Chimeric and Pseudotyped Parvoviruses Minimize the Contamination of Recombinant Stocks with Replication-Competent Viruses and Identify a DNA Sequence That Restricts Parvovirus H-1 in Mouse Cells

Abstract

Recent studies demonstrated the ability of the recombinant autonomous parvoviruses MVMp (fibrotropic variant of the minute virus of mice) and H-1 to transduce therapeutic genes in tumor cells. However, recombinant vector stocks are contaminated by replication-competent viruses (RCVs) generated during the production procedure. To reduce the levels of RCVs, chimeric recombinant vector genomes were designed by replacing the right-hand region of H-1 virus DNA with that of the closely related MVMp virus DNA and conversely. Recombinant H-1 and MVMp virus pseudotypes were also produced with this aim. In both cases, the levels of RCVs contaminating the virus stocks were considerably reduced (virus was not detected in pseudotyped virus stocks, even after two amplification steps), while the yields of vector viruses produced were not affected. H-1 virus could be distinguished from MVMp virus by its restriction in mouse cells at an early stage of infection prior to detectable viral DNA replication and gene expression. The analysis of the composite viruses showed that this restriction could be assigned to a specific genomic determinant(s). Unlike MVMp virus, H-1 virus capsids were found to be a major determinant of the greater permissiveness of various human cell lines for this virus.

MCP‐3 (CCL7) delivered by parvovirus MVMp reduces tumorigenicity of mouse melanoma cells through activation of T lymphocytes and NK cells

“…The enhanced green fluorescent protein-transducing recombinant virus Chi-MVMp/EGFP was previously described. 25 Recombinant molecular clones were verified by sequencing and for their capacity of producing functional transgene products after transfection by biological tests or fluorescence. The recombinant MVMp-derived viruses were produced in 293 T cells by cotransfecting the recombinant parvoviral DNA molecular clone and the helper plasmid expressing the capsid proteins under the control of the CMV promoter.…”

“…The recombinant viral DNA clones pMVMp/MCP-3 and pChiMVMp/MCP-3 were produced by inserting the cDNA encoding the human MCP-3 gene from the plasmid pBKRSV/PCRMCP3B 6 as a BamH1/XhoI fragment (434 bp) in the empty parvoviral DNA clone pMVMpD400 23 or pChi-MVMpD800, 25 respectively. The enhanced green fluorescent protein-transducing recombinant virus Chi-MVMp/EGFP was previously described.…”

“…The recombinant MVMp-derived viruses were produced in 293 T cells by cotransfecting the recombinant parvoviral DNA molecular clone and the helper plasmid expressing the capsid proteins under the control of the CMV promoter. 25 Virus stocks were purified by iodixanol gradient centrifugation. 25,28 …”

“…only the capsid protein-encoding region is replaced by a therapeutic transgene, while the genes coding for the nonstructural (NS) viral proteins NS1 and NS2 are retained to allow viral genome amplification and trans-activation of the second viral promoter in susceptible cells. 18,[22][23][24][25] As a consequence, the capsid-replacement vectors are defective as they fail to produce progeny recombinant virus upon their infection, but are capable to produce transiently high levels of transgene products. 21,26 The fibrotropic variant of the mouse parvovirus minute virus of mice (MVMp) does not seem to induce strong cytotoxic T cell responses (unpublished) or severe inflammatory reactions 20 against incoming virus components, an interesting feature for their use as anticancer agents.…”

Activation of the human immune system via toll‐like receptors by the oncolytic parvovirus H‐1

“…Titration of H-1PV was performed by plaque assays as previously described. 32 The multiplicity of infection (MOI) was given by the number of plaque-forming units (pfu) inoculated per cell. Exponentially growing cell cultures were incubated for 1 hr with H-1PV at a MOI of 20 pfu/cell in complete medium (DMEM for HEK293; X-Vivo for dendritic cell/SK29Mel coculture).…”