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Vaccinia virus (VV) has been utilized in oncolytic virotherapy, but it risks a host antiviral immune response. VV has an extracellular enveloped virus (EEV) form consisting of a normal virion covered with a host-derived outer membrane that enables its spread via circulation while evading host immune mechanisms. However, the immune resistance of EEV is only partial, owing to expression of the surface protein B5R, which has four short consensus repeat (SCR) domains that are targeted by host immune factors. To engineer a more effective virus for oncolytic virotherapy, we developed an enhanced immune-evading oncolytic VV by removing the SCRs from the attenuated strain LC16mO. Although deletion of only the SCRs preserved viral replication, progeny production, and oncolytic activity, deletion of whole B5R led to attenuation of the virus. Importantly, SCR-deleted EEV had higher neutralization resistance than did B5R-wild-type EEV against VV-immunized animal serum; moreover, it retained oncolytic function, thereby prolonging the survival of tumor-bearing mice treated with anti-VV antibody. These results demonstrate that partial SCR deletion increases neutralization escape without affecting the oncolytic potency of VV, making it useful for the treatment of tumors under the anti-virus antibody existence.

Partial Deletion of Glycoprotein B5R Enhances Vaccinia Virus Neutralization Escape while Preserving Oncolytic Function