Investigation of cellular localization of recombinant HSV-2 virions in infected vero cells for enhanced recovery

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Most industrial bioprocesses seek to ensure the elimination of viruses from their process streams. As a result, a significant amount of work has gone into viral clearance research. However, viruses themselves can be very useful products as vaccination agents, oncolytic therapeutics and gene delivery vectors. Although useful, there is a dearth of information available on maximizing recovery of 'active' viruses for large scale applications. This grant application is for a three year study by a PhD student and a post-doctoral fellow on the capture and purification of two viruses: a pseudo-typed baculovirus (a modified insect virus) gene therapy vector, and a therapeutic oncolytic Maraba virus, which has shown tremendous promise in clinical trials as a treatment for cancer. This capture and purification will be done using membrane technologies, which form the basis of Natrix Separations Inc. core business, and that promise to minimize the number of downstream processing steps and ensure recovery of active virus. The wide array of known viruses and their distinct properties can pose major challenges in the development of robust universal downstream strategies for viral preparations - especially those destined for clinical use. These challenges include quantity, purity and potency of the viruses. To minimize the distinct properties, this work focuses on vesicular stomatitis virus envelope glycoprotein G (VSV-G) pseudo-typed baculovirus, and a VSV-related virus, the Maraba virus. Many virus-based vectors are pseudo-typed with VSV-G to increase their potency. Basing our purification strategies on the VSV-G, will allow a comprehensive strategy to purify a number of leading therapeutic viral candidates using the Natrix technology platform. Two major strategies will be examined in parallel. First, with Natrix, a heparin-functionalized membrane will be made for affinity capture of viruses with vesicular stomatitis-like glycoproteins. Second, Natrix ion exchange membranes will be evaluated. Both strategies will be evaluated for their effect on virus recovery, purity and potency.