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Cowpox virus (CPXV) strain GRI-90 contains six genes encoding kelch-like proteins. All six proteins contain both, the N-terminal BTB domain and the C-terminal kelch domain. We constructed mutant variants of a CPXV strain with targeted deletions of one to four genes of the kelch family, namely D11L, C18L, G3L, and A57R. As kelch genes are located in terminal variable regions of the CPXV genome, we studied the relationship of these genes with integral biological characteristics such as virulence, host range, reproduction in vitro and in ovo (in chicken embryos). It was demonstrated that the following effects occurred in a gene dose dependent manner with an increase of the number of genes deleted: (1) range of sensitive cells altered--deletion mutants lacking three genes displayed a considerably decreased ability to reproduce in MDCK cells; mutants lacking four genes lost this ability completely; (2) analysis of pocks formed by mutants with deletion of three and four kelch-like genes on chorioallantoic membranes of chicken embryos demonstrated that pock size and virus yield were significantly decreased; (3) light microscopic analysis of the pocks revealed impaired proliferation and reduced vascularisation in the pock region. More alterations were detected by electron microscopic analysis: the reproduction of mutants results in a reduction of the number of mature virions formed, and in many cells this process was arrested at the stage of assembly of immature virions; and (4) the evaluation of LD(50) and body weight loss in BALB/c mice infected intranasally with CPXVs revealed a reduction of the virulence of the deletion mutants, which became statistically significant when four kelch-like genes were excised.

Vaccinia virus (VACV) oncolytic therapy has been successful in a number of tumor models. In this study our goal was to generate a double recombinant vaccinia virus (VV-GMCSF-Lact) with enhanced antitumor activity that expresses exogenous proteins: the antitumor protein lactaptin and human granulocyte-macrophage colony-stimulating factor (GM-CSF). Lactaptin has previously been demonstrated to act as a tumor suppressor in mouse hepatoma as well as MDA-MB-231 human adenocarcinoma cells grafted into SCID mice. VV-GMCSF-Lact was engineered from Lister strain (L-IVP) vaccinia virus and has deletions of the viral thymidine kinase and vaccinia growth factor genes. Cell culture experiments revealed that engineered VV-GMCSF-Lact induced the death of cultured cancer cells more efficiently than recombinant VACV coding only GM-CSF (VV-GMCSF-dGF). Normal human MCF-10A cells were resistant to both recombinants up to 10 PFU/cell. The selectivity index for breast cancer cells measured in pair cultures MCF-7/MCF-10A was 200 for recombinant VV-GMCSF-Lact coding lactaptin and 100 for VV-GMCSF-dGF. Using flow cytometry we demonstrated that both recombinants induced apoptosis in treated cells but that the rate in the cells with active caspase −3 and −7 was higher after treatment with VV-GMCSF-Lact than with VV-GMCSF-dGF. Tumor growth inhibition and survival outcomes after VV-GMCSF-Lact treatment were estimated using immunodeficient and immunocompetent mice models. We observed that VV-GMCSF-Lact efficiently delays the growth of sensitive and chemoresistant tumors. These results demonstrate that recombinant VACVs coding an apoptosis-inducing protein have good therapeutic potential against chemoresistant tumors. Our data will also stimulate further investigation of coding lactaptin double recombinant VACV in clinical settings.