Type I interferon-sensitive recombinant newcastle disease virus for oncolytic virotherapy

Abstract

Newcastle disease virus (NDV), an avian paramyxovirus, is tumor selective and intrinsically oncolytic because of its potent ability to induce apoptosis. Several studies have demonstrated that NDV is selectively cytotoxic to tumor cells but not normal cells due to defects in the interferon (IFN) antiviral responses of tumor cells. Many naturally occurring strains of NDV have an intact IFN-antagonistic function and can still replicate in normal human cells. To avoid potential toxicity issues with NDV, especially in cancer patients with immunosuppression, safe NDV-oncolytic vectors are needed. We compared the cell killing abilities of (i) a recombinant NDV (rNDV) strain, Beaudette C, containing an IFN-antagonistic, wild-type V protein (rBC), (ii) an isogenic recombinant virus with a mutant V protein (rBC-Edit virus) that induces increased IFN in infected cells and whose replication is restricted in normal human cells, and (iii) a recombinant LaSota virus with a virulent F protein cleavage site that is as interferon sensitive as rBC-Edit virus (LaSota V.F. virus). Our results indicated that the tumor-selective replication of rNDV is determined by the differential regulation of IFN-alpha and downstream antiviral genes induced by IFN-alpha, especially through the IRF-7 pathway. In a nude mouse model of human fibrosarcoma, we show that the IFN-sensitive NDV variants are as effective as IFN-resistant rBC virus in clearing the tumor burden. In addition, mice treated with rNDV exhibited no signs of toxicity to the viruses. These findings indicate that augmentation of innate immune responses by NDV results in selective oncolysis and offer a novel and safe virotherapy platform.

FIG. 1.

Levels of virus replication and spread of NDV differ in normal human epithelial cells and HuTu80 tumor cells. SVHUC1, normal, immortalized human uroepithelial cells, and HuTu80 intestinal epithelial tumor cells were infected with rBC-EGFP virus at an MOI of 0.01. (A to D) Left panels indicate SVHUC1 cells, and right panels indicate HuTu80 cells. (A) Mock-infected cells; (B) expression of EGFP in a few virus-infected normal cells compared with extensive EGFP fluorescence in virus-infected HuTu80 cells by 12 h postinfection; (C) absence of virus spread and replication in normal cells compared with extensive EGFP and virus spread in tumor cells by 24 h postinfection; (D) expression of EGFP in a few cells, with absence of viral spread in normal human cells at 48 h and complete destruction of a monolayer, with clumps of dead cells expressing EGFP in HuTu80 tumor cells.

FIG. 2.

Virus replication and type I interferon induction by rNDV in normal and human tumor cells. (A) Interferon sensitivity of rNDV. Normal human (SVHUC1) and tumor (HuTu80) cells in duplicate were treated with 10 pg/ml of human IFN-α for 24 h, and virus yield was examined by plaque assay on Vero cells at 24, 48, and 72 h postinfection with an rNDV multiplicity of infection of 0.01. (B) DF1 chicken embryo fibroblast cells and normal and human tumor cells were either mock infected or infected with rNDV at an MOI of 10 without IFN pretreatment. Culture supernatants were tested for IFN-β and IFN-α by ELISA. IFN-β production in rNDV-infected cells at 48 h postinfection. (C) IFN-α production at 48 h postinfection. (D) Time course analysis of IFN-β production in HuTu80 cells with an MOI of 0.01. (E) Time course analysis of IFN-α production in HuTu80 cells with an MOI of 0.01. (F) Multicycle virus replication of rNDV in normal and HuTu80 cells without IFN pretreatment. Cells were infected with an rNDV MOI of 0.01. Kinetics of the virus yield of rNDV in HuTu80 tumor cells was assessed in Vero cells. Results represent mean values plus standard errors of the means from two independent experiments.

FIG. 3.

Failure to activate IRF-7 enhances the replication and spread of NDV. SVHUC1, 2fTGH, U3A, and U6A human fibrosarcoma cells were either mock infected or infected with the rLaSota V.F., rBC, or rBC-Edit strain of NDV at an MOI of 10. Virus replication and spread indicated by EGFP expression in SVHUC1 (IRF-7⁺) (A) and IRF-7-hypermethylated wild-type and mutant human fibrosarcoma cells (B to D). (B) 2fTGH cells; (C) U3A cells; (D) U6A cells.

FIG. 4.

IFN-α-responsive genes induced after rNDV infection of normal and tumor cells. The failure of secondary and tertiary transcriptional responses (2°TR and 3°TR, respectively) to NDV aids in tumor selectivity and oncolysis. The primary response to viral infection is mediated by IRF-3, leading to stimulation of the IFN-β promoter. IFN-β is then translated and secreted to stimulate, in an autocrine fashion, JAK/STAT signaling to form ISGF3 complexes in the nucleus, which mediate the induction of the secondary transcriptional responses. Without the consequent expression of IRF-7 in cells infected with rNDV, the tertiary transcriptional wave, which includes almost all IFN-α genes and its downstream antiviral mediators, could not take place. In the absence of IFN-α and IFN-α-responsive antiviral genes, virus replication is enabled in tumor cells. In normal human cells, the transcriptional events proceed unhampered, resulting in a robust antiviral state preventing virus replication. IFN-α-responsive antiviral genes were detected by RT-PCR of cell lysates infected with rNDV at 48 h postinfection. Fold increases of different antiviral mediators, such as ISG15, 2′,5′ A, IRF-1, and ISG 6-16 mRNAs, compared to levels in mock-infected cells are shown. Values represent average fold increases over levels in mock-infected cells from two independent experiments. RT-PCR products of 2′,5′ A and ISG15 analyzed in 2% agarose gels are shown, along with β-actin for comparison.

FIG. 5.

Interferon-sensitive NDV strains regress xenotransplanted tumors in nude mice as efficiently as an interferon-antagonistic NDV strain. BALB/c nude mice were subcutaneously implanted with HT1080 human fibrosarcoma tumor cells. When tumors reached a size of >5 mm in any one plane, recombinant NDV strains were administered intratumorally as a single injection (2 × 10⁷ PFU/ml in 50 μl). (A) Mock-infected mice with tumors on day 31; (B) tumor-bearing mice treated with rBC-EGFP virus; (C) mice treated with rBC-Edit virus; (D) mice treated with rLaSota V.F. virus on day 31 posttreatment. (E) Calculated tumor volumes for each virus strain were plotted against days postinoculation. Results are mean values plus standard errors of the means from three BALB/c mice.

FIG. 6.

Live imaging of rBC-EGFP virus in tumor-bearing BALB/c nude mice. HT1080 tumor-bearing mice were injected intratumorally with 100 μl of 2.0 × 10⁷-PFU/ml rBC-EGFP virus, and the virus distribution was imaged using an IVIS live imager at 72 h postinoculation. Several views of two tumor-bearing, rBC-EGFP-infected mice are shown. sr, steridian; bkg, background.