Replication efficiency of oncolytic vaccinia virus in cell cultures prognosticates the virulence and antitumor efficacy in mice

Nanhai G Chen,Aladar A Szalay,Yong A Yu,Qian Zhang

doi:10.1186/1479-5876-9-164

Abstract

BackgroundWe have shown that insertion of the three vaccinia virus (VACV) promoter-driven foreign gene expression cassettes encoding Renilla luciferase-Aequorea GFP fusion protein, β-galactosidase, and β-glucuronidase into the F14.5L, J2R, and A56R loci of the VACV LIVP genome, respectively, results in a highly attenuated mutant strain GLV-1h68. This strain shows tumor-specific replication and is capable of eradicating tumors with little or no virulence in mice. This study aimed to distinguish the contribution of added VACV promoter-driven transcriptional units as inserts from the effects of insertional inactivation of three viral genes, and to determine the correlation between replication efficiency of oncolytic vaccinia virus in cell cultures and the virulence and antitumor efficacy in miceMethodsA series of recombinant VACV strains was generated by replacing one, two, or all three of the expression cassettes in GLV-1h68 with short non-coding DNA sequences. The replication efficiency and tumor cell killing capacity of these newly generated VACV strains were compared with those of the parent virus GLV-1h68 in cell cultures. The virus replication efficiency in tumors and antitumor efficacy as well as the virulence were evaluated in nu/nu (nude) mice bearing human breast tumor xenografts.Resultswe found that virus replication efficiency increased with removal of each of the expression cassettes. The increase in virus replication efficiency was proportionate to the strength of removed VACV promoters linked to foreign genes. The replication efficiency of the new VACV strains paralleled their cytotoxicity in cell cultures. The increased replication efficiency in tumor xenografts resulted in enhanced antitumor efficacy in nude mice. Similarly, the enhanced virus replication efficiency was indicative of increased virulence in nude mice.ConclusionsThese data demonstrated that insertion of VACV promoter-driven transcriptional units into the viral genome for the purpose of insertional mutagenesis did modulate the efficiency of virus replication together with antitumor efficacy as well as virulence. Replication efficiency of oncolytic VACV in cell cultures can predict the virulence and therapeutic efficacy in nude mice. These findings may be essential for rational design of safe and potent VACV strains for vaccination and virotherapy of cancer in humans and animals.

Highlights

We have shown that insertion of the three vaccinia virus (VACV) promoter-driven foreign gene expression cassettes encoding Renilla luciferase-Aequorea GFP fusion protein, b-galactosidase, and b-glucuronidase into the F14.5L, J2R, and A56R loci of the VACV LIVP genome, respectively, results in a highly attenuated mutant strain GLV-1h68
Generation of the derivatives of GLV-1h68 by replacing the foreign gene expression cassettes with short noncoding DNA sequences Previously, we have shown that insertion of the foreign marker gene expression cassettes into the F14.5L, J2R, and A56R loci of the VACV LIVP genome disrupted the open-reading frames (ORFs) F14.5L, J2R, and A56R, resulting in the recombinant VACV GLV-1h68 with enhanced tumor colonization specificity as well as reduced virulence in mice [10,11]
To determine the contributions of individual foreign gene expression to virus attenuation, in addition to viral gene inactivation, we constructed a series of the derivatives of GLV-1h68 by replacing one, two, or all three of these expression cassettes in the genome of GLV-1h68 with short non-coding DNA sequences while the original ORFs F145L, J2R, and A56R remain disrupted in all the derivatives

Summary

Introduction

We have shown that insertion of the three vaccinia virus (VACV) promoter-driven foreign gene expression cassettes encoding Renilla luciferase-Aequorea GFP fusion protein, b-galactosidase, and b-glucuronidase into the F14.5L, J2R, and A56R loci of the VACV LIVP genome, respectively, results in a highly attenuated mutant strain GLV-1h68. This strain shows tumor-specific replication and is capable of eradicating tumors with little or no virulence in mice. Oncolytic virotherapy can effectively complement conventional cancer therapies, such as chemo- and radiotherapy

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