Funding for this project was provided in part by National Institutes of Health Grant Nos. U19AI107792 and R01AI107159 (S.C.). J.W.W. was supported by an NIH Ruth Kirschstein Postdoctoral Fellowship F32GM109661.
AcknowledgementsWe thank members of the Crosson lab for helpful discussions, Dr. Lauriane Quenee and Dr. Lois Zitzow for help with animal experiments, the University of Chicago Integrated Light Microscopy Core Facility, and Heather Marlatt at Nationwide Histology.
Oncolytic virus; Vaccinia virus; Submicron-sized particles; Extracellular vesicles; Microparticles; Flow cytometry; Flow virometry; Viral sorting; Quality control; Vaccine
Viruses are best known as harmful pathogens that cause disease. However, some viruses are uniquely capable of promoting anti-tumor activity and cancer remission, these are called oncolytic viruses (OVs). Cancer gaba antagonist commonly display defects in interferon signaling which is a critical mechanism for antiviral defense. OVs exploit this deficiency to infect and destroy cancer cells specifically  and . Vaccinia virus (VV) is an enveloped double-stranded DNA virus of the poxviridae family that is best known as the active ingredient in the smallpox vaccine used for global eradication of the disease. The potential of VV as an OV was revealed by showing that it displayed a natural tropism for a broad range of tumors when injected in experimental animals , , ,  and . Further engineering of VV to increase safety and tumor-specificity was achieved by removing its genes coding for thymidine kinase (TK, J2R) and Vaccinia growth factor (VGF, C11R). The resulting double-deleted VV (VVDD) is unable to replicate in healthy cells or induce the proliferation of nearby cells . Replication of VV in cancer cells leads to tumor regression, tumor antigen exposure, and stimulation of host anti-tumor immunity, thereby positioning it as a promising anti-cancer biotherapeutic agent ,  and . The US Food and Drug Administration has recently approved the first OV treatment for melanoma, with an increasing number of OVs in clinical development  and .
In contrast to some viruses that are released from infected cells in large quantities, the infectious pool of VV particles exists primarily as intracellular mature virions (IMVs), also sometimes referred to as mature virions (MVs)  and . However, small amounts of VV particles also bud from infected cells as cell-associated enveloped viruses (CEV) and extracellular enveloped viruses (EEVs)  and . To prepare VV stocks, IMVs are harvested by lysing infected cells and then purifying the virus by density gradient centrifugation or tangential flow filtration  and . Lysis of infected cells releases large amounts of cellular debris and molecules that may bind and co-purify with the virus if not properly eliminated. Additionally, it has long been established that VV self-aggregates, thereby altering the biophysical properties of the virus during the purification process  and . As such, a reliable, accurate and rapid method to assess OV sample purity and integrity would be of significant value, especially for clinical and therapeutic applications.