Recombinant Anti-PD-1-Expressing oncolytic influenza virus combined with radiotherapy synergistically inhibits hepatocellular carcinoma growth through systemic T-Cell activation

WANG Ya; ZHANG Yuan; CHEN Shuhui; ZENG Guineng; YANG Penghui

doi:10.16016/j.2097-0927.202511074

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Recombinant Anti-PD-1-Expressing oncolytic influenza virus combined with radiotherapy synergistically inhibits hepatocellular carcinoma growth through systemic T-Cell activation

Basic Medicine | 更新时间：2026-03-30

- Recombinant Anti-PD-1-Expressing oncolytic influenza virus combined with radiotherapy synergistically inhibits hepatocellular carcinoma growth through systemic T-Cell activation
- Journal of Army Medical University Vol. 48, Issue 6, Pages: 733-744(2026)
- 作者机构：
  
  1.解放军医学院，北京
  2.中国人民解放军总医院第一医学中心肝胆胰外科医学部研究所，北京
  3.内蒙古医科大学基础医学院，内蒙古呼和浩特
  4.南开大学医学院，天津
- 作者简介：
  
  YANG Penghui， E-mail： ypenghuiamms@hotmail.com
- 基金信息：
- DOI：10.16016/j.2097-0927.202511074
  CLC： R735.7;R730.51;R730.55
- Received：14 November 2025，
  
  Revised：2026-01-22，
  
  Published：30 March 2026
- 稿件说明：
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王雅，章元，陈淑慧，等.表达PD1抗体的重组溶瘤流感病毒联合放疗通过激活全身T细胞免疫协同抑制肝癌细胞生长［J］.陆军军医大学学报，2026，48（06）：733-744.DOI：10.16016/j.2097-0927.202511074.

WANG Y，ZHANG Y，CHEN S H，et al.Recombinant Anti-PD-1-Expressing oncolytic influenza virus combined with radiotherapy synergistically inhibits hepatocellular carcinoma growth through systemic T-Cell activation［J］.J Army Med Univ，2026，48（06）：733-744.DOI：10.16016/j.2097-0927.202511074.
王雅，章元，陈淑慧，等.表达PD1抗体的重组溶瘤流感病毒联合放疗通过激活全身T细胞免疫协同抑制肝癌细胞生长［J］.陆军军医大学学报，2026，48（06）：733-744.DOI：10.16016/j.2097-0927.202511074. DOI：

WANG Y，ZHANG Y，CHEN S H，et al.Recombinant Anti-PD-1-Expressing oncolytic influenza virus combined with radiotherapy synergistically inhibits hepatocellular carcinoma growth through systemic T-Cell activation［J］.J Army Med Univ，2026，48（06）：733-744.DOI：10.16016/j.2097-0927.202511074. DOI：

摘要

目的

探讨表达人程序性细胞死亡蛋白1（programmed cell death protein 1，PD-1）抗体的重组溶瘤流感病毒（OVFlu-αhPD1）联合放疗（radiotherapy，RT）对肝癌的协同抑瘤作用及其免疫学机制。

方法

前期利用反向遗传学（reversed genetics，RG）技术成功拯救获得

重组溶瘤病毒，命名为OVFlu-αhPD1，通过鸡红细胞凝集实验，并在人源肝癌细胞Huh7与鼠源肝癌细胞Hepa1-6中检测病毒复制动力学进行全面鉴定。体外实验采用CCK8法检测不同感染复数（MOI=0.1、1、3）的OVFlu-αhPD1单独或联合RT（8 Gy）治疗对肝癌细胞（Huh7、Hepa1-6）与正常肝细胞（LX2、AML12）活力的影响，并通过细胞克隆形成实验评估OVFlu-αhPD1联合放疗治疗对Huh7细胞增殖的抑制作用。体内实验选用6~8周龄体质量17~19 g雌性C57BL/6小鼠（

=45），建立Hepa1-6皮下荷瘤模型，随机分为5组（

=9）：PBS对照组、野生型病毒PR8组、OVFlu-αhPD1组、RT组（24 Gy，分3次）及OVFlu-αhPD1+RT组评价联合治疗的抗肿瘤效果。治疗期间每2天测量肿瘤体积，并于末次治疗后采集脾脏进行流式细胞术分析CD4⁺CD69⁺与CD8⁺CD69⁺ T细胞比例；取心、肝、肺、肾、脑进行HE染色观察组织病理变化；采集血清检测谷草转氨酶（aspartate aminotransferase，AST）与谷丙转氨酶（alanine aminotransferase，ALT）活性水平，评估溶瘤病毒治疗的安全性。

结果

重组溶瘤流感病毒OVFlu-αhPD1的血凝效价为2

，且可在Huh7和Hepa1-6细胞中高效复制，感染72 h时病毒滴度可达7~8 lgTCID₅₀/mL。CCK8实验显示，OVFlu-αhPD1以剂量依赖方式抑制肝癌细胞活力（MOI=3时，Huh7与Hepa1-6细胞活力明显下降，

0.01），联合RT后抑制效果进一步增强（

0.01），而对正常肝细胞无明显毒性。克隆形成实验表明联合处理显著减少Huh7细胞克隆数量（

0.01）。在肝癌荷瘤小鼠模型中，联合治疗组肿瘤体积显著低于各单药组（

0.01），脾脏中CD4⁺CD69⁺与CD8⁺CD69⁺ T细胞比例均显著升高（

0.01）。HE染色结果显示主要脏器未见明显病理学改变；小鼠血清中的ALT和AST水平均未观察到统计学上的显著差异（

0.05）。

结论

溶瘤流感病毒联合放疗体外可显著增强对肝癌细胞的杀伤作用，体内明显增强肝癌荷瘤小鼠抑瘤效果并激活机体抗肿瘤免疫应答，显现出良好的协同抗肿瘤效应，为溶瘤病毒联合治疗临床应用提供了理论依据。

Abstract

Objective

To investigate the synergistic antitumor effect and immunological mechanisms of a recombinant oncolytic influenza virus carrying programmed cell death 1 (PD-1) antibody (OVFlu-αhPD1) combined with radiotherapy (RT) in hepatocellular carcinoma (HCC).

Methods

A recombinant oncolytic virus was successfully rescued in the early stage using reverse genetics (RG) technology

and designated OVFlu-αhPD1

which was comprehensively characterized

via

chicken red blood cell hemagglutination assay and detection of replication kinetics in human hepatocellular carcinoma cells Huh7 and murine hepatocellular carcinoma cells Hepa1-6. In vitro

CCK-8 ass

ays were used to assess the effects of OVFlu-αhPD1 at different multiplicities of infection (MOI=0.1

and 3)

alone or combined with radiotherapy (RT; 8 Gy)

on the viability of hepatoma cells (Huh7 and Hepa1-6) and normal hepatocytes (LX2 and AML12)

and a colony formation assay was performed to evaluate the inhibitory effect of OVFlu-αhPD1 plus RT on Huh7 cell proliferation. For

in vivo

experiments

female C57BL/6 mice (6 to 8 weeks old

weighing 17 to19 g;

=45) were subjected to establish a subcutaneous tumor-bearing model of Hepa1-6 cells

and then the model mice were randomized into 5 groups (

=9): PBS

wild-type PR8

OVFlu-αhPD1

RT (24 Gy in 3 fractions)

and OVFlu-αhPD1+RT to evaluate the antitumor efficacy of the combination therapy. Tumor volumes were measured every 2 days during treatment. After the final treatment

the spleens were havested for CD4⁺CD69⁺ and CD8⁺CD69⁺ T-cell frequencies by flow cytometry. Major organs (heart

liver

lung

kidneys

and brain) were harvested for HE staining to assess histopathological changes

and the activities of serum AST and ALT were measured to evaluate the safety of oncolytic virus therapy.

Results

The recombinant oncolytic influenza virus OVFlu-αhPD1 showed a hemagglutination titer of 2⁷ to 2⁸

and replicated efficiently in Huh7 and Hepa1-6 cells

with viral titers reaching 7 to 8 lgTCID₅₀/mL at 72 h post-infection. CCK-8 assay demonstrated that OVFlu-αhPD1 inhibited HCC cell viability in a dose-dependent manner (MOI=3 significantly decreased viability in Huh7 and Hepa1-6 cells

0.01)

and the inhibitory effect was further enhanced after combination with RT (

0.01). Whereas no significant toxicity to normal hepatocytes was observed. Colony formation assay indicated that combined treatment significantly reduced colony numbers of Huh7 cells (

0.01). In the mouse tumor-bearing model

the combination group exhibited sig

nificantly smaller tumor volumes than any monotherapy group (

0.01)

accompanied by significantly increased proportions of splenic CD4⁺CD69⁺ and CD8⁺CD69⁺ T cells in spleen (

0.01). HE staining revealed no evident pathological changes in major organs

and serum ALT and AST levels did not differ significantly among groups (

0.05).

Conclusion

The combination of oncolytic influenza virus and radiotherapy significantly enhances the killing effect against HCC cells

in vitro

and markedly improves tumor suppression in HCC-bearing mice by activating antitumor immune responses

demonstrating a favorable synergistic antitumor effect and providing a theoretical basis for the clinical application of oncolytic virus-based combination therapy.

关键词

Keywords

references

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