Oncolytic HSV-1 expressing GM-CSF and IL-12 enhances anti-tumor efficacy in immunocompetent murine melanoma model

Han Xiao; Qiran Yin; Jia Liu; Hengrui Hu; Jiang Li; Manli Wang; Zhihong Hu

doi:10.1016/j.virs.2025.11.002

December 2025

Citation: Han Xiao, Qiran Yin, Jia Liu, Hengrui Hu, Jiang Li, Manli Wang, Zhihong Hu. Oncolytic HSV-1 expressing GM-CSF and IL-12 enhances anti-tumor efficacy in immunocompetent murine melanoma model .VIROLOGICA SINICA, 2025, 40(6) : 977-988. http://dx.doi.org/10.1016/j.virs.2025.11.002

Oncolytic HSV-1 expressing GM-CSF and IL-12 enhances anti-tumor efficacy in immunocompetent murine melanoma model

Han Xiao ^a,b ,
Qiran Yin ^a,b ,
Jia Liu ^a ,
Hengrui Hu ^a ,
Jiang Li ^a ,
Manli Wang ^{a,b
,,} ,
Zhihong Hu ^{a
,,}

a. Key Laboratory of Virology and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, 430071, China;
b. University of Chinese Aademy of Sciences, Beijing, 100049, China

Corresponding author: Manli Wang, wangml@wh.iov.cn
Zhihong Hu, huzh@wh.iov.cn
Received Date: 24 July 2025
Accepted Date: 05 November 2025
Available online: 11 November 2025

Abstract

Oncolytic virus therapy is a promising strategy for cancer treatment. Herpes simplex virus type 1 (HSV-1) has been successfully used in oncolytic virotherapy. In the present research, we applied an HSV-1 synthetic genomics platform to construct two oncolytic viruses, oHSV-1.1 and oHSV-1.2. oHSV-1.1 had the virulence gene ICP34.5 and ICP47 deleted for attenuation, and oHSV-1.2 was additionally armed with murine granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-12 (IL-12). The oncolytic viruses were evaluated in vitro and in an immunocompetent murine melanoma model. The animal experiments confirmed that both oncolytic viruses displayed antitumor efficacy, including inhibiting tumor growth and prolonging overall survival. Compared with oHSV-1.1, oHSV-1.2 demonstrated superior tumor growth suppression and enhanced antitumor efficacies, as evidenced by increased tumor cell apoptosis, cytotoxic T cells and macrophages infiltration, IFN-γ production, and upregulation of inflammatory-related gene expression. Our research highlights the potential of oncolytic HSV-1 expressing both GM-CSF and IL-12 for melanoma therapy, and provides a promising strategy for further development of oncolytic virotherapy.
- Herpes simplex virus type I (HSV-1)
- , Oncolytic virus
- , Synthetic genomics
- , Melanoma
- , GM-CSF
- , IL-12

Electronic Supplementary Material

10.1016j.virs.2025.11.002-ESM1.docx
References
1. Achuthan, A.A., Lee, K.M.C., Hamilton, J.A., 2021. Targeting GM-CSF in inflammatory and autoimmune disorders. Seminars in Immunology, 54, 101523.
2. Aldrak, N., Alsaab, S., Algethami, A., Bhere, D., Wakimoto, H., Shah, K., Alomary, M.N., Zaidan, N., 2021. Oncolytic Herpes Simplex Virus-Based Therapies for Cancer. Cells, 10, 1541.
3. Cheema, T.A., Wakimoto, H., Fecci, P.E., Ning, J., Kuroda, T., Jeyaretna, D.S., Martuza, R.L., Rabkin, S.D., 2013. Multifaceted oncolytic virus therapy for glioblastoma in an immunocompetent cancer stem cell model. Proceedings of the National Academy of Sciences, 110, 12006-12011.
4. Chiocca, E.A., Nakashima, H., Kasai, K., Fernandez, S.A., Oglesbee, M., 2020. Preclinical Toxicology of rQNestin34.5v.2: An Oncolytic Herpes Virus with Transcriptional Regulation of the ICP34.5 Neurovirulence Gene. Molecular Therapy-Methods & Clinical Development, 17, 871-893.
5. Chou, J., Roizman, B., 1994. Herpes simplex virus 1 gamma(1)34.5 gene function, which blocks the host response to infection, maps in the homologous domain of the genes expressed during growth arrest and DNA damage. Proceedings of the National Academy of Sciences, 91, 5247-5251.
6. Chowaniec, H., Slubowska, A., Mroczek, M., Borowczyk, M., Braszka, M., Dworacki, G., Dobosz, P., Wichtowski, M., 2024. New hopes for the breast cancer treatment: perspectives on the oncolytic virus therapy. Volume 15 - 2024.
7. Dudley, W.N., Wickham, R., Coombs, N., 2016. An Introduction to Survival Statistics: Kaplan-Meier Analysis. J Adv Pract Oncol, 7, 91-100.
8. Faustino-Rocha, A., Oliveira, P.A., Pinho-Oliveira, J., Teixeira-Guedes, C., Soares-Maia, R., Da Costa, R.G., Colaco, B., Pires, M.J., Colaco, J., Ferreira, R., Ginja, M., 2013. Estimation of rat mammary tumor volume using caliper and ultrasonography measurements. Lab Animal, 42, 217-224.
9. Ferrucci, P.F., Pala, L., Conforti, F., Cocorocchio, E., 2021. Talimogene Laherparepvec (T-VEC): An Intralesional Cancer Immunotherapy for Advanced Melanoma. Cancers, 13, 1383.
10. Frampton, J.E., 2022. Teserpaturev/G47Δ: First Approval. BioDrugs, 36, 667-672.
11. Fukuhara, H., Martuza, R.L., Rabkin, S.D., Ito, Y., Todo, T., 2005. Oncolytic herpes simplex virus vector g47delta in combination with androgen ablation for the treatment of human prostate adenocarcinoma. Clin Cancer Res, 11, 7886-7890.
12. Grosche, L., Dohner, K., Duthorn, A., Hickford-Martinez, A., Steinkasserer, A., Sodeik, B., 2019. Herpes Simplex Virus Type 1 Propagation, Titration and Single-step Growth Curves. Bio Protoc, 9, e3441.
13. Haines, B.B., Denslow, A., Grzesik, P., Lee, J.S., Farkaly, T., Hewett, J., Wambua, D., Kong, L.X., Behera, P., Jacques, J., Goshert, C., Ball, M., Colthart, A., Finer, M.H., Hayes, M.W., Feau, S., Kennedy, E.M., Lerner, L., Queva, C., 2021. ONCR-177, an Oncolytic HSV-1 Designed to Potently Activate Systemic Antitumor Immunity. Cancer Immunology Research, 9, 291-308.
14. Harrington, K., Freeman, D.J., Kelly, B., Harper, J., Soria, J.C., 2019. Optimizing oncolytic virotherapy in cancer treatment. Nature Reviews Drug Discovery, 18, 689-706.
15. Helft, J., Bottcher, J., Chakravarty, P., Zelenay, S., Huotari, J., Schraml, Barbara u., Goubau, D., Reis e sousa, C., 2015. GM-CSF Mouse Bone Marrow Cultures Comprise a Heterogeneous Population of CD11c+MHCII+ Macrophages and Dendritic Cells. Immunity, 42, 1197-1211.
16. Hong, B., Sahu, U., Mullarkey, M.P., Kaur, B., 2022. Replication and Spread of Oncolytic Herpes Simplex Virus in Solid Tumors. Viruses, 14, 118.
17. Hou, Z., Zhou, Z., Wang, Z., Xiao, G., 2016. Assembly of long DNA sequences using a new synthetic Escherichia coli-yeast shuttle vector. Virologica Sinica, 31, 160-167.
18. Hu, H., Wu, G., Shu, Z., Yu, D., Nan, N., Yuan, F., Liu, X., Wang, H., 2020. ICP6 Prevents RIP1 Activation to Hinder Necroptosis Signaling. Front. Cell Dev. Biol, 8, 2020.
19. Jones, F.E., Smibert, C.A., Smiley, J.R., 1995. Mutational analysis of the herpes simplex virus virion host shutoff protein: evidence that vhs functions in the absence of other viral proteins. J Virol, 69, 4863-4871.
20. Kanai, R., Zaupa, C., Sgubin, D., Antoszczyk, S.J., Martuza, R.L., Wakimoto, H., Rabkin, S.D., 2012. Effect of γ34.5 Deletions on Oncolytic Herpes Simplex Virus Activity in Brain Tumors. Journal of Virology, 86, 4420-4431.
21. Kang, S., Mansurov, A., Kurtanich, T., Chun, H.R., Slezak, A.J., Volpatti, L.R., Chang, K., Wang, T., Alpar, A.T., Refvik, K.C., et al., 2024. Engineered GM-CSF polarizes protumorigenic tumor-associated macrophages to an antitumorigenic phenotype and potently synergizes with IL-12 immunotherapy. J Immunother Cancer, 12, e009541.
22. Kaufman, H.L., Kohlhapp, F.J., Zloza, A., 2015. Oncolytic viruses: a new class of immunotherapy drugs. Nature Reviews Drug Discovery, 14, 642-662.
23. Kim, K.J., Moon, D., Kong, S.J., Lee, Y.S., Yoo, Y., Kim, S., Kim, C., Chon, H.J., Kim, J.H., Choi, K.J., 2021. Antitumor effects of IL-12 and GM-CSF co-expressed in an engineered oncolytic HSV-1. Gene Ther, 28, 186-198.
24. Koch, M., Lawler, S., Chiocca, E., 2020. HSV-1 Oncolytic Viruses from Bench to Bedside: An Overview of Current Clinical Trials. Cancers, 12, 3514.
25. Kodama, T., Takeda, K., Shimozato, O., Hayakawa, Y., Atsuta, M., Kobayashi, K., Ito, M., Yagita, H., Okumura, K., 1999. Perforin-dependent NK cell cytotoxicity is sufficient for anti-metastatic effect of IL-12. 29, 1390-1396.
26. Langmead, B., Salzberg, S.L., 2012. Fast gapped-read alignment with Bowtie 2. Nature Methods, 9, 357-U354.
27. Lawler, S.E., Speranza, M.-C., Cho, C.-F., Chiocca, E.A., 2017. Oncolytic Viruses in Cancer Treatment. JAMA Oncology, 3, 841.
28. Li, X., Cheng, Z.P., 2024. Oncolytic Viruses in Cancer Immunotherapy. Advanced Therapeutics, 7, 20.
29. Linehan, M.M., Richman, S., Krummenacher, C., Eisenberg, R.J., Cohen, G.H., Iwasaki, A., 2004. In Vivo Role of Nectin-1 in Entry of Herpes Simplex Virus Type 1 (HSV-1) and HSV-2 through the Vaginal Mucosa. 78, 2530-2536.
30. Ling, A.L., Solomon, I.H., Landivar, A.M., Nakashima, H., Woods, J.K., Santos, A., Masud, N., Fell, G., Mo, X., Yilmaz, A.S., et al., 2023. Clinical trial links oncolytic immunoactivation to survival in glioblastoma. Nature, 623, 157-166.
31. Ma, R., Li, Z., Chiocca, E.A., Caligiuri, M.A., Yu, J., 2023. The emerging field of oncolytic virus-based cancer immunotherapy. Trends in Cancer, 9, 122-139.
32. Marintcheva, B., Weller, S.K., 2001. Residues within the conserved helicase motifs of UL9, the origin-binding protein of herpes simplex virus-1, are essential for helicase activity but not for dimerization or origin binding activity. Journal of Biological Chemistry, 276, 6605-6615.
33. Milne, I., Stephen, G., Bayer, M., Cock, P.J.A., Pritchard, L., Cardle, L., Shaw, P.D., Marshall, D., 2012. Using Tablet for visual exploration of second-generation sequencing data. Briefings in Bioinformatics, 14, 193-202.
34. Mullins-Dansereau, V., Myre, M.L., Bardoul, A., Geoffroy, K., Pita, M.J.R., Beland, D., Desaulniers, K.L., Roy, D.G., Bourgeois-Daigneault, M.C., 2025. Oncolytic VSV-IL-2 has enhanced anticancer vaccination adjuvant abilities. Journal for Immunotherapy of Cancer, 13, 14.
35. Nabi, R., Chouljenko, V.N., Musarrat, F., Davis, M.E., Mohan, H., Ghavimi, R., Stanfield, B., Dutta, O., Kousoulas, K.G., 2025. The Novel Oncolytic Herpes Simplex Virus Type-1 (HSV-1) Vaccine Strain VC2 Constitutively Expressing GM-CSF Causes Increased Intratumoral T Cell Infiltration and Inhibition of Tumor Metastasis in the 4T1/Balb/c Mouse Model of Stage Four Breast Cancer. Journal of Medical Virology, 97, e70220.
36. Nabi, R., Musarrat, F., Lima, J., Langohr, I.M., Chouljenko, V.N., Kousoulas, K.G., 2023. The Oncolytic herpes simplex virus type-1 (HSV-1) vaccine strain VC2 causes intratumor infiltration of functionally active T cells and inhibition of tumor metastasis and pro-tumor genes VEGF and PDL1 expression in the 4T1/Balb/c mouse model of stage four breast cancer. Frontiers in Molecular Biosciences, 10, 13.
37. Nguyen, H.-M., Guz-Montgomery, K., Saha, D., 2020. Oncolytic Virus Encoding a Master Pro-Inflammatory Cytokine Interleukin 12 in Cancer Immunotherapy. 9, 400.
38. Ni, L.Q., Li, Y., Wu, K., Deng, F., Wang, H.L., Ning, Y.J., 2022. Antitumor efficacy of CRISPR/Cas9-engineered ICP6 mutant herpes simplex viruses in a mouse xenograft model for lung adenocarcinoma. Journal of Medical Virology, 94, 6000-6015.
39. Omole, R.K., Oluwatola, O., Akere, M.T., Eniafe, J., Agboluaje, E.O., Daramola, O.B., Ayantunji, Y.J., Omotade, T.I., Torimiro, N., Ayilara, M.S., Adeyemi, O.I., Salinsile, O.S., 2022. Comprehensive assessment on the applications of oncolytic viruses for cancer immunotherapy. Frontiers in Pharmacology, 13, 1082797.
40. Perng, Ghiasi, H., Kaiwar, R., Nesburn, A.B., Wechsler, S.L., 1994. AN IMPROVED METHOD FOR CLONING PORTIONS OF THE REPEAT REGIONS OF HERPES-SIMPLEX VIRUS TYPE-1. Journal of Virological Methods, 46, 111-116.
41. Perng, Thompson, R.L., Sawtell, N.M., Taylor, W.E., Slanina, S.M., Ghiasi, H., Kaiwar, R., Nesburn, A.B., Wechsler, S.L., 1995. An avirulent ICP34.5 deletion mutant of herpes simplex virus type 1 is capable of in vivo spontaneous reactivation. Journal of Virology, 69, 3033-3041.
42. Peters, C., Grandi, P., Nigim, F., 2019. Updates on Oncolytic Virus Immunotherapy for Cancers. Molecular Therapy Oncolytics, 12, 259-262.
43. Polcicova, K., Goldsmith, K., Rainish, B.L., Wisner, T.W., Johnson, D.C., 2005. The extracellular domain of herpes simplex virus gE is indispensable for efficient cell-to-cell spread: evidence for gE/gI receptors. J Virol, 79, 11990-12001.
44. Raman, S.S., Hecht, J.R., Chan, E., 2019. Talimogene laherparepvec: review of its mechanism of action and clinical efficacy and safety. Immunotherapy, 11, 705-723.
45. Schietinger, A., Philip, M., Liu, R.B., Schreiber, K., Schreiber, H., 2010. Bystander killing of cancer requires the cooperation of CD4(+) and CD8(+) T cells during the effector phase. J Exp Med, 207, 2469-2477.
46. Shen, Y., Bai, X., Zhang, Q., Liang, X., Jin, X., Zhao, Z., Song, W., Tan, Q., Zhao, R., Jia, W., et al., 2025. Oncolytic virus VG161 in refractory hepatocellular carcinoma. Nature, 10.1038/s41586-025-08717-5.
47. Tang, G., Wang, D., Zhao, X., Feng, Z., Chen, Q., Shen, Y., 2023. The Dilemma of HSV-1 Oncolytic Virus Delivery: The Method Choice and Hurdles. International Journal of Molecular Sciences, 24, 3681.
48. Tian, Y., Xie, D., Yang, L., 2022. Engineering strategies to enhance oncolytic viruses in cancer immunotherapy. Signal Transduction and Targeted Therapy, 7, 117.
49. Todo, T., Martuza, R.L., Rabkin, S.D., Johnson, P.A., 2001. Oncolytic herpes simplex virus vector with enhanced MHC class I presentation and tumor cell killing. Proceedings of the National Academy of Sciences, 98, 6396-6401.
50. Uche, I.K., Fowlkes, N., Vu, L., Watanabe, T., Carossino, M., Nabi, R., Del Piero, F., Rudd, J.S., Kousoulas, K.G., Rider, P.J.F., 2021. Novel Oncolytic Herpes Simplex Virus 1 VC2 Promotes Long-Lasting, Systemic Anti-melanoma Tumor Immune Responses and Increased Survival in an Immunocompetent B16F10-Derived Mouse Melanoma Model. Journal of Virology, 95, 14.
51. Volovat, S.R., Scripcariu, D.V., Vasilache, I.A., Stolniceanu, C.R., Volovat, C., Augustin, I.G., Volovat, C.C., Ostafe, M.-R., Andreea-Voichița, S.-G., Bejusca-Vieriu, T., Lungulescu, C.V., Sur, D., Boboc, D., 2024. Oncolytic Virotherapy: A New Paradigm in Cancer Immunotherapy. 25, 1180.
52. Whitley, R.J., Roizman, B., 2001. Herpes simplex virus infections. The Lancet, 357, 1513-1518.
53. Xiao, H., Hu, H., Guo, Y., Li, J., Wen, L., Zeng, W.B., Wang, M., Luo, M.H., Hu, Z., 2023. Construction and characterization of a synthesized herpes simplex virus H129-Syn-G2. Virologica Sinica, 38, 373-379.
54. Xiao, H., Hu, H., Guo, Y., Li, J., Zeng, W.B., Luo, M.H., Wang, M., Hu, Z., 2024. Efficient Strategy for Synthesizing Vector-Free and Oncolytic Herpes Simplex Type 1 Viruses. ACS Synth Biol, 13, 3268-3280.
55. York, I.A., Roop, C., Andrews, D.W., Riddell, S.R., Graham, F.L., Johnson, D.C., 1994. A cytosolic herpes simplex virus protein inhibits antigen presentation to CD8+ T lymphocytes. Cell, 77, 525-535.
56. Zamarin, D., Holmgaard, R.B., Subudhi, S.K., Park, J.S., Mansour, M., Palese, P., Merghoub, T., Wolchok, J.D., Allison, J.P., 2014. Localized Oncolytic Virotherapy Overcomes Systemic Tumor Resistance to Immune Checkpoint Blockade Immunotherapy. Science Translational Medicine, 6, 12.
57. Zhou, M., Shen, Z.Y., 2025. Advanced progress in the genetic modification of the oncolytic HSV-1 virus. Frontiers in Oncology, 14, 12.
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