Single-particle characterization of oncolytic vaccinia virus by flow virometry.
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.
University of Ottawa Flow Cytometry and Virometry Core Facility, Canada.
Vera A
VA
Tang
Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Canada.
Tyler M
TM
Renner
Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Canada; Centre for Innovative Cancer Therapeutics, Ottawa Hospital Research Institute, Canada.
Oliver
O
Varette
Centre for Innovative Cancer Therapeutics, Ottawa Hospital Research Institute, Canada.
Fabrice
F
Le Boeuf
Centre for Innovative Cancer Therapeutics, Ottawa Hospital Research Institute, Canada.
Jiahu
J
Wang
Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Canada; Centre for Innovative Cancer Therapeutics, Ottawa Hospital Research Institute, Canada.
Jean-Simon
JS
Diallo
Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Canada; Centre for Innovative Cancer Therapeutics, Ottawa Hospital Research Institute, Canada.
John C
JC
Bell
University of Ottawa Flow Cytometry and Virometry Core Facility, Canada; Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Canada. Electronic address: langlois@uottawa.ca.
Marc-André
MA
Langlois
D000067128:Extracellular Vesicles; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D050130:Oncolytic Virotherapy; D050504:Oncolytic Viruses / Q000302:isolation & purification; D014616:Vaccinia virus / Q000302:isolation & purification; D014771:Virion / Q000302:isolation & purification*; D014779:Virus Replication
Extracellular vesicles; Flow cytometry; Flow virometry; Microparticles; Oncolytic virus; Quality control; Submicron-sized particles; Vaccine; Vaccinia virus; Viral sorting