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. 2009 Oct;282(4):417-35.
doi: 10.1007/s00438-009-0475-1. Epub 2009 Aug 22.

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

Qian Zhang  1 , Chunguang Liang, Yong A Yu, Nanhai Chen, Thomas Dandekar, Aladar A Szalay
Affiliations

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

Qian Zhang et al. Mol Genet Genomics. 2009 Oct.

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-null 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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Figures

Fig. 1
Fig. 1
Phylogenetic tree constructed from orthopoxvirus genomes. The unrooted tree was generated by neighbour-joining, comparing highly conserved 82 kb nucleotide sequence among 16 OPV genomes. Bootstrapping values (percentage; 1,000 trials) are shown beside the nodes. Please see the list of abbreviations and accession numbers for virus identification
Fig. 2
Fig. 2
GLV-1h68 genome map. Predicted ORFs and their lengths are represented by colored arrows. Sixty-one ORFs involved in host interactions and immune modulation are shown in blue. Located mostly in the central conserved region are genes involved in: viral structure and assembly (49 ORFs, shown in pink), metabolism (41 ORFs, shown in orange) and DNA replication, RNA transcription and modifications (28 ORFs, in green). The functions of 58 ORFs remain uncertain (marked with clear arrows). The three foreign insertions are marked in dark grey. The genome size is indicated on the right (nucleotides)
Fig. 3
Fig. 3
The PCR results indicate that the parental strain of GLV-1h68 was a unique isolate from LIVP. a The genomic DNAs from wt-LIVP, GLV-1d27, and GLV-1h68 viruses were used as the templates for PCR amplification of the specific crmE gene fragment and the conserved F13L gene fragment. The PCR results show that, while wt-LIVP population was crmE positive, GLV-1d27 and GLV-1h68 were not; b The genomic DNAs from wt-LIVP and GLV-1h68 were used as the templates for PCR with primers corresponding to mO257L and mORTR02R in LC16mO or GL272 and GL285 in GLV-1h68, respectively. The PCR product for GLV-1h68 was 10,971 bps as predicted. The major PCR product for wt-LIVP was much smaller, similar to the size predicted for LC16mO (5,866 bps). This further confirms that GLV-1h68 was derived from a unique isolate of LIVP
Fig. 4
Fig. 4
GLV-1h68 showed a unique genetic makeup that was distinct from its parental LIVP strain. Genes outlined by navy blue have corresponding paralogs in the left terminus. Genes in light blue are unique for WR strain, and those in light orange are unique for GLV-1h68. The orthologous genes are linked by dashed lines. GL277 (encoding interleukin-18-binding protein) in GLV-1h68 had an ortholog WR013 in the left terminus of WR genome. The majority of the wt-LIVP population had sequences in the CrmE (mO259R) and v-GAAP (mO261R) gene segments that are similar to other Lister strains
Fig. 5
Fig. 5
Replication and pathogenicity of different VACV strains. a Genetic constructs for different VACV strains (reproduced from Zhang et al. 2007). A single base substitution created a premature stop codon; therefore, the TK gene in LIVP wt or GLV-1d27 was naturally inactivated. pSEL, VACV synthetic early late promoter; p7.5 and p11K, VACV 7.5 early/late and 11k promoters; TFR, human transferrin receptor. b The replication of different VACV strains was studied in various cell cultures. c The pathogenicity was evaluated by change in body weight after a single, intravenous injection of each individual virus at 107 pfu per mouse, in nude mice bearing C6 glioma, and C57BL/6 mice bearing B16-F10 melanoma. Change of body weight (%) was calculated as follows: [(b′ − t′) − (b − t)] × 100/(b − t), where b and t are the body weight and tumor weight (estimated by tumor size; 1 cm3 = 1 g) on the day of virus injection, and b′ and t′ are the corresponding weights on the day of monitoring. The average weight change for each group is presented
Fig. 6
Fig. 6
Contribution of an expression cassette in the F14.5L locus on the attenuation of GLV-1h68. a The schematic presentation of the F14.5L-null and revertant viruses derived from GLV-1h68. b The replication of the F14.5L-null and revertant viruses was studied in CV-1 cells, and compared to that of GLV-1h68. The virulence of the F14.5L-null 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
Fig. 7
Fig. 7
Sequence alignment of crmC gene for selected poxvirus strains. The ORFs for CrmC in selected poxvirus strains were translated into amino acids and their sequences were aligned using ClustalX. GLV-1h68 exhibits an intact crmC gene (GL241), which is highly identical to List172 in Lister isolate VACV107, and A56 gene in Cowpox virus, all of which contain a typical signal peptide sequence predicted by SignalP 3 software. Lister strain LC16mO seems to have a full length protein, but its N-terminal sequence suggests that this protein is not secreted. The crmC genes in VACV strains Copenhagen and WR are truncated
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