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Updated: Aug 5, 2026
Growth, Purification, and Titration of Oncolytic Herpes Simplex Virus
Published on: May 13, 2021
Development and Validation of a Rapid Titer Assay for the Oncolytic Virus oHSV2 Expressing a PD-L1/CD3 Bispecific
Shengjie Zhang1, Qingrui Song2, Runyang Wang1
1National "111" Center for Cellular Regulation and Molecular Pharmaceutics, Key Laboratory of Fermentation Engineering (Ministry of Education), Cooperative Innovation Center of Industrial Fermentation (Ministry of Education & Hubei Province), School of Life and Health Sciences, Hubei University of Technology, Wuhan 430068, China.
Abstract:
Oncolytic viruses represent a promising class of anticancer therapeutics, and rapid, accurate quantification of viral titers is critical for ensuring both efficacy and safety during clinical development. Conventional viral titering methods, such as 50% cell culture infectious dose (CCID50), are time-consuming and limited in sensitivity, thereby restricting their application in real-time clinical monitoring. This study aimed to develop and validate a rapid titer assay for oHSV2-PD-L1/CD3-BsAb, an oncolytic herpes simplex virus expressing a PD-L1/CD3 bispecific antibody, to support preclinical and clinical monitoring. A dual-reporter cell system was established using Vero-PD-L1-GFP (Vero cells expressing PD-L1 and GFP) cells as target cells and Jurkat-NFAT-Fluc (Jurkat cells expressing NFAT and Fluc) cells as effector cells. Viral infection activates the NFAT signaling pathway, driving Fluc expression, thereby enabling rapid quantification of infectious virus. The assay was evaluated for specificity, limit of detection (LOD), and lower limit of quantification (LLOQ), and compared with the conventional CCID50 method. Its applicability was further assessed using clinical simulation samples, including PBMCs and swabs. The rapid titer assay accurately quantified virus at 103 CCID50/mL after 8 h of incubation, consistent with CCID50 results, while extending the incubation to 18 h improved the LLOQ to 102.5 CCID50/mL, demonstrating enhanced sensitivity. The assay exhibited high reproducibility and stability in both PBMC and swab samples, enabling reliable quantification of low-titer virus in complex biological matrices. Compared with CCID50, the method substantially reduced assay time (from 3-5 days to 8-18 h) while improving sensitivity and specificity. The developed rapid titer assay for oHSV2-PD-L1/CD3-BsAb provides a sensitive and specific platform for viral quantification. It offers a valuable tool for oncolytic virus development, production quality control, and clinical monitoring, facilitating efficient safety evaluation and risk management in ongoing and future clinical applications.
Insights
A new rapid assay accurately quantifies oncolytic virus titers, improving on traditional methods. This faster, more sensitive method supports oncolytic virus development and clinical monitoring for enhanced safety.
Area of Science:
- Oncolytic virotherapy
- Viral quantification
- Biotechnology
Background:
- Oncolytic viruses are promising anticancer agents, but their clinical development requires rapid, sensitive viral titer quantification.
- Conventional methods like CCID50 are slow and lack sensitivity, hindering real-time monitoring.
- oHSV2-PD-L1/CD3-BsAb is an oncolytic herpes simplex virus engineered to express a bispecific antibody for enhanced therapeutic effects.
Purpose of the Study:
- To develop and validate a rapid, sensitive assay for quantifying oHSV2-PD-L1/CD3-BsAb viral titers.
- To support preclinical and clinical monitoring of this oncolytic virus.
- To compare the novel assay's performance against the conventional CCID50 method.
Main Methods:
- Established a dual-reporter cell system using Vero-PD-L1-GFP and Jurkat-NFAT-Fluc cells.
- Viral infection triggers NFAT signaling, leading to Fluc expression for rapid quantification.
- Assessed specificity, LOD, LLOQ, reproducibility, and stability using clinical simulation samples (PBMCs, swabs).
Main Results:
- The rapid assay quantified virus at 10^3 CCID50/mL within 8 hours, consistent with CCID50.
- Extended incubation to 18 hours improved LLOQ to 10^2.5 CCID50/mL, demonstrating enhanced sensitivity.
- The assay showed high reproducibility and stability in complex biological matrices like PBMCs and swabs.
Conclusions:
- The developed rapid titer assay provides a sensitive, specific, and significantly faster alternative to CCID50 for oncolytic virus quantification.
- This assay is a valuable tool for oncolytic virus development, quality control, and clinical monitoring.
- Facilitates efficient safety evaluation and risk management in oncolytic virus-based therapies.
