melanomas. CONCLUSION: The patient had a complete local tumor response at both the primary lesion as well as the skip lesion with the local injections. She had no side effects and maintained a high quality of life during treatment.

Dual­regulated oncolytic adenovirus carrying ERCC1­siRNA gene possesses potent antitumor effect on ovarian cancer cells.

Ovarian cancer is a multifactorial and deadly disease. Despite significant advancements in ovarian cancer therapy, its incidence is on the rise and the molecular mechanisms underlying ovarian cancer invasiveness, metastasis and drug resistance remain largely elusive, resulting in poor prognosis. Oncolytic viruses armed with therapeutic transgenes of interest offer an attractive alternative to chemical drugs, which often face innate and acquired drug resistance. The present study constructed a novel oncolytic adenovirus carrying ERCC1 short interfering (si)RNA, regulated by hTERT and HIF promoters, termed Ad­siERCC1. The findings demonstrated that this oncolytic adenovirus effectively inhibits the proliferation, migration and invasion of ovarian cancer cells. Furthermore, the downregulation of ERCC1 expression by siRNA ameliorates drug resistance to cisplatin (DDP) chemotherapy. It was found that Ad­siERCC1 blocks the cell cycle in the G1 phase and enhances apoptosis through the PI3K/AKT­caspase­3 signaling pathways in SKOV3 cells. The results of the present study highlighted the critical effect of oncolytic virus Ad­siERCC1 in inhibiting the survival of ovarian cancer cells and increasing chemotherapy sensitivity to DDP. These findings underscore the potent antitumor effect of Ad­siERCC1 on ovarian cancers in vivo.

Identification of restrictive molecules involved in oncolytic virotherapy using genome-wide CRISPR screening.

Oncolytic viruses (OVs) offer a novel approach to treat solid tumors; however, their efficacy is frequently suboptimal due to various limiting factors. To address this challenge, we engineered an OV containing targets for neuron-specific microRNA-124 and Granulocyte-macrophage colony-stimulating factor (GM-CSF), significantly enhancing its neuronal safety while minimally compromising its replication capacity. Moreover, we identified PARP1 as an HSV-1 replication restriction factor using genome-wide CRISPR screening. In models of glioblastoma (GBM) and triple-negative breast cancer (TNBC), we showed that the combination of OV and a PARP inhibitor (PARPi) exhibited superior efficacy compared to either monotherapy. Additionally, single-cell RNA sequencing (scRNA-seq) revealed that this combination therapy sensitized TNBC to immune checkpoint blockade, and the incorporation of an immune checkpoint inhibitor (ICI) further increased the survival rate of tumor-bearing mice. The combination of PARPi and ICI synergistically enhanced the ability of OV to establish durable tumor-specific immune responses. Our study effectively overcomes the inherent limitations of OV therapy, providing valuable insights for the clinical treatment of TNBC, GBM, and other malignancies.

Tumor microenvironment-modulating oncolytic adenovirus combined with GSK-3ß inhibitor enhances antitumor immune response against bladder cancer.

Bladder cancer is a common type of cancer around the world, and the majority of patients are diagnosed with non-muscle-invasive bladder cancer (NMIBC). Although low-risk NMIBC has a good prognosis, the disease recurrence rate and development of treatment-refractory disease remain high in intermediate- to high-risk NMIBC patients. To address these challenges for the treatment of NMIBC, a novel combination therapy composed of an oncolytic adenovirus (oAd) co-expressing interleukin (IL)-12, granulocyte-macrophage colony-stimulating factor (GM-CSF), and relaxin (RLX; HY-oAd) and a clinical-stage glycogen synthase kinase (GSK)-3ß inhibitor (9-ING-41; elraglusib) was investigated in the present report. Our findings demonstrate that HY-oAd and 9-ING-41 combination therapy (HY-oAd+9-ING-41) exerted superior inhibition of tumor growth compared with respective monotherapy in a syngeneic NMIBC tumor model. HY-oAd+9-ING-41 induced high-level tumor extracellular matrix (ECM) degradation and a more potent antitumor immune response than the respective monotherapy. In detail, HY-oAd+9-ING-41 induced superior accumulation of intratumoral T cells, prevention of immune cell exhaustion, and induction of tumor-specific adaptive immune response compared to either monotherapy. Collectively, these results demonstrate that the combination of HY-oAd and 9-ING-41 may be a promising approach to elicit a potent antitumor immune response against bladder cancer.

Cytokine-armed oncolytic herpes simplex viruses: a game-changer in cancer immunotherapy?

Cytokines are small proteins that regulate the growth and functional activity of immune cells, and several have been approved for cancer therapy. Oncolytic viruses are agents that mediate antitumor activity by directly killing tumor cells and inducing immune responses. Talimogene laherparepvec is an oncolytic herpes simplex virus type 1 (oHSV), approved for the treatment of recurrent melanoma, and the virus encodes the human cytokine, granulocyte-macrophage colony-stimulating factor (GM-CSF). A significant advantage of oncolytic viruses is the ability to deliver therapeutic payloads to the tumor site that can help drive antitumor immunity. While cytokines are especially interesting as payloads, the optimal cytokine(s) used in oncolytic viruses remains controversial. In this review, we highlight preliminary data with several cytokines and chemokines, including GM-CSF, interleukin 12, FMS-like tyrosine kinase 3 ligand, tumor necrosis factor α, interleukin 2, interleukin 15, interleukin 18, chemokine (C-C motif) ligand 2, chemokine (C-C motif) ligand 5, chemokine (C-X-C motif) ligand 4, or their combinations, and show how these payloads can further enhance the antitumor immunity of oHSV. A better understanding of cytokine delivery by oHSV can help improve clinical benefit from oncolytic virus immunotherapy in patients with cancer.

Cancer therapy with the viral and bacterial pathogens: The past enemies can be considered the present allies.

Cancer continues to be one of the leading causes of mortality worldwide despite significant advancements in cancer treatment. Many difficulties have arisen as a result of the detrimental consequences of chemotherapy and radiotherapy as a common cancer therapy, such as drug inability to penetrate deep tumor tissue, and also the drug resistance in tumor cells continues to be a major concern. These obstacles have increased the need for the development of new techniques that are more selective and effective against cancer cells. Bacterial-based therapies and the use of oncolytic viruses can suppress cancer in comparison to other cancer medications. The tumor microenvironment is susceptible to bacterial accumulation and proliferation, which can trigger immune responses against the tumor. Oncolytic viruses (OVs) have also gained considerable attention in recent years because of their potential capability to selectively target and induce apoptosis in cancer cells. This review aims to provide a comprehensive summary of the latest literature on the role of bacteria and viruses in cancer treatment, discusses the limitations and challenges, outlines various strategies, summarizes recent preclinical and clinical trials, and emphasizes the importance of optimizing current strategies for better clinical outcomes.

Nanoparticle-Mediated Synergistic Chemoimmunotherapy for Cancer Treatment.

Until now, there has been a lack of effective strategies for cancer treatment. Immunotherapy has high potential in treating several cancers but its efficacy is limited as a monotherapy. Chemoimmunotherapy (CIT) holds promise to be widely used in cancer treatment. Therefore, identifying their involvement and potential synergy in CIT approaches is decisive. Nano-based drug delivery systems (NDDSs) are ideal delivery systems because they can simultaneously target immune cells and cancer cells, promoting drug accumulation, and reducing the toxicity of the drug. In this review, we first introduce five current immunotherapies, including immune checkpoint blocking (ICB), adoptive cell transfer therapy (ACT), cancer vaccines, oncolytic virus therapy (OVT) and cytokine therapy. Subsequently, the immunomodulatory effects of chemotherapy by inducing immunogenic cell death (ICD), promoting tumor killer cell infiltration, down-regulating immunosuppressive cells, and inhibiting immune checkpoints have been described. Finally, the NDDSs-mediated collaborative drug delivery systems have been introduced in detail, and the development of NDDSs-mediated CIT nanoparticles has been prospected.

Multimodal Therapy Approaches for NUT Carcinoma by Dual Combination of Oncolytic Virus Talimogene Laherparepvec with Small Molecule Inhibitors.

NUT (nuclear-protein-in-testis) carcinoma (NC) is a highly aggressive tumor disease. Given that current treatment regimens offer a median survival of six months only, it is likely that this type of tumor requires an extended multimodal treatment approach to improve prognosis. In an earlier case report, we could show that an oncolytic herpes simplex virus (T-VEC) is functional in NC patients. To identify further combination partners for T-VEC, we have investigated the anti-tumoral effects of T-VEC and five different small molecule inhibitors (SMIs) alone and in combination in human NC cell lines. Dual combinations were found to result in higher rates of tumor cell reductions when compared to the respective monotherapy as demonstrated by viability assays and real-time tumor cell growth monitoring. Interestingly, we found that the combination of T-VEC with SMIs resulted in both stronger and earlier reductions in the expression of c-Myc, a main driver of NC cell proliferation, when compared to T-VEC monotherapy. These results indicate the great potential of combinatorial therapies using oncolytic viruses and SMIs to control the highly aggressive behavior of NC cancers and probably will pave the way for innovative multimodal clinical studies in the near future.

OV Modulators of the Paediatric Brain TIME: Current Status, Combination Strategies, Limitations and Future Directions.

Oncolytic viruses (OVs) are characterised by their preference for infecting and replicating in tumour cells either naturally or after genetic modification, resulting in oncolysis. Furthermore, OVs can elicit both local and systemic anticancer immune responses while specifically infecting and lysing tumour cells. These characteristics render them a promising therapeutic approach for paediatric brain tumours (PBTs). PBTs are frequently marked by a cold tumour immune microenvironment (TIME), which suppresses immunotherapies. Recent preclinical and clinical studies have demonstrated the capability of OVs to induce a proinflammatory immune response, thereby modifying the TIME. In-depth insights into the effect of OVs on different cell types in the TIME may therefore provide a compelling basis for using OVs in combination with other immunotherapy modalities. However, certain limitations persist in our understanding of oncolytic viruses' ability to regulate the TIME to enhance anti-tumour activity. These limitations primarily stem from the translational limitations of model systems, the difficulties associated with tracking reliable markers of efficacy throughout the course of treatment and the role of pre-existing viral immunity. In this review, we describe the different alterations observed in the TIME in PBTs due to OV treatment, combination therapies of OVs with different immunotherapies and the hurdles limiting the development of effective OV therapies while suggesting future directions based on existing evidence.

Tuning cellular metabolism for cancer virotherapy.

Oncolytic viruses (OVs) represent an emerging immunotherapeutic strategy owing to their capacity for direct tumor lysis and induction of antitumor immunity. However, hurdles like transient persistence and moderate efficacy necessitate innovative approaches. Metabolic remodeling has recently gained prominence as a strategic intervention, wherein OVs or combination regimens could reprogram tumor and immune cell metabolism to enhance viral replication and oncolysis. In this review, we summarize recent advances in strategic reprogramming of tumor and immune cell metabolism to enhance OV-based immunotherapies. Specific tactics include engineering viruses to target glycolytic, glutaminolytic, and nucleotide synthesis pathways in cancer cells, boosting viral replication and tumor cell death. Additionally, rewiring T cell and NK cell metabolism of lipids, amino acids, and carbohydrates shows promise to enhance antitumor effects. Further insights are discussed to pave the way for the clinical implementation of metabolically enhanced oncolytic platforms, including balancing metabolic modulation to limit antiviral responses while promoting viral persistence and tumor clearance.

VP5 protein of oncolytic herpes simplex virus type 2 induces apoptosis in A549 cells through TP53I3 protein.

Oncolytic virotherapy stands out as a burgeoning and promising therapeutic paradigm, harnessing the intrinsic cytotoxicity of oncolytic viruses for selective replication and dissemination within tumors. The primary mode of action revolves around the direct eradication of tumor cells. In our previous investigations, we formulated an oncolytic herpes simplex virus type 2 (OH2) and substantiated its anti-tumor efficacy both in vivo and in vitro. Subsequently, we embarked on a phase I/II clinical trial in China (NMPA, 2018L02743) and the USA (FDA, IND 27137) to assess OH2's safety, biodistribution, and anti-tumor activity as a standalone agent in patients with advanced solid tumors. In this investigation, our primary focus was to comprehend the influence of the major capsid protein VP5 of OH2 on its efficacy as an antitumor agent. Our findings underscore that the VP5 protein significantly amplifies OH2's oncolytic impact on A549 cells. Additionally, we observed that VP5 actively promotes the induction of apoptosis in A549 cells, both in vivo and in vitro. Through comprehensive transcriptional sequencing, we further authenticated that the VP5 protein triggers apoptosis-related signaling pathways and Gene Ontology (GO) terms in A549 cells. Moreover, we scrutinized differentially expressed genes in the p53-dependent apoptosis pathway and conducted meticulous in vitro validation of these genes. Subsequently, we delved deeper into unraveling the functional significance of the TP53I3 gene and conclusively affirmed that the VP5 protein induces apoptosis in A549 cells through the TP53I3 gene. These revelations illuminate the underlying mechanisms of OH2's antitumor activity and underscore the pivotal role played by the VP5 protein. The outcomes of our study harbor promising implications for the formulation of effective oncolytic virotherapy strategies in cancer treatment.