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The highly attenuated oncolytic recombinant vaccinia virus GLV-1h68: comparative genomic features and the contribution of F14.5L inactivation.

Zhang Q, Liang C, Yu YA, Chen N, Dandekar T, Szalay AA - Mol. Genet. Genomics (2009)

Bottom Line: The reduced pathogenicity of GLV-1h68 is confirmed in male mice bearing C6 rat glioma and in immunocompetent mice bearing B16-F10 murine melanoma.The contribution of foreign gene expression cassettes in the F14.5L, J2R and A56R loci is analyzed, in particular the contribution of F14.5L inactivation to the reduced virulence is demonstrated by comparing the virulence of GLV-1h68 with its F14.5L- and revertant viruses.GLV-1h68 is a promising engineered VACV variant for anticancer therapy with tumor-specific replication, reduced pathogenicity and benign tissue tropism.

View Article: PubMed Central - PubMed

Affiliation: Genelux Corporation, San Diego Science Center, 3030 Bunker Hill St., Ste. 310, San Diego, CA 92109, USA.

ABSTRACT
As a new anticancer treatment option, vaccinia virus (VACV) has shown remarkable antitumor activities (oncolysis) in preclinical studies, but potential infection of other organs remains a safety concern. We present here genome comparisons between the de novo sequence of GLV-1h68, a recombinant VACV, and other VACVs. The identified differences in open reading frames (ORFs) include genes encoding host-range selection, virulence and immune modulation proteins, e.g., ankyrin-like proteins, serine proteinase inhibitor SPI-2/CrmA, tumor necrosis factor (TNF) receptor homolog CrmC, semaphorin-like and interleukin-1 receptor homolog proteins. Phylogenetic analyses indicate that GLV-1h68 is closest to Lister strains but has lost several ORFs present in its parental LIVP strain, including genes encoding CrmE and a viral Golgi anti-apoptotic protein, v-GAAP. The reduced pathogenicity of GLV-1h68 is confirmed in male mice bearing C6 rat glioma and in immunocompetent mice bearing B16-F10 murine melanoma. The contribution of foreign gene expression cassettes in the F14.5L, J2R and A56R loci is analyzed, in particular the contribution of F14.5L inactivation to the reduced virulence is demonstrated by comparing the virulence of GLV-1h68 with its F14.5L- and revertant viruses. GLV-1h68 is a promising engineered VACV variant for anticancer therapy with tumor-specific replication, reduced pathogenicity and benign tissue tropism.

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Contribution of an expression cassette in the F14.5L locus on the attenuation of GLV-1h68. a The schematic presentation of the F14.5L- and revertant viruses derived from GLV-1h68. b The replication of the F14.5L- and revertant viruses was studied in CV-1 cells, and compared to that of GLV-1h68. The virulence of the F14.5L- and revertant viruses was investigated in nude mice by an intranasal application of 2 × 106 pfu of individual virus per mouse. The survival and body weight of the mice were monitored for 10 weeks and presented in c and d
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Fig6: Contribution of an expression cassette in the F14.5L locus on the attenuation of GLV-1h68. a The schematic presentation of the F14.5L- and revertant viruses derived from GLV-1h68. b The replication of the F14.5L- and revertant viruses was studied in CV-1 cells, and compared to that of GLV-1h68. The virulence of the F14.5L- and revertant viruses was investigated in nude mice by an intranasal application of 2 × 106 pfu of individual virus per mouse. The survival and body weight of the mice were monitored for 10 weeks and presented in c and d

Mentions: Given the promising data on GLV-1h68 from infection studies and genome comparisons, direct experimental tests probed next the pathogenicity effect of individual genes. Unlike the well known TK or HA gene in the VACV genome, the F14.5L is relatively new and less studied. We therefore focused our direct experimental tests on whether the insertion of an expression cassette in the F14.5L contributed to the attenuation of GLV-1h68. We constructed an F14.5L-revertant virus GLV-1e135, in which the F14.5L was restored to its wild-type LIVP sequence (Fig. 6a). To differentiate the contribution of the inactivation of F14.5L by itself from the contribution by the RUC-GFP expression, GLV-1h71, an F14.5L- virus, was used as a control in which a short nonsense sequence was inserted into the F14.5L locus to interrupt the F14.5L gene (Fig. 6a). GLV-1e135 was first studied in CV-1 cells, and its infection and/or replication capacities were compared to GLV-1h68 or GLV-1h71. There appeared to be an increase in virus infection and/or replication for GLV-1e135 and GLV-1h71 compared to GLV-1h68 (Fig. 6b). At 24 h after the infection, the viral titer recovered from the infected CV-1 cells was the highest for GLV-1e135 (6.9 × 106 pfu/106 cells, p < 0.001 vs. GLV-1h71 or GLV-1h68), followed by GLV-1h71 (2.5 × 106 pfu/106 cells), which was also significantly (p = 0.002) higher than that of GLV-1h68 (1.0 × 106 pfu/106 cells).Fig. 6


The highly attenuated oncolytic recombinant vaccinia virus GLV-1h68: comparative genomic features and the contribution of F14.5L inactivation.

Zhang Q, Liang C, Yu YA, Chen N, Dandekar T, Szalay AA - Mol. Genet. Genomics (2009)

Contribution of an expression cassette in the F14.5L locus on the attenuation of GLV-1h68. a The schematic presentation of the F14.5L- and revertant viruses derived from GLV-1h68. b The replication of the F14.5L- and revertant viruses was studied in CV-1 cells, and compared to that of GLV-1h68. The virulence of the F14.5L- and revertant viruses was investigated in nude mice by an intranasal application of 2 × 106 pfu of individual virus per mouse. The survival and body weight of the mice were monitored for 10 weeks and presented in c and d
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2746888&req=5

Fig6: Contribution of an expression cassette in the F14.5L locus on the attenuation of GLV-1h68. a The schematic presentation of the F14.5L- and revertant viruses derived from GLV-1h68. b The replication of the F14.5L- and revertant viruses was studied in CV-1 cells, and compared to that of GLV-1h68. The virulence of the F14.5L- and revertant viruses was investigated in nude mice by an intranasal application of 2 × 106 pfu of individual virus per mouse. The survival and body weight of the mice were monitored for 10 weeks and presented in c and d
Mentions: Given the promising data on GLV-1h68 from infection studies and genome comparisons, direct experimental tests probed next the pathogenicity effect of individual genes. Unlike the well known TK or HA gene in the VACV genome, the F14.5L is relatively new and less studied. We therefore focused our direct experimental tests on whether the insertion of an expression cassette in the F14.5L contributed to the attenuation of GLV-1h68. We constructed an F14.5L-revertant virus GLV-1e135, in which the F14.5L was restored to its wild-type LIVP sequence (Fig. 6a). To differentiate the contribution of the inactivation of F14.5L by itself from the contribution by the RUC-GFP expression, GLV-1h71, an F14.5L- virus, was used as a control in which a short nonsense sequence was inserted into the F14.5L locus to interrupt the F14.5L gene (Fig. 6a). GLV-1e135 was first studied in CV-1 cells, and its infection and/or replication capacities were compared to GLV-1h68 or GLV-1h71. There appeared to be an increase in virus infection and/or replication for GLV-1e135 and GLV-1h71 compared to GLV-1h68 (Fig. 6b). At 24 h after the infection, the viral titer recovered from the infected CV-1 cells was the highest for GLV-1e135 (6.9 × 106 pfu/106 cells, p < 0.001 vs. GLV-1h71 or GLV-1h68), followed by GLV-1h71 (2.5 × 106 pfu/106 cells), which was also significantly (p = 0.002) higher than that of GLV-1h68 (1.0 × 106 pfu/106 cells).Fig. 6

Bottom Line: The reduced pathogenicity of GLV-1h68 is confirmed in male mice bearing C6 rat glioma and in immunocompetent mice bearing B16-F10 murine melanoma.The contribution of foreign gene expression cassettes in the F14.5L, J2R and A56R loci is analyzed, in particular the contribution of F14.5L inactivation to the reduced virulence is demonstrated by comparing the virulence of GLV-1h68 with its F14.5L- and revertant viruses.GLV-1h68 is a promising engineered VACV variant for anticancer therapy with tumor-specific replication, reduced pathogenicity and benign tissue tropism.

View Article: PubMed Central - PubMed

Affiliation: Genelux Corporation, San Diego Science Center, 3030 Bunker Hill St., Ste. 310, San Diego, CA 92109, USA.

ABSTRACT
As a new anticancer treatment option, vaccinia virus (VACV) has shown remarkable antitumor activities (oncolysis) in preclinical studies, but potential infection of other organs remains a safety concern. We present here genome comparisons between the de novo sequence of GLV-1h68, a recombinant VACV, and other VACVs. The identified differences in open reading frames (ORFs) include genes encoding host-range selection, virulence and immune modulation proteins, e.g., ankyrin-like proteins, serine proteinase inhibitor SPI-2/CrmA, tumor necrosis factor (TNF) receptor homolog CrmC, semaphorin-like and interleukin-1 receptor homolog proteins. Phylogenetic analyses indicate that GLV-1h68 is closest to Lister strains but has lost several ORFs present in its parental LIVP strain, including genes encoding CrmE and a viral Golgi anti-apoptotic protein, v-GAAP. The reduced pathogenicity of GLV-1h68 is confirmed in male mice bearing C6 rat glioma and in immunocompetent mice bearing B16-F10 murine melanoma. The contribution of foreign gene expression cassettes in the F14.5L, J2R and A56R loci is analyzed, in particular the contribution of F14.5L inactivation to the reduced virulence is demonstrated by comparing the virulence of GLV-1h68 with its F14.5L- and revertant viruses. GLV-1h68 is a promising engineered VACV variant for anticancer therapy with tumor-specific replication, reduced pathogenicity and benign tissue tropism.

Show MeSH
Related in: MedlinePlus