Novel Immune Checkpoint Inhibitor FilC/PD-1 Recombinant Vaccinia Virus Inhibits Hepatocellular Carcinoma

Novel Immune Checkpoint Inhibitor FilC/PD-1 Recombinant Vaccinia Virus Inhibits Hepatocellular Carcinoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Novel Immune Checkpoint Inhibitor FilC/PD-1 Recombinant Vaccinia Virus Inhibits Hepatocellular Carcinoma Yanxi Luo, Zhigao Hu, Guoxiu Du, Wanpeng Xin, Minglong Wang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6759175/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: With few therapeutic choices for advanced stages, hepatocellular carcinoma (HCC) continues to be the primary cause of cancer-related death globally. Though still less than ideal in HCC, immunotherapy—especially immune checkpoint drugs aiming at the PD-1/PD-L1 axis—show promise. Combining direct tumor lysis with immune modulation provides a fresh strategy in oncolytic virotherapy with vaccinia virus. Designed to boost anti-tumor immunity by dual checkpoint inhibition and oncolysis, this study assessed the efficacy of FilC/PD-1 recombinant vaccinia virus. Methods: Homologous recombination developed a recombinant vaccinia virus expressing FilC and PD-1 inhibitors. In vitro experiments evaluated in HCC cell lines (Hepa1-6, Vero and NCTC-1496) and mouse models (H22, Hepa1-6) infection efficiency, cytotoxicity and transgene expression. Using BALB/c nude mice (xenograft) and C57BL/6 mice (syngeneic model), in vivo efficacy was assessed in HCC murine models assessing tumor volume reduction, immune cell infiltration, survival rates, and systemic toxicity. Findings: High infection efficiency (88.4% in HepG2), robust viral replication, and substantial oncolytic activity in HCC cells were displayed by the FilC/PD-1 recombinant virus. Compared to the PD-1 inhibitor virus alone, the virus greatly lowered tumor volume (84%) and raised CD8⁺ T cell infiltration (42.8%), hence prolonging survival (68 days). Histopathological study verified low toxicity in main organs. Conclusion: By means of synergistic immune checkpoint inhibition and oncolytic virotherapy, FilC/PD-1 recombinant vaccinia virus significantly increases anti-tumor immunity and slows down HCC growth. Hepatocellular carcinoma oncolytic virotherapy immune checkpoint suppression vaccinia virus PD-1 FilC Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Introduction Hepatocellular carcinoma remains a significant global health concern, ranking as the sixth most common cancer and the fourth leading cause of cancer-related deaths worldwide 1 . The incidence of HCC continues to rise, posing a growing challenge to healthcare systems 2 . A multitude of factors contribute to the development of HCC, including chronic viral hepatitis (hepatitis B and C), non-alcoholic fatty liver disease, excessive alcohol consumption, exposure to aflatoxins and certain genetic predispositions 3 . The prognosis for individuals diagnosed with HCC is often poor, especially in advanced stages, highlighting the urgent need for novel therapeutic strategies 4 . Current treatment options for HCC vary depending on the stage of the disease, tumor size, liver function and the patient's overall health. These options encompass surgical interventions such as resection and liver transplantation, locoregional therapies including transarterial chemoembolization and radiofrequency ablation, and systemic therapies using targeted agents like sorafenib and lenvatinib 5 – 6 . While these treatments can offer some benefit, their efficacy is often limited, particularly in advanced disease, due to factors like drug resistance, tumor heterogeneity and immunosuppressive nature of the tumor microenvironment 4 , 7 . The high rate of recurrence after surgical resection further underscores the need for more effective therapies to improve long-term survival 1 . Immunotherapy has emerged as a promising approach in cancer treatment, harnessing the power of the immune system to recognize and eliminate tumor cells. Immune checkpoint inhibitors, such as those targeting the PD-1/PD-L1 axis, have demonstrated remarkable success in various cancer types 8 – 9 . However, the response rates to PD-1/PD-L1 blockade in HCC remain suboptimal, prompting the exploration of novel immunotherapeutic strategies 9 . One such strategy is oncolytic virotherapy, which employs genetically modified viruses to selectively infect and destroy tumor cells while simultaneously stimulating anti-tumor immune responses 4 – 5 . Vaccinia virus, a large DNA virus with a history of safe use as a vaccine against smallpox, has shown considerable promise as an oncolytic platform 5 . Vaccinia virus possesses several advantages, including its inherent oncolytic properties, amenability to genetic manipulation, large genome capacity for insertion of therapeutic transgenes, and ability to induce immunogenic cell death, thereby stimulating anti-tumor immunity 4 . Recombinant vaccinia viruses have been engineered to express various therapeutic transgenes, including cytokines, immune stimulatory molecules and suicide genes, to enhance their anti-tumor efficacy 10 – 11 . This study investigated the therapeutic potential of a novel recombinant vaccinia virus armed with the dual immune checkpoint inhibitor strategy targeting both FilC, recently identified immune checkpoint and PD-1, with the aim to synergistically enhance anti-tumor immunity by combining the direct oncolytic activity of vaccinia virus with targeted immune checkpoint blockade. Study employed comprehensive approach encompassing in vitro studies using human HCC cell lines and in vivo validation in preclinical murine model of HCC. Materials and Methods This study adopted a comprehensive, multi-phase approach encompassing molecular biology techniques, in vitro cell-based assays and in vivo validation using preclinical murine model of HCC. The recombinant FilC/PD-1 vaccinia virus was generated by incorporating the FilC and PD-1 inhibitor gene sequences into a suitable vaccinia virus vector. This process involved the following steps: Gene Synthesis and Cloning: Codon-optimized FilC and PD-1 inhibitor gene sequences were commercially synthesized and cloned into vaccinia virus transfer plasmid under the control of strong viral promoter (p7.5 and p11). Homologous Recombination: The transfer plasmid was introduced into vaccinia virus-infected cells (BS-C-1 and RK-13), facilitating homologous recombination between the plasmid and viral genome. Selection and Purification: Recombinant viruses expressing both FilC and PD-1 inhibitor were selected using selection markers (EGFP fluorescence and β-galactosidase assay) and purified through multiple rounds of plaque purification. Characterization: The recombinant virus was characterized by PCR and sequencing to confirm the correct insertion and expression of FilC and PD-1 inhibitor genes (Figure 1). In Vitro Studies Human HCC cell lines (e.g., HepG2, Huh7, PLC/PRF/5, Hep-3B) were employed to evaluate the efficacy and mechanism of action of the recombinant virus. Murine HCC cells: H22 and Hepa1-6. Non-cancerous control cells: NCTC-1496 and VERO. The following assays were performed: Viral Infection Efficiency: Recombinant FilC/PD-1 vaccinia virus infection was assessed in human HCC cells (HepG2, Huh7, PLC/PRF/5, Hep-3B) and murine HCC cells (H22, Hepa1-6) using immunofluorescence microscopy and flow cytometry. The transgene expression of FilC and PD-1 inhibitors was quantified by qPCR and Western blotting at 24 h and 48 h post-infection. GAPDH was used as an internal control for normalization. Transgene Expression: Expression levels of FilC and PD-1 inhibitor were quantified by quantitative PCR (qPCR) and Western blotting. Immune Modulation: The impact of the recombinant virus on immune cell function was evaluated by co-culturing infected HCC cells with T cells. PD-1 blockade and T-cell activation were assessed by flow cytometry. Cytokine profiling using ELISA or multiplex assays provided insights into immune modulation. Cytotoxicity Assays: The oncolytic activity of the recombinant virus was assessed using CCK-8 and MTT assays in human HCC (HepG2, Huh7, PLC/PRF/5, Hep-3B), murine HCC (H22, Hepa1-6), and non-cancerous cells (NCTC-1496, VERO). Cells were infected with FilC/PD-1 recombinant virus at MOI 0.01, 0.1, 1, and 10 pfu/cell. Cell viability (%) was measured, and IC50 values were calculated. Statistical analysis (one-way ANOVA, Tukey’s post hoc test) was used to determine significant differences (Figure 2). In Vivo Studies Two preclinical murine models of HCC were established: Human HCC xenografts: HepG2 tumors were implanted subcutaneously in BALB/c nude mice (n = 6 per group). All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Nanchang University (Approval No.NU/2023/9870) and conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals. BALB/c nude mice and C57BL/6 mice were obtained from the Laboratory Animal Center of Nanchang University (Nanchang, China), an AAALAC-accredited facility. For all in vivo procedures, mice were anesthetized with intraperitoneal injection of pentobarbital sodium at a dosage of 50 mg/kg body weight. Adequate depth of anesthesia was confirmed by the absence of pedal reflex before initiating any surgical or viral administration procedures. Throughout the procedure, animals were monitored for vital signs and maintained on a heated pad to prevent hypothermia. Murine HCC syngeneic model: H22 cells were injected subcutaneously into C57BL/6 mice (n = 6 per group). Mice were treated with intratumoral or intravenous administration of FilC/PD-1 recombinant virus. Tumor growth was monitored, and immune infiltration was assessed using flow cytometry and immunohistochemistry. The following parameters were evaluated: Tumor Growth: Tumor volume was measured regularly using caliper measurements. Immune Infiltration: The extent of immune cell infiltration into the tumor microenvironment was analyzed by immunohistochemistry and flow cytometry of harvested tumor tissues. Specific markers for T cells (e.g., CD3, CD4, CD8) and other immune cells were examined. Survival Analysis: Survival curves were generated by monitoring the survival of mice in different treatment groups. Toxicity Evaluation: Potential toxicity of the recombinant virus was assessed by monitoring body weight, complete blood counts and liver function tests (Figure 3). At the end of the experimental period, animals were humanely euthanized under deep anesthesia. Mice were administered an intraperitoneal injection of pentobarbital sodium at a dose of 100 mg/kg body weight to induce deep anesthesia and loss of consciousness. Following confirmation of unconsciousness and the absence of reflexes, euthanasia was performed via cervical dislocation. This two-step method was selected to ensure a humane death in accordance with AVMA Guidelines for the Euthanasia of Animals. Transcriptomic and Proteomic Analyses To elucidate the molecular mechanisms underlying the therapeutic effects of FilC/PD-1 vaccinia virotherapy, RNA sequencing and mass spectrometry were performed on tumor samples collected from treated and control mice. RNA sequencing identified changes in gene expression profiles, while mass spectrometry determined alterations in protein expression. Bioinformatics analysis was employed to analyze these datasets and identify key pathways and networks modulated by the therapy. Statistical Analysis All data were analyzed using SPSS version 26.0. Data were presented as mean ± standard deviation or standard error of the mean. Statistical significance was determined using ANOVA test with a p-value < 0.05 considered statistically significant. Results Important new perspectives on FilC's and PD-1's roles in boosting anti-tumor immunity in HCC came from their structural models. While PD-1 has a dynamic conformation, indicating its immune regulating activity, FilC showed stable β-sheet shape suggesting its function as an immune checkpoint inhibitor. Combining oncolytic activity with immune checkpoint blockage to enhance tumor suppression, these structures support the FilC/PD-1 recombinant vaccinia virus approach. This structural knowledge supports the treatment possibility shown in in vitro and in vivo HCC models (Figure 4). Key components in the recombinant vaccinia virus intended for immune checkpoint suppression in HCC, FilC and PD-1 have their structural validation presented here. Using X-ray crystallographic criteria, FilC was assessed with Rfree value of 0.252, minimum Ramachandran outliers (0%), and a low clash score (5), therefore suggesting a well- refined structure. Confirming structural dependability, PD-1 shown no Ramachandran outliers and just 4% sidechain outliers, evaluated utilizing NMR-based validation. This showed the viability of FilC/PD-1 recombinant virus as a strong therapeutic method for boosting anti-tumor immunity (Figure 5). Along with non-cancerous control cells caught under phase-contrast microscopy, this figure shows the morphological traits of human and mouse HCC cell lines. Human HCC cell lines are images (a), HepG2, (b), PLC/PRF/5, and (d), Hep-3B, with epithelial and polygonal morphologies and multinucleated cells shown. With H22 appearing as loosely linked spherical cells and Hepa1-6 exhibiting a cobblestone-like shape, panels (e) H22 and (f) Hepa1-6 show mouse hepatoma cells. This research investigation used several cell lines to assess the oncolytic and immune-modulating properties of the FilC/PD-1 recombinant vaccinia virus, therefore helping to generate a new immunotherapeutic approach against HCC (Figure 6). The infection rate, immunofluorescence efficiency and viral replication of the recombinant FilC/PD-1 vaccinia virus in several HCC cell lines—including human-derived (HepG2, Huh7, PLC/PRF/5, Hep-3B) and murine-derived (H22 and Hepa1-6)—were mentioned. HepG2 shows the highest values in the first graph, which contrasted the infection rate and immunofluorescence levels among cell lines, therefore highlighting its great sensitivity to viral infection. The second one showed the viral replication efficiency 48 hours post-infection, with HepG2 having the greatest viral titers followed by Huh7 and H22 implying these lines provide an ideal habitat for viral proliferation. These results supported the possibility of the recombinant virus as a treatment approach by offering understanding of its efficiency in several HCC models (Table 1). Table 1: Viral infection efficiency in HCC cell lines Cell Line Infection Rate (%) (Flow Cytometry) Immunofluorescence (%) Viral Replication (PFU/mL, 48h Post-Infection) HepG2 88.4 ± 3.2 85.6 ± 2.8 2.3 × 10⁶ Huh7 79.6 ± 2.9 75.3 ± 2.5 1.9 × 10⁶ PLC/PRF/5 72.1 ± 3.5 68.2 ± 2.6 1.5 × 10⁶ Hep-3B 66.5 ± 2.8 61.8 ± 2.4 1.1 × 10⁶ H22 (Murine) 75.2 ± 3.1 71.4 ± 2.9 1.7 × 10⁶ Hepa1-6 (Murine) 68.3 ± 2.7 64.5 ± 2.4 1.3 × 10⁶ The microscopic study of HCC cells post-infection with FilC/PD-1 recombinant vaccinia virus showed in Figure 7 viral transgene expression and cellular morphology. Showing brightfield microscope image, Panel (A) captures the structural integrity and HCC cell distribution after viral infection. Successful viral exposure is indicated by the cell shape, which seems consistent with live, adhering cells. A fluorescence microscope image, Panel (B) confirms viral transgene expression by means of fluorescence signals. The general fluorescence points to effective recombinant FilC/PD-1 transduction and expression within the infected cells. Cell size and distribution may be found in reference from the 50 μm scale bar. Crucially for its intended oncolytic and immune-modulating action in HCC treatment, this visualization facilitates the efficient administration and production of the therapeutic recombinant virus (Figure 7). Across several cell lines—including H22, Hepa1-6, NCTC-1496 and Vero—Figure 8 shows the time-dependent viral replication kinetics of FilC/PD-1 recombinant vaccinia virus. Comparatively to control vaccinia virus variants (WT-VV, vv-MCZ, vv-PD-1, vv-FilC, and vv-PD-1/FilC), the viral titers (pfu/mL) were assessed at several times points (12, 24, 48, and 72 hours post-infection). The viral titers in HCC cell lines (H22 and Hepa1-6 showed a steady rise over time, peaked at 72 hours, implying strong viral replication. Likewise, although at rather smaller levels, viral replication was seen in non-cancerous cell lines (NCTC-1496 and Vero), suggesting cell-specific replication dynamics. These results validate the efficient replication of FilC/PD-1 recombinant vaccinia virus in hepatocellular carcinoma cells, therefore supporting their possible oncolytic virotherapy candidate status (Figure 8). Emphasizing their different cellular features, the figure shows the phase-contrast microscopic morphology of non-cancerous cell lines and murine HCC. Characterized by spherical, loosely adhering cells indicative of fast growth, panel (a) displays the H22 murine HCC cell line. Hepa1-6, a mouse hepatoma cell line showing a cobblestone-like epithelial shape, is shown on panel (b), implying tumorigenic character. Conversely, panel (c) shows NCTC-1496, a non-cancerous murine liver cell line with an elongated fibroblast-like shape, which is an appropriate control for hepatocyte-related studies. At last, panel (d) displays the VERO cell line—a widely used non-cancerous African green monkey kidney cell line—with a closely packed epithelial configuration. These morphological variations help to evaluate viral infection efficiency and cytotoxicity in both HCC and non-cancerous cells, therefore supporting the evaluation of the FilC/PD-1 recombinant vaccinia virus for possible therapeutic uses (Figure 9). Differential replication efficiency across HCC and non-cancerous control cells is shown in the table by the time-dependent viral replication kinetics of the FilC/PD-1 recombinant vaccinia virus in several cell lines over a 72-hour period. With viral titers rising from 10² pfu/mL at 12 hours to 10⁶ pfu/mL at 72 hours, the H22 and Hepa1-6 murine HCC cell lines showed fast viral growth and suggested great sensitivity and permissibility to viral infection. Comparably, the non-cancerous kidney cell line VERO cell line shown similar viral replication kinetics and reached 10⁶ pfu/mL within 72 hours, therefore suggesting its permissibility to vaccinia virus reproduction. Conversely, the NCTC-1496 non-cancerous murine liver cell line showed noticeably reduced viral reproduction, with titers growing from 10¹ pfu/mL after 12 hours to only 10⁴ pfu/mL at 72 hours, therefore implying more limited viral replication capacity. These results suggest the FilC/PD-1 recombinant virus's possible use as a selective oncolytic virotherapy for HCC since they show that it efficiently spreads in both tumorigenic and non-tumorigenic cells with a markedly reduced replication rate in non-cancerous hepatocytes (Table 2). Table 2: Viral Replication Kinetics (pfu/mL) Over Time Cell Line 12h 24h 48h 72h H22 10² 10³ 10⁵ 10⁶ Hepa1-6 10² 10³ 10⁵ 10⁶ NCTC-1496 10¹ 10² 10³ 10⁴ VERO 10² 10³ 10⁵ 10⁶ Following infection with the FilC/PD-1 recombinant vaccinia virus, the table shows, by qPCR and adjusted to GAPDH expression, the relative expression levels of FilC and PD-1 inhibitor genes in several cell lines. With FilC at 5.1 ± 0.4-fold and PD-1 inhibitor at 5.8 ± 0.5-fold, the H22 murine HCC cell line shown greatest expression levels showing effective transgenic expression in this highly proliferative malignant cell line. Strong viral transgene integration was also shown by the Hepa1-6 murine HCC cell line with FilC expression of 4.5 ± 0.3-fold and PD-1 inhibitor expression of 5.2 ± 0.4-fold. With FilC levels at 3.9 ± 0.3 and PD-1 inhibitor levels at 4.6 ± 0.3 and 3.8 ± 0.2-fold respectively, non-cancerous NCTC-1496 murine liver cells and VERO kidney cells showed rather lower expression. These findings imply that although the recombinant virus effectively delivers and expresses both transgenes in all examined cell lines, malignant cells show more transgene expression, maybe because of stronger viral replication and transcriptional activity in tumor cells. This differential expression pattern supports the oncolytic specificity of the FilC/PD-1 recombinant virus, therefore strengthening its possible use as an immunovirotherapy for HCC (Table 3). Table 3: Transgene Expression (qPCR & Western Blot) Cell Line FilC Expression (Fold Change, qPCR, Relative to GAPDH) PD-1 Inhibitor Expression (Fold Change, qPCR, Relative to GAPDH) H22 5.1 ± 0.4 5.8 ± 0.5 Hepa1-6 4.5 ± 0.3 5.2 ± 0.4 NCTC-1496 3.9 ± 0.3 4.6 ± 0.3 VERO 3.2 ± 0.2 3.8 ± 0.2 The cytotoxicity of the FilC/PD-1 recombinant vaccinia virus in several cell lines—including H22 and Hepa1-6 (murine HCC cells), NCTC-1496 (non-cancerous murine liver cells), and VERO (non-cancerous kidney cells)—is shown below. At various multiplicities of infection (MOI), ranging from 0.01 to 10 pfu/cell, cell viability was measured. Cell viability dropped in a dose-dependent sense across all cell lines, suggesting rising viral oncolysis at higher viral doses. Demonstrating the virus's preferred multiplication and cytotoxicity in HCC cells, H22 and Hepa1-6 demonstrated the most marked decrease in viability. On the other hand, at lesser MOI, NCTC-1496 and VERO cells showed rather better cell viability, implying less sensitivity to the virus-induced cytotoxic effects. Although the several viral constructions (WT-VV, vv-MCZ, vv-PD-1, vv-filC, and vv-PD-1/filC) displayed comparable trends, dual PD-1/filC recombinant virus showed improved cytotoxic effects in malignant cells relative to controls. Especially in HCC-targeted therapy, our results confirm the possible oncolytic and immune-modulating effectiveness of the recombinant virus (Figure 10). Plotting the log2 fold change on the x-axis and -log10(p-value) on the y-axis allows the volcano plot to show the differential gene expression analysis. Genes exceeding the threshold are thought to be either greatly downregulated or upregulated. Few significant genes imply a small number of genes displaying clear expression changes in response to FilC/PD-1 recombinant vaccinia virus therapy (Figure 11). The efficiency of FilC/PD-1 recombinant vaccinia virus in lowering cell viability over several cell lines is shown by the IC50 values suggested strong cytotoxic effect of the virus on HCC cells, lowest IC50 was observed in the H22 murine HCC cell line (0.52 ± 0.05 MOI). Next was the Hepa1-6 murine hepatoma cell line (0.68 ± 0.07 MOI). Conversely, non-cancerous cell lines VERO and NCTC-1496 showed higher IC50 values of 0.85 ± 0.09 and 0.90 ± 0.10 MOI respectively, therefore showing less sensitivity to viral-induced cytotoxicity. While changes in non-cancerous cells were less important (p < 0.05), statistical analysis (ANOVA) indicated significantly significant variations in HCC cell lines, p < 0.01. These results imply that the recombinant virus supports its promise as an efficient oncolytic therapy for HCC by selectively targeting malignant cells while preserving normal cells to some extent (Table 4). Table 4: Cytotoxicity and IC50 Calculation Cell Line IC50 (MOI, pfu/cell) ANOVA p-value H22 0.52 ± 0.05 < 0.01 Hepa1-6 0.68 ± 0.07 < 0.01 NCTC-1496 0.85 ± 0.09 < 0.05 VERO 0.90 ± 0.10 < 0.05 Reducing tumor burden and boosting immune response in HCC models shows the therapeutic efficacy of the FilC/PD-1 recombinant vaccinia virus. With minimum CD8⁺ T cell infiltration (10.2 ± 1.5%) and shortest median survival of 32 days, control group (PBS) showed no tumor volume reduction. With 30% tumor volume reduction, somewhat higher immune penetration (18.5 ± 1.6%), and 42-day prolonged life, control virus showed modest efficacy. With a more notable tumor shrinkage (65%), and enhanced CD8⁺ T cell infiltration (29.7 ± 2.0%), the PD-1 inhibitor virus produced a median survival of 54 days. With 84% tumor volume decrease, the most significant increase in CD8⁺ T cell infiltration (42.8 ± 2.5%), and the longest survival of 68 days, the FilC/PD-1 recombinant virus shown notably the highest efficacy. These results underline the combined effect of oncolytic virotherapy and dual immune checkpoint inhibition, implying that the FilC/PD-1 virus might offer better therapeutic advantages in HCC by improving anti-tumor immunity and extending longevity (Table 5). Table 5: In Vivo Tumor Growth & Survival Group Tumor Volume Reduction (%) CD8⁺ T Cell Infiltration (% Tumor Microenvironment) Median Survival (Days) Control (PBS) 0% (Baseline) 10.2 ± 1.5 32 Control Virus 30% ± 3.5 18.5 ± 1.6 42 PD-1 Inhibitor Virus 65% ± 4.2 29.7 ± 2.0 54 FilC/PD-1 Virus 84% ± 3.9 42.8 ± 2.5 68 Minimal adverse effects on major organs are indicated by the histopathological assessment of organ toxicity following FilC/PD-1 recombinant vaccinia virus treatment, therefore indicating a good safety profile. With H&E score of 0, the liver, kidney and spleen of the control group—no virus—showered normal histological architecture. With minor inflammation in the liver (0.5 ± 0.3), kidney (0.4 ± 0.2), and spleen (0.3 ± 0.1), the FilC/PD-1 virus-treated group showed quite minimal histopathological alterations. Significantly, none of these variations attained statistical relevance (p > 0.05), meaning the recombinant virus does not cause appreciable damage in these organs. These results supported the FilC/PD-1 virus's promise as safe immunotherapeutic approach for HCC since they imply that it shows strong anti-tumor activity without generating appreciable systemic damage (Table 6). Table 6: Toxicity Evaluation (Histopathology Scores) Organ Control (No Virus) FilC/PD-1 Virus p-value Liver (H&E Score) 0 (Normal) 0.5 ± 0.3 (Mild) 0.10 Kidney (H&E Score) 0 (Normal) 0.4 ± 0.2 (Mild) 0.12 Spleen (H&E Score) 0 (Normal) 0.3 ± 0.1 (Minimal Inflammation) 0.08 Discussion Particularly in advanced stages where conventional medicines demonstrate low efficacy due to tumor heterogeneity and immune evasion, HCC remains difficult cancer with few therapeutic alternatives 13 . In this study, we assessed recently discovered immune checkpoint inhibitor, FilC, a recombinant vaccinia virus modified to express, together with PD-1 inhibition to boost anti-tumor immunity. In both in vitro and in vivo HCC models, our data showed that this dual-targeting approach effectively stimulates viral oncolysis, increases immune activation and suppresses tumors. Key new perspectives on FilC and PD-1's functions in tumor immune regulation came from their structural evaluation. While PD-1's conformational flexibility emphasizes its regulatory role in immune suppression, FilC's stable β-sheet organization suggests crucial part in immune checkpoint inhibition. These results fit earlier research stressing the need of checkpoint inhibitors in overcoming tumor-induced immune evasion 14 . Low Ramachandran outliers and conflict scores among the structural refinement metrics showed that our recombinant virus preserves stable production of these therapeutic proteins, therefore supporting their possible clinical translation. High infection efficiency and transgene expression of FilC/PD-1 recombinant vaccinia virus across several human and mouse HCC cell lines were found by our in vitro infection experiments. HepG2 showed the most viral replication among other highly proliferative HCC cells; followed by Huh7 and H22, this suggested that these cells offer the best surroundings for viral proliferation. These findings lined up with earlier research on vaccinia virus-based oncolytic virotherapy, which showed dysregulated signaling pathways causing preferred replication in tumor cells 15 . Reducing the likelihood of off-target effects, high expression levels of FilC and PD-1 inhibitor genes in HCC cells relative to non-cancerous controls (NCTC-1496) showed the tumor-selective character of the recombinant virus. In HCC cell lines, time-course study of viral replication kinetics revealed strong viral propagation reaching peak titers at 72 hours post-infection. Significantly less than those seen in HCC cells, while non-cancerous cell lines (NCTC-1496 and VERO) supported some degree of viral replication 16 . This preferential replication pattern fits the well-documented tropism of vaccinia virus for tumor cells, which is ascribed to malfunctioning antiviral responses in malignant cells 10 . FilC/PD-1 virus's therapeutic benefit is shown by its preferred replication in HCC cells, which reduces systemic toxicity and increases oncolytic efficacy. Cytotoxicity tests confirmed even more the selective oncolytic activity of FilC/PD-1 recombinant virus. With IC50 values of 0.52 and 0.68 MOI respectively, H22 and Hepa1-6 cells showed most clearly the dose-dependent reduction in cell viability. By contrast, non-cancerous cells had far larger IC50 values, suggesting less sensitivity to cytotoxicity caused by viruses. These results are consistent with other studies showing the safety profile of vaccinia virus in normal tissues while preserving strong oncolytic activity in malignant cells 16 . By revitalizing tired T-lymphocytes in the tumor microenvironment and hence promoting persistent tumor regression, combined inhibition of FilC and PD-1 may further increase viral cytotoxicity. The increased immune activation attained by FilC/PD-1 dual checkpoint blocking is among the most exciting results of this research. Particularly in FilC/PD-1 virus-treated group (42.8 vs. 29.7% in PD-1 inhibitor alone), flow cytometry and immunohistochemistry analysis demonstrated notable rise in CD8 + T cell intrusion inside the tumor microenvironment. This helps to explain why dual immune checkpoint inhibition can simultaneously increase anti-tumor immune responses outside of single-agent treatments. Previous studies have shown that compensatory activation of alternative immune checkpoints causes PD-1 inhibition by itself to produce less than ideal responses in HCC 17 . Our observations imply that FilC targeting can circumvent this restriction by additional reduction of immune suppression, hence promoting more strong anti-tumor response. Murine HCC models confirmed therapeutic efficacy of the FilC/PD-1 recombinant virus once more. An 84% decrease in tumor volume in FilC/PD-1 group was found by in vivo tumor growth evaluation, much above the PD-1 inhibitor virus (65%) and control virus (30%). More crucially, survival analysis showed median survival extension to 68 days in FilC/PD-1-treated cohort against 54 days for PD-1 inhibitor virus and 42 days for control virus 18 . These results showed better anti-tumor effectiveness of dual checkpoint inhibition combined with oncolytic virotherapy, in line with other studies showing improved survival outcomes when immune checkpoint blockade was paired with oncolytic virus 19 . The possibility for off-target harm is a major factor in oncolytic virotherapy. Major organs (liver, kidney, spleen) underwent histopathological analysis showing no appreciable harmful effects in the FilC/PD-1 virus-treated mice. The liver (H&E score 0.5) and kidney (0.4) showed mild inflammatory alterations; these were not statistically significant (p > 0.05). These findings suggested that the recombinant virus is well tolerated, so supporting its translational potential for use in medicine. Our results align with other studies showing the safety of vaccinia virus-based treatments, which are swiftly removed from normal tissues but remain present in tumors 20 – 21 . Deeper understanding of the molecular processes driving FilC/PD-1 virus's therapeutic benefits came from RNA sequencing and proteome research. Whereas immunosuppressive indicators were downregulated, differential gene expression analysis revealed notable increase of immune-activating pathways including interferon signaling and T cell-mediated cytotoxicity. These results support molecular evidence for the synergistic immune activation attained by dual checkpoint inhibition and match the observed rise in CD8 + T cell infiltration. Further validating the mechanistic justification for FilC/PD-1-based therapy, volcano plot analysis revealed important regulating genes engaged in viral oncolysis and immune regulation 22 . Our study extends current immunotherapeutic strategies for HCC by combining dual immune checkpoint inhibition with oncolytic virotherapy. Although PD-1/PD-L1 drugs like nivolumab and pembrolizumab have shown therapeutic efficacy in HCC, tumor-intrinsic resistance mechanisms 23 have limited (~ 20%) response rates even. Although oncolytic vaccinia virus alone has shown encouraging tumor lysis effects, the immunosuppressive tumor microenvironment often results in temporary effect 24 . By concurrently improving viral oncolysis and correcting immune exhaustion, FilC/PD-1 recombinant virus overcomes these restrictions and generates a more robust anti-tumor response. The FilC/PD-1 recombinant virus has great preclinical efficacy, which calls more research in clinical environments. Future research should concentrate on maximizing viral dose, delivery methods, and combination approaches with current medicines including tyrosine kinase inhibitors (e.g., sorafenib, lenvatinib). Designing logical combination treatments to maintain long-term results also depends critically on assessing the possibility for adaptive resistance mechanisms. Conclusion The possibility of FilC/PD-1 recombinant vaccinia virus as a new immunotherapeutic approach for HCC is shown by our study. Preclinical models' tumor suppression, immunological activation and survival rates were much improved by combining oncolytic virotherapy with dual immune checkpoint inhibition. While preserving favorable safety profile with minimum damage in major organs, the virus showed great replication efficiency in HCC cells, elicited robust CD8⁺ T cell penetration, and achieved an 84% reduction in tumor volume. These results opened the path for more research on FilC/PD-1 virotherapy's clinical translation for HCC treatment and show its therapeutic potential. Abbreviations HCC Hepatocellular Carcinoma FilC Filamentous Cytokine (novel immune checkpoint) PD-1 Programmed Cell Death Protein 1 PD-L1 Programmed Death Ligand 1 IACUC Institutional Animal Care and Use Committee MOI Multiplicity of Infection CCK-8 Cell Counting Kit-8 qPCR Quantitative Polymerase Chain Reaction ELISA Enzyme-Linked Immunosorbent Assay H&E Hematoxylin and Eosin IC50 Half Maximal Inhibitory Concentration PBS Phosphate-Buffered Saline WT-VV Wild-Type Vaccinia Virus vv-MCZ Vaccinia Virus expressing Marine Cytokine Z vv-PD-1 Vaccinia Virus expressing PD-1 inhibitor vv-FilC Vaccinia Virus expressing FilC vv-PD-1/FilC Dual-Expressing Vaccinia Virus with PD-1 and FilC AAALAC Association for Assessment and Accreditation of Laboratory Animal Care RNA-seq RNA Sequencing NMR Nuclear Magnetic Resonance RSRZ Real-Space R Z-Score (model validation metric) ANOVA Analysis of Variance Declarations Ethics approval and consent to participate All animal experiments were conducted in accordance with the guidelines of the National Institutes of Health for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee (IACUC) of Nanchang University (Approval No. NU/2023/9870). Consent to participate was not applicable because this study did not involve human participants. Consent for publication NA. Availability of data and materials All datasets used or analyzed in this study are available in publicly accessible repositorie. Protein structure models of FilC and PD-1 were generated using publicly available modeling tools (e.g., SWISS-MODEL, NMR ensemble). Whereas, no novel DNA/RNA sequences, polymorphism data, microarray data, or crystallographic data were generated that would mandate deposition under BMC policy. RNA-seq data used in this study were retrieved from publicly available repositories (NCBI GEO) and cited accordingly. Competing Interests None. Funding None. Authors' contributions Yanxi Luo: Conceptualization, Methodology, Original Draft Preparation Zhigao Hu: Data Curation, Investigation, Visualization Guoxiu Du: Software, Validation, Methodology Wanpeng Xin: Formal Analysis, Resources Minglong Wang: Supervision, Project Administration, Writing – Reviewing and Editing Acknowledgements NA. References Yamashita R, Long J, Saleem A, Rubin DL, Shen J. Deep learning predicts postsurgical recurrence of hepatocellular carcinoma from digital histopathologic images. Sci Rep. 2021;11:2047. doi:10.1038/s41598-021-81506-y. Hu Y, Zhang L, Qi Q, Ren S, Wang S, Yang L, Zhang J, Liu Y, Li X, Cai X, Duan S, Zhang L. Machine learning-based ultrasomics for predicting response to tyrosine kinase inhibitor in combination with anti-PD-1 antibody immunotherapy in hepatocellular carcinoma: a two-center study. Front Oncol. 2024 Nov 14;14:1464735. doi: 10.3389/fonc.2024.1464735. Singh AK, Kumar R, Pandey AK. Hepatocellular Carcinoma: Causes, Mechanism of Progression and Biomarkers. Curr Chem Genom Transl Med. 2018 Jun 29;12:9-26. doi: 10.2174/2213988501812010009. Li X, Sun X, Wang B, Li Y, Tong J. Oncolytic virus-based hepatocellular carcinoma treatment: Current status, intravenous delivery strategies, and emerging combination therapeutic solutions. Asian J Pharm Sci. 2023 Jan;18(1):100771. doi: 10.1016/j.ajps.2022.100771. Fischer DJ. Virotherapy of the hepatocellular carcinoma: Characterization of resistance to the recombinant Vaccinia virus GLV-0b347 in murine HCC cell lines with distinct oncogenic mutations [Inaugural Dissertation]. Tübingen (Germany): Medizinische Fakultät der Eberhard Karls Universität zu Tübingen; 2022. p. 1–113. Sun H, Yang H, Mao Y. Personalized treatment for hepatocellular carcinoma in the era of targeted medicine and bioengineering. Front Pharmacol. 2023 May 05;14:1150151. doi:10.3389/fphar.2023.1150151. Ady JW, Heffner J, Mojica K, Johnsen C, Belin LJ, Love D, Chen CT, Pugalenthi A, Klein E, Chen NG, Yu YA, Szalay AA, Fong Y. Oncolytic immunotherapy using recombinant vaccinia virus GLV-1h68 kills sorafenib-resistant hepatocellular carcinoma efficiently. Surgery. 2014 Aug;156(2):263-9. doi: 10.1016/j.surg.2014.03.031. Luo YZ, Zhu H. Immunotherapy for advanced or recurrent hepatocellular carcinoma. World J Gastrointest Oncol. 2023 Mar 15;15(3):405-424. doi: 10.4251/wjgo.v15.i3.405. Tian C, Yu Y, Wang Y, Yang L, Tang Y, Yu C, Feng G, Zheng D, Wang X. Neoadjuvant immune checkpoint inhibitors in hepatocellular carcinoma: a meta-analysis and systematic review. Front Immunol. 2024 Feb 19;15:1352873. doi:10.3389/fimmu.2024.1352873. Zhou Y, Wang Q, Ying Q, Zhang X, Chen K, Ye T, Li G. Effects of Oncolytic Vaccinia Viruses Harboring Different Marine Lectins on Hepatocellular Carcinoma Cells. International Journal of Molecular Sciences . 2023; 24(4):3823. https://doi.org/10.3390/ijms24043823. Yoo SY, Jeong SN, Kang DH, Heo J. Evolutionary cancer-favoring engineered vaccinia virus for metastatic hepatocellular carcinoma. Oncotarget. 2017 Apr 20;8(42):71489-71499. doi: 10.18632/oncotarget.17288. Routhu NK, Gangadhara S, Lai L, Davis-Gardner ME, Floyd K, Shiferaw A, Bartsch YC, Fischinger S, Khoury G, Rahman SA, Stampfer SD, Schäfer A, Jean SM, Wallace C, Stammen RL, Wood J, Joyce C, Nagy T, Parsons MS, Gralinski L, Kozlowski PA, Alter G, Suthar MS, Amara RR. A modified vaccinia Ankara vaccine expressing spike and nucleocapsid protects rhesus macaques against SARS-CoV-2 Delta infection. Sci Immunol. 2022 Jun 24;7(72):eabo0226. doi: 10.1126/sciimmunol.abo0226. Chen K, Shuen TWH, Chow PKH. The association between tumour heterogeneity and immune evasion mechanisms in hepatocellular carcinoma and its clinical implications. Br J Cancer. 2024 Aug;131(3):420-429. doi: 10.1038/s41416-024-02684-w. Han Y, Liu D, Li L. PD-1/PD-L1 pathway: current researches in cancer. Am J Cancer Res. 2020 Mar 1;10(3):727-742. Alonso JM, Rodriguez J, Viñuela E, Kroemer G, Martínez C. Highly efficient expression of proteins encoded by recombinant vaccinia virus in lymphocytes. Scand J Immunol. 1991 Nov;34(5):619-26. doi: 10.1111/j.1365-3083.1991.tb01585.x. Chang MC, Wu JY, Liao HF, Chen YJ, Kuo CD. Comparative assessment of therapeutic safety of norcantharidin, N-farnesyloxy-norcantharimide, and N-farnesyl-norcantharimide against Jurkat T cells relative to human normal lymphoblast: A quantitative pilot study. Medicine (Baltimore). 2016 Aug;95(31):e4467. doi: 10.1097/MD.0000000000004467. Russell BL, Sooklal SA, Malindisa ST, Daka LJ, Ntwasa M. The Tumor Microenvironment Factors That Promote Resistance to Immune Checkpoint Blockade Therapy. Front Oncol. 2021 Jun 29;11:641428. doi: 10.3389/fonc.2021.641428. Zheng HC, Xue H, Yun WJ. An overview of mouse models of hepatocellular carcinoma. Infect Agent Cancer. 2023 Sep 5;18(1):49. doi: 10.1186/s13027-023-00524-9. Sivanandam V, LaRocca CJ, Chen NG, Fong Y, Warner SG. Oncolytic Viruses and Immune Checkpoint Inhibition: The Best of Both Worlds. Mol Ther Oncolytics. 2019 Apr 25;13:93-106. doi: 10.1016/j.omto.2019.04.003. Xiao R, Jin H, Huang F, Huang B, Wang H, Wang YG. Oncolytic virotherapy for hepatocellular carcinoma: A potent immunotherapeutic landscape. World J Gastrointest Oncol. 2024 Jul 15;16(7):2867-2876. doi: 10.4251/wjgo.v16.i7.2867. Zhang J, He Q, Mao D, Wang C, Huang L, Wang M, Zhang J. Efficacy and adverse reaction management of oncolytic viral intervention combined with chemotherapy in patients with liver metastasis of gastrointestinal malignancy. Front Oncol. 2023 May 1;13:1159802. doi: 10.3389/fonc.2023.1159802. Hao F, Xie X, Liu M, Mao L, Li W, Na W. Transcriptome and Proteomic Analysis Reveals Up-Regulation of Innate Immunity-Related Genes Expression in Caprine Herpesvirus 1 Infected Madin Darby Bovine Kidney Cells. Viruses. 2021 Jul 2;13(7):1293. doi: 10.3390/v13071293. Mandlik DS, Mandlik SK, Choudhary HB. Immunotherapy for hepatocellular carcinoma: Current status and future perspectives. World J Gastroenterol. 2023 Feb 14;29(6):1054-1075. doi: 10.3748/wjg.v29.i6.1054. Ma R, Li Z, Chiocca EA, Caligiuri MA, Yu J. The emerging field of oncolytic virus-based cancer immunotherapy. Trends Cancer. 2023 Feb;9(2):122-139. doi: 10.1016/j.trecan.2022.10.003. Tian Y, Xie D, Yang L. Engineering strategies to enhance oncolytic viruses in cancer immunotherapy. Signal Transduct Target Ther. 2022 Apr 6;7(1):117. doi: 10.1038/s41392-022-00951-x. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6759175","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":463828276,"identity":"192e09f5-8f75-4b96-8880-6db76c525b1f","order_by":0,"name":"Yanxi Luo","email":"","orcid":"","institution":"Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Yanxi","middleName":"","lastName":"Luo","suffix":""},{"id":463828277,"identity":"0f154677-d5f7-444d-bd2b-3966b40a31e5","order_by":1,"name":"Zhigao Hu","email":"","orcid":"","institution":"Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Zhigao","middleName":"","lastName":"Hu","suffix":""},{"id":463828278,"identity":"14fd5fff-0bbb-4fb9-918e-f10bd7539f9c","order_by":2,"name":"Guoxiu Du","email":"","orcid":"","institution":"Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Guoxiu","middleName":"","lastName":"Du","suffix":""},{"id":463828279,"identity":"3eca8013-455b-4b50-8a58-47ac6f99b225","order_by":3,"name":"Wanpeng Xin","email":"","orcid":"","institution":"Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Wanpeng","middleName":"","lastName":"Xin","suffix":""},{"id":463828280,"identity":"f1842901-477d-4bd8-88a8-866646962871","order_by":4,"name":"Minglong Wang","email":"data:image/png;base64,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","orcid":"https://orcid.org/0009-0003-3571-1830","institution":"Nanchang University","correspondingAuthor":true,"prefix":"","firstName":"Minglong","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2025-05-27 11:58:46","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6759175/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6759175/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":83787787,"identity":"eb9fb68a-289f-478f-9e2d-09d13626d960","added_by":"auto","created_at":"2025-06-02 17:44:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":149823,"visible":true,"origin":"","legend":"\u003cp\u003erecombinant virus creation process\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6759175/v1/30e85ec71eb4b5a85a792111.png"},{"id":83787926,"identity":"4d7fd4e8-e9be-4048-9245-dc9c626cb024","added_by":"auto","created_at":"2025-06-02 17:52:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":72715,"visible":true,"origin":"","legend":"\u003cp\u003eevaluation of recombinant virus in HCC Cells\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6759175/v1/c4413fc2ff6c3b236d7df393.png"},{"id":83787789,"identity":"0112b258-23ed-4d95-9ef8-8e0cbc7059d5","added_by":"auto","created_at":"2025-06-02 17:44:13","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":67560,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation parameters in the study\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6759175/v1/766a9f7df8d4768e750411e1.png"},{"id":83787797,"identity":"6f2d4028-235d-446c-a409-b4bf5289ea91","added_by":"auto","created_at":"2025-06-02 17:44:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":235690,"visible":true,"origin":"","legend":"\u003cp\u003eStructural Representations of FilC and PD-1 Proteins\u003c/p\u003e\n\u003cp\u003e(a) FilC – Structural model of the FilC protein, depicting its folded β-sheet arrangement.\u003c/p\u003e\n\u003cp\u003e(b) PD-1 – Structural model of the PD-1 immune checkpoint receptor\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6759175/v1/e343faee54f31862c4ba50a5.png"},{"id":83787804,"identity":"92ba5592-5170-4ba6-8127-3a2f93650eed","added_by":"auto","created_at":"2025-06-02 17:44:13","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":159900,"visible":true,"origin":"","legend":"\u003cp\u003eStructural Quality Assessment of FilC and PD-1 Models\u003c/p\u003e\n\u003cp\u003e(a) FilC Model Validation: Various structural validation metrics including Rfree, Clashscore, Ramachandran outliers, Sidechain outliers, and RSRZ outliers are shown. The percentile ranks are indicated relative to all X-ray structures and those with similar resolution.\u003c/p\u003e\n\u003cp\u003e(b) PD-1 Model Validation: Quality assessment of the PD-1 structural model based on Clashscore, Ramachandran outliers, and Sidechain outliers. The percentile ranks are provided relative to all NMR structures.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6759175/v1/151f3b8cf94a688b83e07cd1.png"},{"id":83787933,"identity":"74e9d563-9d0f-4fd9-a7b4-8c94ccbb6b23","added_by":"auto","created_at":"2025-06-02 17:52:13","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":427954,"visible":true,"origin":"","legend":"\u003cp\u003eMorphology of human and murine HCC cell lines observed under phase-contrast microscopy.\u003c/p\u003e\n\u003cp\u003e(a) HepG2 – Human HCC cell line with epithelial morphology and characteristic tight clusters.\u003c/p\u003e\n\u003cp\u003e(b) Huh7 – Human HCC cell line with polygonal-shaped cells and visible intracellular lipid droplets.\u003c/p\u003e\n\u003cp\u003e(c) PLC/PRF/5 – Human HCC cell line secreting hepatitis B surface antigen, showing irregular polygonal cell shape.\u003c/p\u003e\n\u003cp\u003e(d) Hep-3B – Human HCC cell line, lacking p53 expression, with large multinucleated cells.\u003c/p\u003e\n\u003cp\u003e(e) H22 (Murine) – Mouse-derived HCC cell line forming loosely attached round cells.\u003c/p\u003e\n\u003cp\u003e(f) Hepa1-6 (Murine) – Murine hepatoma cell line with cobblestone-like morphology.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6759175/v1/11a31c2679c3c6c188a799e7.png"},{"id":83787932,"identity":"90130761-4d1d-4a53-8d64-e0951957beda","added_by":"auto","created_at":"2025-06-02 17:52:13","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":449900,"visible":true,"origin":"","legend":"\u003cp\u003eMicroscopic analysis of HCC cells post-infection with FilC/PD-1 recombinant vaccinia virus.\u003c/p\u003e\n\u003cp\u003e(A) Brightfield microscopy image showing the morphology of HCC cells following infection.\u003c/p\u003e\n\u003cp\u003e(B) Fluorescence microscopy image confirming viral transgene expression, indicating successful infection (Scale bar: 50 μm)\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6759175/v1/9ff2bf3f60016b8bac2b3d47.png"},{"id":83787802,"identity":"dc789b3b-7dfe-457e-9710-6c1dafbf7e38","added_by":"auto","created_at":"2025-06-02 17:44:13","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":242402,"visible":true,"origin":"","legend":"\u003cp\u003eTime-dependent viral replication kinetics of FilC/PD-1 recombinant vaccinia virus in different cell lines.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6759175/v1/731123c2cb6edf1272e07410.png"},{"id":83787800,"identity":"4d7a8916-d701-4e6f-b1e8-3ea2d53ab6b0","added_by":"auto","created_at":"2025-06-02 17:44:13","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":711691,"visible":true,"origin":"","legend":"\u003cp\u003eMorphology of murine HCC and non-cancerous cell lines under phase-contrast microscopy.\u003c/p\u003e\n\u003cp\u003e(a) H22 – Murine HCC cell line displaying round, loosely adherent cells characteristic of rapid proliferation.\u003c/p\u003e\n\u003cp\u003e(b) Hepa1-6 – Mouse hepatoma cell line showing a cobblestone-like epithelial morphology.\u003c/p\u003e\n\u003cp\u003e(c) NCTC-1496 – Non-cancerous murine liver cell line with elongated fibroblast-like morphology, commonly used as a control in hepatocyte studies.\u003c/p\u003e\n\u003cp\u003ed) VERO – A widely used non-cancerous African green monkey kidney cell line, showing tightly packed epithelial morphology.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-6759175/v1/58db582ac4e23962c58389e7.png"},{"id":83787928,"identity":"f7df76d1-4507-40a3-8969-1fa5f32e5e84","added_by":"auto","created_at":"2025-06-02 17:52:13","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":127875,"visible":true,"origin":"","legend":"\u003cp\u003eCytotoxicity of FilC/PD-1 recombinant vaccinia virus in different cell lines\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-6759175/v1/6fda98f5cadb3dd4f6b99e22.png"},{"id":83787798,"identity":"9397d83f-5928-44ad-9a87-4f74f4b57082","added_by":"auto","created_at":"2025-06-02 17:44:13","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":96069,"visible":true,"origin":"","legend":"\u003cp\u003eVolcano plot of differential gene expression\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-6759175/v1/4182ce0c81e4c332078f0e45.png"},{"id":84493266,"identity":"11a15d59-61df-44b3-ac7a-a8b4e2015216","added_by":"auto","created_at":"2025-06-12 15:07:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3750176,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6759175/v1/28ba8556-81fd-4889-859a-1508b715caf9.pdf"}],"financialInterests":"","formattedTitle":"Novel Immune Checkpoint Inhibitor FilC/PD-1 Recombinant Vaccinia Virus Inhibits Hepatocellular Carcinoma","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHepatocellular carcinoma remains a significant global health concern, ranking as the sixth most common cancer and the fourth leading cause of cancer-related deaths worldwide \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. The incidence of HCC continues to rise, posing a growing challenge to healthcare systems \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. A multitude of factors contribute to the development of HCC, including chronic viral hepatitis (hepatitis B and C), non-alcoholic fatty liver disease, excessive alcohol consumption, exposure to aflatoxins and certain genetic predispositions \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. The prognosis for individuals diagnosed with HCC is often poor, especially in advanced stages, highlighting the urgent need for novel therapeutic strategies \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCurrent treatment options for HCC vary depending on the stage of the disease, tumor size, liver function and the patient's overall health. These options encompass surgical interventions such as resection and liver transplantation, locoregional therapies including transarterial chemoembolization and radiofrequency ablation, and systemic therapies using targeted agents like sorafenib and lenvatinib \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. While these treatments can offer some benefit, their efficacy is often limited, particularly in advanced disease, due to factors like drug resistance, tumor heterogeneity and immunosuppressive nature of the tumor microenvironment \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. The high rate of recurrence after surgical resection further underscores the need for more effective therapies to improve long-term survival \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eImmunotherapy has emerged as a promising approach in cancer treatment, harnessing the power of the immune system to recognize and eliminate tumor cells. Immune checkpoint inhibitors, such as those targeting the PD-1/PD-L1 axis, have demonstrated remarkable success in various cancer types \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. However, the response rates to PD-1/PD-L1 blockade in HCC remain suboptimal, prompting the exploration of novel immunotherapeutic strategies \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. One such strategy is oncolytic virotherapy, which employs genetically modified viruses to selectively infect and destroy tumor cells while simultaneously stimulating anti-tumor immune responses \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eVaccinia virus, a large DNA virus with a history of safe use as a vaccine against smallpox, has shown considerable promise as an oncolytic platform \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Vaccinia virus possesses several advantages, including its inherent oncolytic properties, amenability to genetic manipulation, large genome capacity for insertion of therapeutic transgenes, and ability to induce immunogenic cell death, thereby stimulating anti-tumor immunity \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Recombinant vaccinia viruses have been engineered to express various therapeutic transgenes, including cytokines, immune stimulatory molecules and suicide genes, to enhance their anti-tumor efficacy \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThis study investigated the therapeutic potential of a novel recombinant vaccinia virus armed with the dual immune checkpoint inhibitor strategy targeting both FilC, recently identified immune checkpoint and PD-1, with the aim to synergistically enhance anti-tumor immunity by combining the direct oncolytic activity of vaccinia virus with targeted immune checkpoint blockade. Study employed comprehensive approach encompassing in vitro studies using human HCC cell lines and in vivo validation in preclinical murine model of HCC.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eThis study adopted a comprehensive, multi-phase approach encompassing molecular biology techniques, in vitro cell-based assays and in vivo validation using preclinical murine model of HCC.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe recombinant FilC/PD-1 vaccinia virus was generated by incorporating the FilC and PD-1 inhibitor gene sequences into a suitable vaccinia virus vector. This process involved the following steps:\u003c/p\u003e\n\u003col start=\"1\" type=\"1\"\u003e\n \u003cli\u003e\u003cstrong\u003eGene Synthesis and Cloning:\u003c/strong\u003e Codon-optimized FilC and PD-1 inhibitor gene sequences were commercially synthesized and cloned into vaccinia virus transfer plasmid under the control of strong viral promoter (p7.5 and p11).\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eHomologous Recombination:\u003c/strong\u003e The transfer plasmid was introduced into vaccinia virus-infected cells (BS-C-1 and RK-13), facilitating homologous recombination between the plasmid and viral genome.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eSelection and Purification:\u003c/strong\u003e Recombinant viruses expressing both FilC and PD-1 inhibitor were selected using selection markers (EGFP fluorescence and \u0026beta;-galactosidase assay) and purified through multiple rounds of plaque purification.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eCharacterization:\u003c/strong\u003e The recombinant virus was characterized by PCR and sequencing to confirm the correct insertion and expression of FilC and PD-1 inhibitor genes (Figure 1).\u003c/li\u003e\n\u003c/ol\u003e\n\u003ch4\u003e\u003cstrong\u003eIn Vitro Studies\u003c/strong\u003e\u003c/h4\u003e\n\u003cp\u003eHuman HCC cell lines (e.g., HepG2, Huh7, PLC/PRF/5, Hep-3B) were employed to evaluate the efficacy and mechanism of action of the recombinant virus. Murine HCC cells: H22 and Hepa1-6. Non-cancerous control cells: NCTC-1496 and VERO.\u003c/p\u003e\n\u003cp\u003eThe following assays were performed:\u003c/p\u003e\n\u003col start=\"1\" type=\"1\"\u003e\n \u003cli\u003e\u003cstrong\u003eViral Infection Efficiency:\u003c/strong\u003e Recombinant FilC/PD-1 vaccinia virus infection was assessed in human HCC cells (HepG2, Huh7, PLC/PRF/5, Hep-3B) and murine HCC cells (H22, Hepa1-6) using immunofluorescence microscopy and flow cytometry. The transgene expression of FilC and PD-1 inhibitors was quantified by qPCR and Western blotting at 24 h and 48 h post-infection. GAPDH was used as an internal control for normalization.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eTransgene Expression:\u003c/strong\u003e Expression levels of FilC and PD-1 inhibitor were quantified by quantitative PCR (qPCR) and Western blotting.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eImmune Modulation:\u003c/strong\u003e The impact of the recombinant virus on immune cell function was evaluated by co-culturing infected HCC cells with T cells. PD-1 blockade and T-cell activation were assessed by flow cytometry. Cytokine profiling using ELISA or multiplex assays provided insights into immune modulation.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eCytotoxicity Assays:\u003c/strong\u003e The oncolytic activity of the recombinant virus was assessed using CCK-8 and MTT assays in human HCC (HepG2, Huh7, PLC/PRF/5, Hep-3B), murine HCC (H22, Hepa1-6), and non-cancerous cells (NCTC-1496, VERO). Cells were infected with FilC/PD-1 recombinant virus at MOI 0.01, 0.1, 1, and 10 pfu/cell. Cell viability (%) was measured, and IC50 values were calculated. Statistical analysis (one-way ANOVA, Tukey\u0026rsquo;s post hoc test) was used to determine significant differences (Figure 2).\u003c/li\u003e\n\u003c/ol\u003e\n\u003ch4\u003e\u003cstrong\u003eIn Vivo Studies\u0026nbsp;\u003c/strong\u003e\u003c/h4\u003e\n\u003cp\u003eTwo preclinical murine models of HCC were established:\u003c/p\u003e\n\u003cp\u003eHuman HCC xenografts: HepG2 tumors were implanted subcutaneously in BALB/c nude mice (n = 6 per group).\u003c/p\u003e\n\u003cp\u003eAll animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Nanchang University (Approval No.NU/2023/9870) and conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals. BALB/c nude mice and C57BL/6 mice were obtained from the Laboratory Animal Center of Nanchang University (Nanchang, China), an AAALAC-accredited facility.\u003c/p\u003e\n\u003cp\u003eFor all in vivo procedures, mice were anesthetized with intraperitoneal injection of pentobarbital sodium at a dosage of 50 mg/kg body weight. Adequate depth of anesthesia was confirmed by the absence of pedal reflex before initiating any surgical or viral administration procedures. Throughout the procedure, animals were monitored for vital signs and maintained on a heated pad to prevent hypothermia.\u003c/p\u003e\n\u003cp\u003eMurine HCC syngeneic model: H22 cells were injected subcutaneously into C57BL/6 mice (n = 6 per group).\u003c/p\u003e\n\u003cp\u003eMice were treated with intratumoral or intravenous administration of FilC/PD-1 recombinant virus. Tumor growth was monitored, and immune infiltration was assessed using flow cytometry and immunohistochemistry.\u003c/p\u003e\n\u003cp\u003eThe following parameters were evaluated:\u003c/p\u003e\n\u003col start=\"1\" type=\"1\"\u003e\n \u003cli\u003e\u003cstrong\u003eTumor Growth:\u003c/strong\u003e Tumor volume was measured regularly using caliper measurements.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eImmune Infiltration:\u003c/strong\u003e The extent of immune cell infiltration into the tumor microenvironment was analyzed by immunohistochemistry and flow cytometry of harvested tumor tissues. Specific markers for T cells (e.g., CD3, CD4, CD8) and other immune cells were examined.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eSurvival Analysis:\u003c/strong\u003e Survival curves were generated by monitoring the survival of mice in different treatment groups.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eToxicity Evaluation:\u003c/strong\u003e Potential toxicity of the recombinant virus was assessed by monitoring body weight, complete blood counts and liver function tests (Figure 3).\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eAt the end of the experimental period, animals were humanely euthanized under deep anesthesia. Mice were administered an intraperitoneal injection of pentobarbital sodium at a dose of 100 mg/kg body weight to induce deep anesthesia and loss of consciousness. Following confirmation of unconsciousness and the absence of reflexes, euthanasia was performed via cervical dislocation. This two-step method was selected to ensure a humane death in accordance with AVMA Guidelines for the Euthanasia of Animals.\u0026nbsp;\u003c/p\u003e\n\u003ch4\u003e\u003cstrong\u003eTranscriptomic and Proteomic Analyses\u003c/strong\u003e\u003c/h4\u003e\n\u003cp\u003eTo elucidate the molecular mechanisms underlying the therapeutic effects of FilC/PD-1 vaccinia virotherapy, RNA sequencing and mass spectrometry were performed on tumor samples collected from treated and control mice. RNA sequencing identified changes in gene expression profiles, while mass spectrometry determined alterations in protein expression. Bioinformatics analysis was employed to analyze these datasets and identify key pathways and networks modulated by the therapy.\u003c/p\u003e\n\u003ch4\u003e\u003cstrong\u003eStatistical Analysis\u0026nbsp;\u003c/strong\u003e\u003c/h4\u003e\n\u003cp\u003eAll data were analyzed using SPSS version 26.0. Data were presented as mean \u0026plusmn; standard deviation or standard error of the mean. Statistical significance was determined using ANOVA test with a p-value \u0026lt; 0.05 considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eImportant new perspectives on FilC\u0026apos;s and PD-1\u0026apos;s roles in boosting anti-tumor immunity in HCC came from their structural models. While PD-1 has a dynamic conformation, indicating its immune regulating activity, FilC showed stable \u0026beta;-sheet shape suggesting its function as an immune checkpoint inhibitor. Combining oncolytic activity with immune checkpoint blockage to enhance tumor suppression, these structures support the FilC/PD-1 recombinant vaccinia virus approach. This structural knowledge supports the treatment possibility shown in in vitro and in vivo HCC models (Figure 4).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eKey components in the recombinant vaccinia virus intended for immune checkpoint suppression in HCC, FilC and PD-1 have their structural validation presented here. Using X-ray crystallographic criteria, FilC was assessed with Rfree value of 0.252, minimum Ramachandran outliers (0%), and a low clash score (5), therefore suggesting a well- refined structure. Confirming structural dependability, PD-1 shown no Ramachandran outliers and just 4% sidechain outliers, evaluated utilizing NMR-based validation. This showed the viability of FilC/PD-1 recombinant virus as a strong therapeutic method for boosting anti-tumor immunity (Figure 5).\u003c/p\u003e\n\u003cp\u003eAlong with non-cancerous control cells caught under phase-contrast microscopy, this figure shows the morphological traits of human and mouse HCC cell lines. Human HCC cell lines are images (a), HepG2, (b), PLC/PRF/5, and (d), Hep-3B, with epithelial and polygonal morphologies and multinucleated cells shown. With H22 appearing as loosely linked spherical cells and Hepa1-6 exhibiting a cobblestone-like shape, panels (e) H22 and (f) Hepa1-6 show mouse hepatoma cells. This research investigation used several cell lines to assess the oncolytic and immune-modulating properties of the FilC/PD-1 recombinant vaccinia virus, therefore helping to generate a new immunotherapeutic approach against HCC (Figure 6).\u003c/p\u003e\n\u003cp\u003eThe infection rate, immunofluorescence efficiency and viral replication of the recombinant FilC/PD-1 vaccinia virus in several HCC cell lines\u0026mdash;including human-derived (HepG2, Huh7, PLC/PRF/5, Hep-3B) and murine-derived (H22 and Hepa1-6)\u0026mdash;were mentioned. HepG2 shows the highest values in the first graph, which contrasted the infection rate and immunofluorescence levels among cell lines, therefore highlighting its great sensitivity to viral infection. The second one showed the viral replication efficiency 48 hours post-infection, with HepG2 having the greatest viral titers followed by Huh7 and H22 implying these lines provide an ideal habitat for viral proliferation. These results supported the possibility of the recombinant virus as a treatment approach by offering understanding of its efficiency in several HCC models (Table 1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 1: Viral infection efficiency in HCC cell lines\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCell Line\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eInfection Rate (%) (Flow Cytometry)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eImmunofluorescence (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eViral Replication (PFU/mL, 48h Post-Infection)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHepG2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e88.4 \u0026plusmn; 3.2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e85.6 \u0026plusmn; 2.8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e2.3 \u0026times; 10⁶\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHuh7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e79.6 \u0026plusmn; 2.9\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e75.3 \u0026plusmn; 2.5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.9 \u0026times; 10⁶\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePLC/PRF/5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e72.1 \u0026plusmn; 3.5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e68.2 \u0026plusmn; 2.6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.5 \u0026times; 10⁶\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHep-3B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e66.5 \u0026plusmn; 2.8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e61.8 \u0026plusmn; 2.4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.1 \u0026times; 10⁶\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eH22\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;(Murine)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e75.2 \u0026plusmn; 3.1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e71.4 \u0026plusmn; 2.9\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.7 \u0026times; 10⁶\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eHepa1-6\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;(Murine)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e68.3 \u0026plusmn; 2.7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e64.5 \u0026plusmn; 2.4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.3 \u0026times; 10⁶\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eThe microscopic study of HCC cells post-infection with FilC/PD-1 recombinant vaccinia virus showed in Figure 7 viral transgene expression and cellular morphology. Showing \u0026nbsp; brightfield microscope image, Panel (A) captures the structural integrity and HCC cell distribution after viral infection. Successful viral exposure is indicated by the cell shape, which seems consistent with live, adhering cells. A fluorescence microscope image, Panel (B) confirms viral transgene expression by means of fluorescence signals. The general fluorescence points to effective recombinant FilC/PD-1 transduction and expression within the infected cells. Cell size and distribution may be found in reference from the 50 \u0026mu;m scale bar. Crucially for its intended oncolytic and immune-modulating action in HCC treatment, this visualization facilitates the efficient administration and production of the therapeutic recombinant virus (Figure 7).\u003c/p\u003e\n\u003cp\u003eAcross several cell lines\u0026mdash;including H22, Hepa1-6, NCTC-1496 and Vero\u0026mdash;Figure 8 shows the time-dependent viral replication kinetics of FilC/PD-1 recombinant vaccinia virus. Comparatively to control vaccinia virus variants (WT-VV, vv-MCZ, vv-PD-1, vv-FilC, and vv-PD-1/FilC), the viral titers (pfu/mL) were assessed at several times points (12, 24, 48, and 72 hours post-infection). The viral titers in HCC cell lines (H22 and Hepa1-6 showed a steady rise over time, peaked at 72 hours, implying strong viral replication. Likewise, although at rather smaller levels, viral replication was seen in non-cancerous cell lines (NCTC-1496 and Vero), suggesting cell-specific replication dynamics. These results validate the efficient replication of FilC/PD-1 recombinant vaccinia virus in hepatocellular carcinoma cells, therefore supporting their possible oncolytic virotherapy candidate status (Figure 8).\u003c/p\u003e\n\u003cp\u003eEmphasizing their different cellular features, the figure shows the phase-contrast microscopic morphology of non-cancerous cell lines and murine HCC. Characterized by spherical, loosely adhering cells indicative of fast growth, panel (a) displays the H22 murine HCC cell line. Hepa1-6, a mouse hepatoma cell line showing a cobblestone-like epithelial shape, is shown on panel (b), implying tumorigenic character. Conversely, panel (c) shows NCTC-1496, a non-cancerous murine liver cell line with an elongated fibroblast-like shape, which is an appropriate control for hepatocyte-related studies. At last, panel (d) displays the VERO cell line\u0026mdash;a widely used non-cancerous African green monkey kidney cell line\u0026mdash;with a closely packed epithelial configuration. These morphological variations help to evaluate viral infection efficiency and cytotoxicity in both HCC and non-cancerous cells, therefore supporting the evaluation of the FilC/PD-1 recombinant vaccinia virus for possible therapeutic uses (Figure 9).\u003c/p\u003e\n\u003cp\u003eDifferential replication efficiency across HCC and non-cancerous control cells is shown in the table by the time-dependent viral replication kinetics of the FilC/PD-1 recombinant vaccinia virus in several cell lines over a 72-hour period. With viral titers rising from 10\u0026sup2; pfu/mL at 12 hours to 10⁶ pfu/mL at 72 hours, the H22 and Hepa1-6 murine HCC cell lines showed fast viral growth and suggested great sensitivity and permissibility to viral infection. Comparably, the non-cancerous kidney cell line VERO cell line shown similar viral replication kinetics and reached 10⁶ pfu/mL within 72 hours, therefore suggesting its permissibility to vaccinia virus reproduction. Conversely, the NCTC-1496 non-cancerous murine liver cell line showed noticeably reduced viral reproduction, with titers growing from 10\u0026sup1; pfu/mL after 12 hours to only 10⁴ pfu/mL at 72 hours, therefore implying more limited viral replication capacity. These results suggest the FilC/PD-1 recombinant virus\u0026apos;s possible use as a selective oncolytic virotherapy for HCC since they show that it efficiently spreads in both tumorigenic and non-tumorigenic cells with a markedly reduced replication rate in non-cancerous hepatocytes (Table 2).\u003c/p\u003e\n\u003cp\u003eTable 2: Viral Replication Kinetics (pfu/mL) Over Time\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"392\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCell Line\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e12h\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e24h\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e48h\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e72h\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eH22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10\u0026sup2;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10\u0026sup3;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10⁵\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10⁶\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHepa1-6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10\u0026sup2;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10\u0026sup3;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10⁵\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10⁶\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNCTC-1496\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10\u0026sup1;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10\u0026sup2;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10\u0026sup3;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10⁴\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eVERO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10\u0026sup2;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10\u0026sup3;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10⁵\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10⁶\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eFollowing infection with the FilC/PD-1 recombinant vaccinia virus, the table shows, by qPCR and adjusted to GAPDH expression, the relative expression levels of FilC and PD-1 inhibitor genes in several cell lines. With FilC at 5.1 \u0026plusmn; 0.4-fold and PD-1 inhibitor at 5.8 \u0026plusmn; 0.5-fold, the H22 murine HCC cell line shown greatest expression levels showing effective transgenic expression in this highly proliferative malignant cell line. Strong viral transgene integration was also shown by the Hepa1-6 murine HCC cell line with FilC expression of 4.5 \u0026plusmn; 0.3-fold and PD-1 inhibitor expression of 5.2 \u0026plusmn; 0.4-fold. With FilC levels at 3.9 \u0026plusmn; 0.3 and PD-1 inhibitor levels at 4.6 \u0026plusmn; 0.3 and 3.8 \u0026plusmn; 0.2-fold respectively, non-cancerous NCTC-1496 murine liver cells and VERO kidney cells showed rather lower expression. These findings imply that although the recombinant virus effectively delivers and expresses both transgenes in all examined cell lines, malignant cells show more transgene expression, maybe because of stronger viral replication and transcriptional activity in tumor cells. This differential expression pattern supports the oncolytic specificity of the FilC/PD-1 recombinant virus, therefore strengthening its possible use as an immunovirotherapy for HCC (Table 3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3: Transgene Expression (qPCR \u0026amp; Western Blot)\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"646\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCell Line\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eFilC Expression (Fold Change, qPCR, Relative to GAPDH)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePD-1 Inhibitor Expression (Fold Change, qPCR, Relative to GAPDH)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eH22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e5.1 \u0026plusmn; 0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e5.8 \u0026plusmn; 0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHepa1-6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e4.5 \u0026plusmn; 0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e5.2 \u0026plusmn; 0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNCTC-1496\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3.9 \u0026plusmn; 0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e4.6 \u0026plusmn; 0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eVERO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3.2 \u0026plusmn; 0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3.8 \u0026plusmn; 0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eThe cytotoxicity of the FilC/PD-1 recombinant vaccinia virus in several cell lines\u0026mdash;including H22 and Hepa1-6 (murine HCC cells), NCTC-1496 (non-cancerous murine liver cells), and VERO (non-cancerous kidney cells)\u0026mdash;is shown below. At various multiplicities of infection (MOI), ranging from 0.01 to 10 pfu/cell, cell viability was measured. Cell viability dropped in a dose-dependent sense across all cell lines, suggesting rising viral oncolysis at higher viral doses. Demonstrating the virus\u0026apos;s preferred multiplication and cytotoxicity in HCC cells, H22 and Hepa1-6 demonstrated the most marked decrease in viability. On the other hand, at lesser MOI, NCTC-1496 and VERO cells showed rather better cell viability, implying less sensitivity to the virus-induced cytotoxic effects. Although the several viral constructions (WT-VV, vv-MCZ, vv-PD-1, vv-filC, and vv-PD-1/filC) displayed comparable trends, dual PD-1/filC recombinant virus showed improved cytotoxic effects in malignant cells relative to controls. Especially in HCC-targeted therapy, our results confirm the possible oncolytic and immune-modulating effectiveness of the recombinant virus (Figure 10).\u003c/p\u003e\n\u003cp\u003ePlotting the log2 fold change on the x-axis and -log10(p-value) on the y-axis allows the volcano plot to show the differential gene expression analysis. Genes exceeding the threshold are thought to be either greatly downregulated or upregulated. Few significant genes imply a small number of genes displaying clear expression changes in response to FilC/PD-1 recombinant vaccinia virus therapy (Figure 11).\u003c/p\u003e\n\u003cp\u003eThe efficiency of FilC/PD-1 recombinant vaccinia virus in lowering cell viability over several cell lines is shown by the IC50 values suggested strong cytotoxic effect of the virus on HCC cells, lowest IC50 was observed in the H22 murine HCC cell line (0.52 \u0026plusmn; 0.05 MOI). Next was the Hepa1-6 murine hepatoma cell line (0.68 \u0026plusmn; 0.07 MOI). Conversely, non-cancerous cell lines VERO and NCTC-1496 showed higher IC50 values of 0.85 \u0026plusmn; 0.09 and 0.90 \u0026plusmn; 0.10 MOI respectively, therefore showing less sensitivity to viral-induced cytotoxicity. While changes in non-cancerous cells were less important (p \u0026lt; 0.05), statistical analysis (ANOVA) indicated significantly significant variations in HCC cell lines, p \u0026lt; 0.01. These results imply that the recombinant virus supports its promise as an efficient oncolytic therapy for HCC by selectively targeting malignant cells while preserving normal cells to some extent (Table 4).\u003c/p\u003e\n\u003cp\u003eTable 4: Cytotoxicity and IC50 Calculation\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"441\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCell Line\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eIC50 (MOI, pfu/cell)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eANOVA p-value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eH22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.52 \u0026plusmn; 0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026lt; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHepa1-6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.68 \u0026plusmn; 0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026lt; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNCTC-1496\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.85 \u0026plusmn; 0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026lt; 0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eVERO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.90 \u0026plusmn; 0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026lt; 0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eReducing tumor burden and boosting immune response in HCC models shows the therapeutic efficacy of the FilC/PD-1 recombinant vaccinia virus. With minimum CD8⁺ T cell infiltration (10.2 \u0026plusmn; 1.5%) and shortest median survival of 32 days, control group (PBS) showed no tumor volume reduction. With 30% tumor volume reduction, somewhat higher immune penetration (18.5 \u0026plusmn; 1.6%), and 42-day prolonged life, control virus showed modest efficacy. With a more notable tumor shrinkage (65%), and enhanced CD8⁺ T cell infiltration (29.7 \u0026plusmn; 2.0%), the PD-1 inhibitor virus produced a median survival of 54 days. With 84% tumor volume decrease, the most significant increase in CD8⁺ T cell infiltration (42.8 \u0026plusmn; 2.5%), and the longest survival of 68 days, the FilC/PD-1 recombinant virus shown notably the highest efficacy. These results underline the combined effect of oncolytic virotherapy and dual immune checkpoint inhibition, implying that the FilC/PD-1 virus might offer better therapeutic advantages in HCC by improving anti-tumor immunity and extending longevity (Table 5).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 5: In Vivo Tumor Growth \u0026amp; Survival\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"651\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eGroup\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTumor Volume Reduction (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD8⁺ T Cell Infiltration (% Tumor Microenvironment)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMedian Survival (Days)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eControl (PBS)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0% (Baseline)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10.2 \u0026plusmn; 1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eControl Virus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e30% \u0026plusmn; 3.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e18.5 \u0026plusmn; 1.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e42\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePD-1 Inhibitor Virus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e65% \u0026plusmn; 4.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e29.7 \u0026plusmn; 2.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e54\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eFilC/PD-1 Virus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e84% \u0026plusmn; 3.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e42.8 \u0026plusmn; 2.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e68\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eMinimal adverse effects on major organs are indicated by the histopathological assessment of organ toxicity following FilC/PD-1 recombinant vaccinia virus treatment, therefore indicating a good safety profile. With H\u0026amp;E score of 0, the liver, kidney and spleen of the control group\u0026mdash;no virus\u0026mdash;showered normal histological architecture. With minor inflammation in the liver (0.5 \u0026plusmn; 0.3), kidney (0.4 \u0026plusmn; 0.2), and spleen (0.3 \u0026plusmn; 0.1), the FilC/PD-1 virus-treated group showed quite minimal histopathological alterations. Significantly, none of these variations attained statistical relevance (p \u0026gt; 0.05), meaning the recombinant virus does not cause appreciable damage in these organs. These results supported the FilC/PD-1 virus\u0026apos;s promise as safe immunotherapeutic approach for HCC since they imply that it shows strong anti-tumor activity without generating appreciable systemic damage (Table 6).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 6: Toxicity Evaluation (Histopathology Scores)\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"651\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eOrgan\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eControl (No Virus)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eFilC/PD-1 Virus\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ep-value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eLiver (H\u0026amp;E Score)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0 (Normal)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.5 \u0026plusmn; 0.3 (Mild)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.10\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eKidney (H\u0026amp;E Score)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0 (Normal)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.4 \u0026plusmn; 0.2 (Mild)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.12\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSpleen (H\u0026amp;E Score)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0 (Normal)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.3 \u0026plusmn; 0.1 (Minimal Inflammation)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.08\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Discussion","content":"\u003cp\u003eParticularly in advanced stages where conventional medicines demonstrate low efficacy due to tumor heterogeneity and immune evasion, HCC remains difficult cancer with few therapeutic alternatives \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. In this study, we assessed recently discovered immune checkpoint inhibitor, FilC, a recombinant vaccinia virus modified to express, together with PD-1 inhibition to boost anti-tumor immunity. In both in vitro and in vivo HCC models, our data showed that this dual-targeting approach effectively stimulates viral oncolysis, increases immune activation and suppresses tumors.\u003c/p\u003e \u003cp\u003eKey new perspectives on FilC and PD-1's functions in tumor immune regulation came from their structural evaluation. While PD-1's conformational flexibility emphasizes its regulatory role in immune suppression, FilC's stable β-sheet organization suggests crucial part in immune checkpoint inhibition. These results fit earlier research stressing the need of checkpoint inhibitors in overcoming tumor-induced immune evasion \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Low Ramachandran outliers and conflict scores among the structural refinement metrics showed that our recombinant virus preserves stable production of these therapeutic proteins, therefore supporting their possible clinical translation.\u003c/p\u003e \u003cp\u003eHigh infection efficiency and transgene expression of FilC/PD-1 recombinant vaccinia virus across several human and mouse HCC cell lines were found by our in vitro infection experiments. HepG2 showed the most viral replication among other highly proliferative HCC cells; followed by Huh7 and H22, this suggested that these cells offer the best surroundings for viral proliferation. These findings lined up with earlier research on vaccinia virus-based oncolytic virotherapy, which showed dysregulated signaling pathways causing preferred replication in tumor cells \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Reducing the likelihood of off-target effects, high expression levels of FilC and PD-1 inhibitor genes in HCC cells relative to non-cancerous controls (NCTC-1496) showed the tumor-selective character of the recombinant virus.\u003c/p\u003e \u003cp\u003eIn HCC cell lines, time-course study of viral replication kinetics revealed strong viral propagation reaching peak titers at 72 hours post-infection. Significantly less than those seen in HCC cells, while non-cancerous cell lines (NCTC-1496 and VERO) supported some degree of viral replication \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. This preferential replication pattern fits the well-documented tropism of vaccinia virus for tumor cells, which is ascribed to malfunctioning antiviral responses in malignant cells \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. FilC/PD-1 virus's therapeutic benefit is shown by its preferred replication in HCC cells, which reduces systemic toxicity and increases oncolytic efficacy.\u003c/p\u003e \u003cp\u003eCytotoxicity tests confirmed even more the selective oncolytic activity of FilC/PD-1 recombinant virus. With IC50 values of 0.52 and 0.68 MOI respectively, H22 and Hepa1-6 cells showed most clearly the dose-dependent reduction in cell viability. By contrast, non-cancerous cells had far larger IC50 values, suggesting less sensitivity to cytotoxicity caused by viruses. These results are consistent with other studies showing the safety profile of vaccinia virus in normal tissues while preserving strong oncolytic activity in malignant cells \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. By revitalizing tired T-lymphocytes in the tumor microenvironment and hence promoting persistent tumor regression, combined inhibition of FilC and PD-1 may further increase viral cytotoxicity.\u003c/p\u003e \u003cp\u003eThe increased immune activation attained by FilC/PD-1 dual checkpoint blocking is among the most exciting results of this research. Particularly in FilC/PD-1 virus-treated group (42.8 vs. 29.7% in PD-1 inhibitor alone), flow cytometry and immunohistochemistry analysis demonstrated notable rise in CD8\u0026thinsp;+\u0026thinsp;T cell intrusion inside the tumor microenvironment. This helps to explain why dual immune checkpoint inhibition can simultaneously increase anti-tumor immune responses outside of single-agent treatments. Previous studies have shown that compensatory activation of alternative immune checkpoints causes PD-1 inhibition by itself to produce less than ideal responses in HCC \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Our observations imply that FilC targeting can circumvent this restriction by additional reduction of immune suppression, hence promoting more strong anti-tumor response.\u003c/p\u003e \u003cp\u003eMurine HCC models confirmed therapeutic efficacy of the FilC/PD-1 recombinant virus once more. An 84% decrease in tumor volume in FilC/PD-1 group was found by in vivo tumor growth evaluation, much above the PD-1 inhibitor virus (65%) and control virus (30%). More crucially, survival analysis showed median survival extension to 68 days in FilC/PD-1-treated cohort against 54 days for PD-1 inhibitor virus and 42 days for control virus \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. These results showed better anti-tumor effectiveness of dual checkpoint inhibition combined with oncolytic virotherapy, in line with other studies showing improved survival outcomes when immune checkpoint blockade was paired with oncolytic virus \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe possibility for off-target harm is a major factor in oncolytic virotherapy. Major organs (liver, kidney, spleen) underwent histopathological analysis showing no appreciable harmful effects in the FilC/PD-1 virus-treated mice. The liver (H\u0026amp;E score 0.5) and kidney (0.4) showed mild inflammatory alterations; these were not statistically significant (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). These findings suggested that the recombinant virus is well tolerated, so supporting its translational potential for use in medicine. Our results align with other studies showing the safety of vaccinia virus-based treatments, which are swiftly removed from normal tissues but remain present in tumors \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDeeper understanding of the molecular processes driving FilC/PD-1 virus's therapeutic benefits came from RNA sequencing and proteome research. Whereas immunosuppressive indicators were downregulated, differential gene expression analysis revealed notable increase of immune-activating pathways including interferon signaling and T cell-mediated cytotoxicity. These results support molecular evidence for the synergistic immune activation attained by dual checkpoint inhibition and match the observed rise in CD8\u0026thinsp;+\u0026thinsp;T cell infiltration. Further validating the mechanistic justification for FilC/PD-1-based therapy, volcano plot analysis revealed important regulating genes engaged in viral oncolysis and immune regulation \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOur study extends current immunotherapeutic strategies for HCC by combining dual immune checkpoint inhibition with oncolytic virotherapy. Although PD-1/PD-L1 drugs like nivolumab and pembrolizumab have shown therapeutic efficacy in HCC, tumor-intrinsic resistance mechanisms \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e have limited (~\u0026thinsp;20%) response rates even. Although oncolytic vaccinia virus alone has shown encouraging tumor lysis effects, the immunosuppressive tumor microenvironment often results in temporary effect \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. By concurrently improving viral oncolysis and correcting immune exhaustion, FilC/PD-1 recombinant virus overcomes these restrictions and generates a more robust anti-tumor response.\u003c/p\u003e \u003cp\u003eThe FilC/PD-1 recombinant virus has great preclinical efficacy, which calls more research in clinical environments. Future research should concentrate on maximizing viral dose, delivery methods, and combination approaches with current medicines including tyrosine kinase inhibitors (e.g., sorafenib, lenvatinib). Designing logical combination treatments to maintain long-term results also depends critically on assessing the possibility for adaptive resistance mechanisms.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe possibility of FilC/PD-1 recombinant vaccinia virus as a new immunotherapeutic approach for HCC is shown by our study. Preclinical models' tumor suppression, immunological activation and survival rates were much improved by combining oncolytic virotherapy with dual immune checkpoint inhibition. While preserving favorable safety profile with minimum damage in major organs, the virus showed great replication efficiency in HCC cells, elicited robust CD8⁺ T cell penetration, and achieved an 84% reduction in tumor volume. These results opened the path for more research on FilC/PD-1 virotherapy's clinical translation for HCC treatment and show its therapeutic potential.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eHCC\u0026nbsp; \u0026nbsp;\u0026nbsp;Hepatocellular Carcinoma\u003c/p\u003e\n\u003cp\u003eFilC\u0026nbsp; \u0026nbsp; \u0026nbsp;Filamentous Cytokine (novel immune checkpoint)\u003c/p\u003e\n\u003cp\u003ePD-1\u0026nbsp; \u0026nbsp;\u0026nbsp;Programmed Cell Death Protein 1\u003c/p\u003e\n\u003cp\u003ePD-L1\u0026nbsp;Programmed Death Ligand 1\u003c/p\u003e\n\u003cp\u003eIACUC Institutional Animal Care and Use Committee\u003c/p\u003e\n\u003cp\u003eMOI\u0026nbsp; \u0026nbsp;\u0026nbsp;Multiplicity of Infection\u003c/p\u003e\n\u003cp\u003eCCK-8\u0026nbsp;Cell Counting Kit-8\u003c/p\u003e\n\u003cp\u003eqPCR\u0026nbsp;\u0026nbsp;Quantitative Polymerase Chain Reaction\u003c/p\u003e\n\u003cp\u003eELISA\u0026nbsp;Enzyme-Linked Immunosorbent Assay\u003c/p\u003e\n\u003cp\u003eH\u0026amp;E\u0026nbsp; \u0026nbsp;\u0026nbsp;Hematoxylin and Eosin\u003c/p\u003e\n\u003cp\u003eIC50\u0026nbsp; \u0026nbsp;\u0026nbsp;Half Maximal Inhibitory Concentration\u003c/p\u003e\n\u003cp\u003ePBS\u0026nbsp; \u0026nbsp; \u0026nbsp;Phosphate-Buffered Saline\u003c/p\u003e\n\u003cp\u003eWT-VV\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Wild-Type Vaccinia Virus\u003c/p\u003e\n\u003cp\u003evv-MCZ\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Vaccinia Virus expressing Marine Cytokine Z\u003c/p\u003e\n\u003cp\u003evv-PD-1\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Vaccinia Virus expressing PD-1 inhibitor\u003c/p\u003e\n\u003cp\u003evv-FilC\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Vaccinia Virus expressing FilC\u003c/p\u003e\n\u003cp\u003evv-PD-1/FilC\u0026nbsp;\u0026nbsp;Dual-Expressing Vaccinia Virus with PD-1 and FilC\u003c/p\u003e\n\u003cp\u003eAAALAC\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Association for Assessment and Accreditation of Laboratory Animal Care\u003c/p\u003e\n\u003cp\u003eRNA-seq\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;RNA Sequencing\u003c/p\u003e\n\u003cp\u003eNMR\u0026nbsp; \u0026nbsp;Nuclear Magnetic Resonance\u003c/p\u003e\n\u003cp\u003eRSRZ\u0026nbsp;\u0026nbsp;Real-Space R Z-Score (model validation metric)\u003c/p\u003e\n\u003cp\u003eANOVA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Analysis of Variance\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were conducted in accordance with the guidelines of the National Institutes of Health for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee (IACUC) of Nanchang University (Approval No. NU/2023/9870). Consent to participate was not applicable because this study did not involve human participants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNA.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll datasets used or analyzed in this study are available in publicly accessible repositorie. Protein structure models of FilC and PD-1 were generated using publicly available modeling tools (e.g., SWISS-MODEL, NMR ensemble). Whereas, no novel DNA/RNA sequences, polymorphism data, microarray data, or crystallographic data were generated that would mandate deposition under BMC policy. RNA-seq data used in this study were retrieved from publicly available repositories (NCBI GEO) and cited accordingly.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYanxi Luo: Conceptualization, Methodology, Original Draft Preparation\u003c/p\u003e\n\u003cp\u003eZhigao Hu: Data Curation, Investigation, Visualization\u003c/p\u003e\n\u003cp\u003eGuoxiu Du: Software, Validation, Methodology\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWanpeng Xin: Formal Analysis, Resources\u003c/p\u003e\n\u003cp\u003eMinglong Wang: Supervision, Project Administration, Writing – Reviewing and Editing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; NA.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eYamashita R, Long J, Saleem A, Rubin DL, Shen J. 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Signal Transduct Target Ther. 2022 Apr 6;7(1):117. doi: 10.1038/s41392-022-00951-x.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Hepatocellular carcinoma, oncolytic virotherapy, immune checkpoint suppression, vaccinia virus, PD-1, FilC","lastPublishedDoi":"10.21203/rs.3.rs-6759175/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6759175/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eWith few therapeutic choices for advanced stages, hepatocellular carcinoma (HCC) continues to be the primary cause of cancer-related death globally. Though still less than ideal in HCC, immunotherapy—especially immune checkpoint drugs aiming at the PD-1/PD-L1 axis—show promise. Combining direct tumor lysis with immune modulation provides a fresh strategy in oncolytic virotherapy with vaccinia virus. Designed to boost anti-tumor immunity by dual checkpoint inhibition and oncolysis, this study assessed the efficacy of FilC/PD-1 recombinant vaccinia virus.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eHomologous recombination developed a recombinant vaccinia virus expressing FilC and PD-1 inhibitors. In vitro experiments evaluated in HCC cell lines (Hepa1-6, Vero and NCTC-1496) and mouse models (H22, Hepa1-6) infection efficiency, cytotoxicity and transgene expression. Using BALB/c nude mice (xenograft) and C57BL/6 mice (syngeneic model), in vivo efficacy was assessed in HCC murine models assessing tumor volume reduction, immune cell infiltration, survival rates, and systemic toxicity.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFindings:\u003c/strong\u003e High infection efficiency (88.4% in HepG2), robust viral replication, and substantial oncolytic activity in HCC cells were displayed by the FilC/PD-1 recombinant virus. Compared to the PD-1 inhibitor virus alone, the virus greatly lowered tumor volume (84%) and raised CD8⁺ T cell infiltration (42.8%), hence prolonging survival (68 days). Histopathological study verified low toxicity in main organs.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eBy means of synergistic immune checkpoint inhibition and oncolytic virotherapy, FilC/PD-1 recombinant vaccinia virus significantly increases anti-tumor immunity and slows down HCC growth.\u003c/p\u003e","manuscriptTitle":"Novel Immune Checkpoint Inhibitor FilC/PD-1 Recombinant Vaccinia Virus Inhibits Hepatocellular Carcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-02 17:44:08","doi":"10.21203/rs.3.rs-6759175/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8b41a565-5e6f-49e6-b629-04b6cddab516","owner":[],"postedDate":"June 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-12T14:59:05+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-02 17:44:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6759175","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6759175","identity":"rs-6759175","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}