Abstract
Vesicular stomatitis virus (VSV) based oncolytic viruses are promising agents against various cancers. We have shown that pancreatic ductal adenocarcinoma (PDAC) cell lines exhibit great diversity in susceptibility and permissibility to VSV. Here, using a directed evolution approach with our two previously described oncolytic VSV recombinants, VSV-p53wt and VSV-p53-CC, we generated novel oncolytic VSVs with an improved ability to replicate in virus-resistant PDAC cell lines. VSV-p53wt and VSV-p53-CC encode a VSV matrix protein (M) with a ΔM51 mutation (M-ΔM51) and one of two versions of a functional human tumor suppressor, p53, fused to a far-red fluorescent protein, eqFP650. Each virus was serially passaged 32 times (which accounts for more than 60 viral replication cycles) on either the SUIT-2 (moderately resistant to VSV) or MIA PaCa-2 (highly permissive to VSV) human PDAC cell lines. While no phenotypic changes were observed for MIA PaCa-2-passaged viruses, both SUIT-2-passaged VSV-p53wt and VSV-p53-CC showed improved replication in SUIT-2 and AsPC-1, another human PDAC cell line also moderately resistant to VSV, while remaining highly attenuated in nonmalignant cells. Surprisingly, two identical VSV glycoprotein (VSV-G) mutations, K174E and E238K, were identified in both SUIT-2-passaged viruses. Additional experiments indicated that the acquired G mutations improved VSV replication, at least in part due to improved virus attachment to SUIT-2 cells. Importantly, no mutations were found in the M-ΔM51 protein, and no deletions or mutations were found in the p53 or eqFP650 portions of virus-carried transgenes in any of the passaged viruses, demonstrating long-term genomic stability of complex VSV recombinants carrying large transgenes.IMPORTANCE Vesicular stomatitis virus (VSV)-based oncolytic viruses are promising agents against pancreatic ductal adenocarcinoma (PDAC). However, some PDAC cell lines are resistant to VSV. Here, using a directed viral evolution approach, we generated novel oncolytic VSVs with an improved ability to replicate in virus-resistant PDAC cell lines, while remaining highly attenuated in nonmalignant cells. Two independently evolved VSVs obtained 2 identical VSV glycoprotein mutations, K174E and E238K. Additional experiments indicated that these acquired G mutations improved VSV replication, at least in part due to improved virus attachment to SUIT-2 cells. Importantly, no deletions or mutations were found in the virus-carried transgenes in any of the passaged viruses. Our findings demonstrate long-term genomic stability of complex VSV recombinants carrying large transgenes and support further clinical development of oncolytic VSV recombinants as safe therapeutics for cancer.