Single-particle characterization of oncolytic vaccinia virus by flow virometry. Issue 42 (30th September 2016)

• Record Type: Journal Article
• Authors: Tang, Vera A.; Renner, Tyler M.; Varette, Oliver; Le Boeuf, Fabrice; Wang, Jiahu; Diallo, Jean-Simon; Bell, John C.; Langlois, Marc-André
• Is Part Of: Vaccine. Volume 34:Issue 42(2016)
• Page Start: 5082
• Page End: 5089
• Publication Date: 2016-09-30
• Subjects: Oncolytic virus -- Vaccinia virus -- Submicron-sized particles -- Extracellular vesicles -- Microparticles -- Flow cytometry -- Flow virometry -- Viral sorting -- Quality control -- Vaccine; Vaccines -- Periodicals; 615.372
• DOI: 10.1016/j.vaccine.2016.08.074
• Languages: English
• ISSNs: 0264-410X

Abstract

Highlights: Flow virometry analysis of Vaccinia virus (VV). Characterization of VV at the single particle level. Characterization VV aggregation. Quantification of submicron-sized particles in an oncolytic virus vaccine. Sorting of VV particle populations by flow virometry. Abstract: Vaccinia virus (VV) is an oncolytic virus that is currently being evaluated as a promising cancer vaccine in several phase I, II and III clinical trials. Although several quality control tests are performed on each new batch of virus, these do not routinely include a systematic characterization of virus particle homogeneity, or relate the infectious titer to the total number of submicron sized particles (SSPs) present in the sample. SSPs are comprised of infectious virus and non-infectious viral particles, but also cell contaminants derived from the virus isolation procedures, such as cellular vesicles and debris. Here we have employed flow virometry (FV) analysis and sorting to isolate and characterize distinct SSP populations in therapeutic oncolytic VV preparations. We show that VV preparations contain SSPs heterogeneous in size and include large numbers of non-infectious VV particles. Furthermore, we used FV to illustrate how VV has a propensity to aggregate over time and under various handling and storage procedures. Accordingly, we find that together the infectious titer, the total number of SSPs, the number of viral genomes and the level of particle aggregation in a sample constitute useful parameters that greatly facilitate inter-sample assessment of physical quality, and also provides a means to monitor sample deterioration over time. Additionally, we have successfully employed FV sorting to further isolate virus from other particles by identifying a lipophilic dye that preferentially stains VV over other SSPs in the sample. Overall, we demonstrate that FV is a fast and effective tool that can be used to perform quality, and consistency control assessments of oncolytic VV vaccine preparations.