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. 2018 Jul 10;10(7):231.
doi: 10.3390/cancers10070231.

Vaccinia Virus Shuffling: deVV5, a Novel Chimeric Poxvirus with Improved Oncolytic Potency

Marine Ricordel  1 , Johann Foloppe  2 , Delphine Antoine  3 , Annie Findeli  4 , Juliette Kempf  5 , Pascale Cordier  6 , Aude Gerbaud  7 , Benoit Grellier  8 , Monika Lusky  9 , Eric Quemeneur  10 , Philippe Erbs  11
Affiliations

Vaccinia Virus Shuffling: deVV5, a Novel Chimeric Poxvirus with Improved Oncolytic Potency

Marine Ricordel et al. Cancers (Basel). .

Abstract

Oncolytic virus (OV) therapy has emerged as a promising approach for cancer treatment with the potential to be less toxic and more efficient than classic cancer therapies. Various types of OVs in clinical development, including Vaccinia virus (VACV)-derived OVs, have shown good safety profiles, but limited therapeutic efficacy as monotherapy in some cancer models. Many different methods have been employed to improve the oncolytic potency of OVs. In this study, we used a directed evolution process, pooling different strains of VACV, including Copenhagen, Western Reserve and Wyeth strains and the attenuated modified vaccinia virus Ankara (MVA), to generate a new recombinant poxvirus with increased oncolytic properties. Through selective pressure, a chimeric VACV, deVV5, with increased cancer cell killing capacity and tumor selectivity in vitro was derived. The chimeric viral genome contains sequences of all parental strains. To further improve the tumor selectivity and anti-tumor activity of deVV5, we generated a thymidine kinase (TK)-deleted chimeric virus armed with the suicide gene FCU1. This TK-deleted virus, deVV5-fcu1 replicated efficiently in human tumor cells, and was notably attenuated in normal primary cells. These studies demonstrate the potential of directed evolution as an efficient way to generate recombinant poxviruses with increased oncolytic potency, and with high therapeutic index to improve cancer therapy.

Keywords: deVV5-fcu1; directed evolution; genome shuffling; vaccinia virus.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The directed evolution process. Representation of the different steps of the directed evolution process used to select a new Poxvirus chimera with improved oncolytic activity.
Figure 2
Figure 2
Oncolytic activities during the different steps of the directed evolution process. (a) Percentage of surviving cells 5 days after infection with passage 6 (LP6), passage 9 (LP9) and Copenhagen stain (COP). Human tumor cells were infected at a MOI of 10–5 and 10–4 with COP and the indicated passages and cell viability was determined by trypan blue exclusion. The parental COP was used as reference. The results are presented as a mean of triplicate experiments ± SD. (b) Percentage of surviving cells after infection of MIA PaCa-2 tumor cell line with the indicated passages at MOI 10–5 and 10–4. Cell viability was determined 5 days after infection. The parental COP was used as reference. The results are presented as a mean of triplicate experiments ± SD. (c) Percentage of surviving cells 5 days after infection of MIA PaCa-2 tumor cell line with the indicated clones at MOI 10–4 and 10–5. The parental COP and MP12 were used as references. The results are presented as a mean of triplicate experiments ± SD.
Figure 3
Figure 3
Homology maps between deVV5 and each parental genome. (a) De novo sequenced viral genomes are shown around the circos plot (COP, orange; MVA, red; WY, blue; WR, green; deVV5, white). Only one ITR is displayed (black). Blast results of deVV5 regions greater than 1 kb with 100% identity are linked by color-coded ribbons to the corresponding hit. (b) deVV5 genome annotated with results from global pairwise alignments. Each annotation corresponds to the longest region with 100% identity to the corresponding parental genome. deVV5 core and single ITR region are also displayed.
Figure 4
Figure 4
Functionality and efficacy of FCU1 expressed by deVV5. (a) Conversion of 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU) and release of 5-FU in the cell culture supernatant. HCT 116 cells were infected with the indicated vector at a MOI of 10–5 and then incubated with 0.1 mM 5-FC from day 2 to day 5 post infection. The relative concentration of 5-FC and 5-FU in the culture supernatant was measured by HPLC. The results are expressed as the percentage of 5-FU released relative to the total amount of 5-FC + 5-FU. Each data point represents the mean of triplicate determinations ± SD. (b) In vitro sensitivities of infected human tumor cells to 5-FC. HCT 116 human tumor cells were mock-infected or infected with deVV5 and deVV5-fcu1 at a MOI of 10–5. After 48 h, cells were treated by increasing concentrations of 5-FC. Cell survival was determined 3 days later as described in Materials and Methods section. Cell viability results are expressed as the percentage of viable cells in the presence and absence of the prodrug. Values are represented as mean ± SD of triplicate determinations.
Figure 5
Figure 5
Both chimeric and recombinant chimera are able to infect and kill cancer cells leaving primary cells intact. (a) Oncolytic activities of COP, deVV5 and deVV5-fcu1 by measuring the cell viability 5 days after infection of different cancer cell lines. Tumor cells were infected with the indicated viruses and cell viability was determined as described in the Materials and Methods section. The MOI used for each cell line was adjusted according to its susceptibility to COP infection. The MOI used for the infection was MOI 10–5 (3 PFU/well) for UM-UC-3, SK-OV-3, CAL33, A549 and Hep G2 cells, MOI 10–4 (30 PFU/well) for OE19, MIA PaCa-2 and HCT 116 cells and MOI 10–3 (300 PFU/well) for KATO III cells. (b) Replication in tumor cell lines and in primary human cells. Tumor cells were infected at MOI 10–5 and harvested 3 days post infection. Human primary hepatocytes were infected with 100 PFU/well and harvested 3 days post infection. 3D Phenion FT skin models were infected with 1.105 PFU and harvested 7 days post infection. Viral progeny production was determined by plaque titration. Results are expressed as viral fold amplification (corresponding to output/input ratio). Asterisks denote statistical significance compared to COP (p < 0.05). (c) Ratio between viral production obtained in Hep G2 hepatocarcinoma cells and hepatocytes for the three viruses: COP, deVV5 and deVV5-fcu1.
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