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Journal of Virology, September 2006, p. 8603-8612, Vol. 80, No. 17
0022-538X/06/$08.00+0     doi:10.1128/JVI.00142-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Rapid Adaptation of a Recombinant Vesicular Stomatitis Virus to a Targeted Cell Line

Yanhua Gao,1 Patricia Whitaker-Dowling,2 Simon C. Watkins,3 Judith A. Griffin,4 and Ira Bergman5*

Department of Pediatrics, University of Pittsburgh School of Medicine,1 Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine,2 Center for Biologic Imaging, University of Pittsburgh School of Medicine,3 Department of Pediatrics, Children's Hospital of Pittsburgh,4 Departments of Pediatrics, Neurology, and Immunology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania5

Received 20 January 2006/ Accepted 7 June 2006

Vesicular stomatitis virus (VSV) is being developed for cancer therapy. We created a recombinant replicating VSV (rrVSV) that preferentially infected Her2/neu-expressing breast cancer cells. This rrVSV did not express the native VSV-G glycoprotein (gp). Instead, it expressed a chimeric Sindbis gp which included a single-chain antibody (SCA) directed to the human Her2/neu receptor. The virus infected mouse mammary carcinoma cells (D2F2/E2) expressing Her2/neu 23-fold better than the parent cells (D2F2). However, viral growth in cultured D2F2/E2 cells was curtailed after several cycles, and viral yield was very poor at 2 x 104 infectious doses (ID)/ml. We performed in vitro serial passage in D2F2/E2 cells to evolve a virus with improved growth that could be used for preclinical therapy trials in mice. Fifteen passes generated an adapted virus that progressed through multiple cycles in cultured D2F2/E2 cells until all cells were infected and had a viral yield of 1 x 108 ID/ml. Sequencing of the entire viral genomes found only 2 mutations in the adapted virus. Both mutations occurred in the gp gene segment coding for the SCA. An additional N-glycosylation site was created by one of the mutations. The adapted virus showed higher density of gp on the viral envelope, improved infectivity, much greater stability, higher burst size, and decreased induction of cellular interferon. The specificity for cells expressing the Her2/neu receptor was unchanged. These studies demonstrate that serial passage can be used to rapidly evolve a VSV genome encoding an improved chimeric glycoprotein.

* Corresponding author. Mailing address: Children's Hospital of Pittsburgh, 3705 Fifth Avenue, Pittsburgh, PA 15213. Phone: (412) 692-6182. Fax: (412) 692-6787. E-mail: ira.bergman{at}chp.edu.

Journal of Virology, September 2006, p. 8603-8612, Vol. 80, No. 17
0022-538X/06/$08.00+0     doi:10.1128/JVI.00142-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.





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