3. Enhancement of RNA viruses in IFN-responsive cells by VV is mediated both by soluble and local factors
4. Spread of RNA viruses in IFN-responsive cells is dependent on the presence and the level of IFN-antagonistic factorsB C D Heikkilä JE, Vähä-Koskela MJ, Ruotsalainen JJ, Martikainen MW, Stanford MM, McCart JA, Bell JC, Hinkkanen AE. Intravenously administered alphavirus vector VA7 eradicates orthotopic human glioma xenografts in nude mice.PLoS One. 2010 Jan 6;5(1):e8603. Lun XQ, Jang JH, Tang N, Deng H, Head R, Bell JC, Stojdl DF, Nutt CL, Senger DL, Forsyth PA, McCart JA. Efficacy of systemically administered oncolytic vaccinia virotherapy for malignant gliomas is enhanced by combination therapy with rapamycin or cyclophosphamide. Clin Cancer Res. 2009 Apr 15;15(8):2777-88. Le Boeuf F, Diallo J-S, McCart A., Thorne S., Falls T., Stanford M.M., Adatia F., Auer R., Brown C., Lichty B., Parato K., Atkins H., Kirn D., Bell J.C. Synergistic Interaction Between Oncolytic Viruses Augments Tumour Killing. Molecular Therapy 2010. Article in press.
![Exploring the interactions between oncolytic viruses and cellular innate defenses Markus J.V. Vähä-Koskela, Fabrice LeBoeuf, Jean-Simon Diallo, Sophie Breton and John C. Bell Experimental Therapeutics group, Ottawa Hospital Research Institute, Ottawa Regional Cancer Centre Oncolytic viruses offer great promise in the treatment of cancer and several new candidate viruses are being studied [1]. Semliki Forest virus (SFV) is a recently characterized oncolytic virus that appears particularly well suited to target brain tumors [2]. As a single stranded RNA virus, SFV is very sensitive to the antiviral effects of type I (alpha/beta) interferons and we have found its oncolytic efficacy is limited by intact type I IFN signaling. The same is true for anothe oncolytic candidate, vesicular stomatitis virus (VSV), a negative sense single stranded RNA virus. On the other hand, vaccinia virus, a large double stranded DNA virus, has also shown great potential in targeting brain tumors [2] but is not nearly as sensitive to type I IFN-mediated antiviral signaling as these RNA viruses. This is in large part due to its soluble IFN scavenger B18R [3]. Here we show that both SFV and VSV replication may be augmented by combining it with vaccinica virus or with recombinant B18R protein. RNA viruses are inhibited by type I IFNs in vitro and in vivo SFV localizes close to VV infection VVdd SFV Overlay Protection against virus by IFN beta in vitro Individual animal tumor responses to SFV in vivo Figure 5. SFV infection is radially enhanced around VV infection centers, but also seems to involve factors that do not spread far from VV infected cells and only enhance SFV locally. U251 human glioma cells were infected with VVdd (MOI 0.001) and SFV (MOI 0.1) and overlayed with agarose. Fluorescence micrographs were taken 72 hours later. SFV Co-infection (GFP) Co-infection (Overlay) Plaque expansion assay demonstrates dynamics of virus interactions Figure 1. Pretreatment of cultured cancer cells (human U251 glioma) with recombinant interferon beta (100 U/ml) abrogates replication of both SFV and VSV but not vaccinia virus. Figure 2. Murine CT26 colon carcinoma clones differ in responsiveness to interferon. Only CT26LacZ tumors (IFN unresponsive) can be cured in vivo (subcutaneous model, virus given intratumorally). Vaccinia can boost SFV in a species-dependent fashion Figure 3. Vaccinia virus is able to overcome antiviral effects of IFN beta in human but not mouse cancer cell lines. Cells were infected with VVdd-mCherry (MOI 0.1) and 6 hours later with VA7-EGFP (MOI 0.1) either in the continous presence or absence of recombinant interferon beta (100 IU/mL). Fluorescence micrographs taken 72 hours post VVdd infection reveal augmentation of SFV replication in the absence of IFN, and partial restoration of SFV replication in co-infected human but not mouse cells. Co-infection (RFP) VVdd SFV + rB18R Figure 6. SFV infection is eliminated over time in U251 human glioma cells. Co-infection with VV or co-administration of recombinant B18R protein (0.1 mg) enables to virus to spread. VV displays an enhancing factor other than B18R, seen as a zone of increased SFV enhancement in the immediate vicinity of VV. Methodology: Monolayers of cancer cells (U251 human glioma) in 6-well plates were overlayed with agarose and then 4 mm diameter wells were made into the agarose using a cut pipet tip. Virus dilutions and rB18R protein were applied into the wells in a small volume. Plates were incubated for six daysbefore imaging. SFV, 5000 PFU VVdd, 0 PFU SFV, 500 PFU VVdd, 5000 PFU SFV, 5000 PFU VVdd, 5000 PFU SFV, 5000 PFU VVdd, 500 PFU Both soluble and local factors produced by vaccinia virus contribute Figure 7. The extent of SFV spread in IFN-responsive cells is dependent on the level of augmenting factors (in this case vaccinia virus). U251 cells (duplicate wells) were fixed and stained against SFV antigen six days post inoculation. A Figure 4. A) SFV localizes close to VV infection centres. Double-infected cells are very rare, indicating mutual interference and exclusion. Supernatant of VV infected cells (containing no VV particles) is able to enhance SFV replication in autologous cancer cells (MOI 0.01), determined under fluorescence microscope (B) and titration of infected cell culture supernatant 48 h post infection (C). E) Enhancement of SFV oncolysis is mediated in part by vaccinia virus B18R gene product, as evidence by cytotoxicity measurements of SFV infected HT29 cells in the presence or absence of either vvDD or recombinant B18R protein. Conclusions ,[object Object]](https://image.slidesharecdn.com/postdocday2010-110210055044-phpapp01/85/Postdoc-day-2010-1-320.jpg)
