Engineering of double recombinant vaccinia virus with enhanced oncolytic potential for solid tumor virotherapy

Abstract

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.

Recombinant Vaccinia Viruses Coding Transgenes of Apoptosis-Inducing Proteins Enhance Apoptosis But Not Immunogenicity of Infected Tumor Cells

“…The Lister strain (L-IVP) of VV was obtained from the State Collection of Viral and Rickettsial Disease Agents of the State Research Center of Virology and Biotechnology “Vector”, Koltsovo, Russia. The recombinant vaccinia viruses, VV-GMCSF-dGF and VV-GMCSF-Lact, were constructed from L-IVP and purified as described previously [ 24 ]. VVdGF-ApoS24/2 was used as the parental virus for homologous recombination of GM-CSF human cDNA into the structural part of the tk locus [ 25 , 28 , 29 ].…”

“…The structure of recombinant VV-GMCSF-Apo was confirmed by sequencing both DNA strains with primers 5′-CAGAATTAATTAGACGAGTTAGACG (TK-flank1 sense) and 5′-TCTCGGTTTCCTCACCCAAT (TK-flank1 as) for the tk region, and 5′-GTAAGCAAAGAATATAAGAATGAAGCGGTAATGAT (Up35) and 5′-CGAGCACAATACCGGGAGATGG (Apa-L22) for the vgf region. The expression of GM-CSF and apoptin was tested by western blot as described in [ 24 , 25 ].…”

“…Tumor cells were seeded at 1.5 × 10 4 cells per well on 96-well plates. The next day, recombinant viruses were added to the cells and incubated for 72 h. During incubation, cells were analyzed by phase-contrast microscopy using ZOE Fluorescent Cell Imager (Bio-Rad) and the final viability was detected by an MTT test, as described earlier [ 24 ].…”

“…Cell populations with the annexin V − /PI − phenotype were designed as living cells, annexin V + /PI − as apoptotic cells, and annexin V + /PI + as secondary necrotic cells. Caspase-3 and caspase-7 activation was detected, as described previously [ 24 ]. In short, all detached and adhesive infected cells were incubated with FLICA working solution from Vibrant FAM Caspase-3 and Caspase-7 Assay Kit (ref.…”

“…Although the vaccinia virus has been shown to preferably trigger programmed necrosis we reported in our previous investigation that the double recombinant vaccinia virus VV-GMCSF-Lact, coding proapoptotic protein lactaptin and human GM-CSF, induced cancer cell death with caspase-3 and caspase-7 activation [ 6 , 24 ]. Nevertheless, the effect of VV-GMCSF-Lact on the other checkpoint elements of the apoptotic cascade, as well as the induction of immunogenic cell death, has not been investigated yet.…”