Enhancement of oncolytic properties of recombinant newcastle disease virus through antagonism of cellular innate immune responses

Abstract

Newcastle disease virus (NDV) has been previously shown to possess oncolytic activity, causing specific lysis of cancerous but not normal cells. Here we show that despite these findings, the oncolytic efficiency of naturally occurring NDV strains can still be relatively low, as many tumors exhibit strong innate immune responses that suppress viral replication and spread. To overcome this problem, we generated a recombinant fusogenic NDV expressing influenza NS1 protein, a protein exhibiting interferon (IFN)-antagonist and antiapoptotic functions in human and mouse cells. Interestingly, the resultant virus was dramatically enhanced in its ability to form syncytia, lyse a variety of human and mouse tumor cell lines, and suppressed the induction of the cellular IFN responses. Using the aggressive syngeneic murine melanoma model, we show that the NDV-NS1 virus is more effective than virus not expressing NS1 in clearing the established footpad tumors and results in higher overall long-term animal survival. In addition, mice treated with NDV-NS1 exhibited no signs of toxicity to the virus and developed tumor-specific cytotoxic T lymphocyte (CTL) responses. These findings demonstrate that modulation of innate immune responses by NDV results in enhancement of its oncolytic properties and warrant further investigation of this strategy in design of oncolytic NDV vectors against human tumors.

Figure 1

Recombinant Newcastle disease virus (NDV) with modified fusion protein induces enhanced cytolysis in multiple human cancer cell lines. (a) Cell lines (5 × 10⁵ cells) were infected at multiplicity of infection (MOI) 0.1 in triplicate and lactate dehydrogenase release assays were performed at 24, 48, and 72 hours. Percentage of cells surviving at 24, 48, and 72 hours is shown. (b) Syncytia formation by the NDV(F3aa) virus. Cell lines tested in a were infected with NDV(B1)-GFP and NDV(F3aa)-GFP at MOI 0.01, stained with Hoechst after 24 hours, and images were taken under fluorescent microscope. Representative images from Panc-1 and Scc-25 cells are shown (green: GFP; blue: Hoechst).

Figure 2

Newcastle disease virus (NDV) induces antiviral response in the infected cancer cells. (a) Schematic diagram of the bioassay for interferon (IFN) production. Panc-1 cells were infected with NDVs at multiplicity of infection (MOI) 0.1. Supernatants were collected at 24 hours, and any virus present was UV inactivated. Fresh Vero cells were treated with the inactivated supernatants and then infected with NDV-GFP at MOI of 0.1. (b) Antiviral activity in the infected Panc-1 supernatants. Supernatants were diluted in fivefold series. Supernatants from noninfected cells and recombinant human IFN-β were used as negative and positive controls respectively. NDV-GFP-infected Vero cells were examined by fluorescence microscopy.

Figure 3

Generation of NDV(F3aa)-NS1 virus. (a) Schematic diagram of the generated NDV(F3aa)-NS1 virus genome. (b) Expression of the NS1 protein in NDV(F3aa)-NS1-infected Vero cells. Cells were infected at multiplicity of infection (MOI) 0.01, fixed at 18 hours postinfection, and stained with Dapi (blue), anti-NDV rabbit polyclonal antibody (green) and anti-NS1 mouse monoclonal antibody (red). NDV(B1) and NDV(F3aa)-infected cells were used as negative controls. (c) Time course of NS1 protein expression. Vero cells were infected with appropriate viruses at MOI 0.1 and collected at the indicated time points. Cells were lysed and analyzed by immunoblotting with antibodies to β-actin, NDV proteins, and influenza NS1. NDV, Newcastle disease virus.

Figure 4

NDV(F3aa)-NS1 virus replicates and induces oncolysis in human and mouse melanoma cell lines. (a) Cytotoxicity of the genetically engineered Newcastle disease virus (NDV) in B16-F10 and SkMel-2 cells. B16-F10 cells (left panel) and SkMel-2 cells (right panel) were infected with NDV(B1), NDV(F3aa), and NDV(F3aa)-NS1 viruses at the indicated MOIs. Cytotoxicity was assessed at 24, 48, and 72 hours by lactate dehydrogenase release assays. Lower MOIs were used in SkMel-2 cells due to higher susceptibility of the cells to NDV. (b) Efficiency of syncytia formation by the recombinant viruses. B16-F10 and SkMel-2 cells were infected with appropriate viruses at MOI 0.001 for 18 hours, and fixed and stained for NDV (green), NS1 (red), and Dapi (blue). (c) Replication of NDV in B16-F10 and SkMel-2 cell lines. Cells were infected at the indicated MOIs and the supernatants were collected at 24, 48, and 72 hours. (d) Interferon-β (IFN-β) induction in B16-F10 and SkMel-2 cells. Cells were infected with the indicated virus at MOI 1 and the supernatants were collected every 2 hours for 24 hours. Levels of IFN-β in the supernatant were assessed by murine and human IFN-β enzyme-linked immunosorbent assay.

Figure 5

NDV(F3aa)-NS1 suppresses tumor growth and promotes mouse survival in a syngeneic murine melanoma model. (a) Short-term tumor growth in B16-F10 melanoma-bearing mice treated with recombinant Newcastle disease viruses (NDVs) at 7 days. Mice were injected in the right posterior foot pad with 10⁵ of cultured B16-F10 cells, and 7 days later were treated with 5 × 10⁶ of the indicated viruses or phosphate-buffered saline (PBS) for a total of four injections. All eight mice from the control group and six randomly chosen mice from each virus group were killed on day 25 for immune studies (see Figure 6). (b) Short-term tumor growth in mice treated at 10 days. Starting on day 10 after tumor cell line injection, the mice were treated every other day with a total of six doses of 5 × 10⁶ pfu of the indicated virus or PBS. When the largest tumors reached 8 mm in length, all of the animals were killed. (c) Long-term tumor growth follow-up in the treated mice. The remaining seven animals from each group in a were continued to be followed for 120 days, with tumor measurements being recorded every 2 days. (d) Summary of 120-day survival of the animals treated in a. Mice were killed when the tumors reached 8 mm in length. For experimental groups, only the mice included in the long-term study (n = 7 for each group) were included in the analysis (^*P < 1 × 10⁻⁶).

Figure 6

NDV treatment leads to tumor lymphocyte infiltration and generation of antimelanoma immune responses. (a) Tumor infiltration with CD4⁺ and CD8⁺ cells. Tumors collected from the animals described in 6D were dissociated into single-cell suspensions and analyzed by flow cytometry for presence of CD4 and CD8 cells. (b) Interferon-γ (IFN-γ) release from stimulated splenocytes. Splenocytes collected from the killed animals in 6a were cocultured with mitomycin-inactivated B16-F10 cells and IFN-γ was measured in the supernatants on day 3 of coculture (^*P < 0.003, ^**P < 0.00006). (c) Melanoma-specific cytotoxicity of the stimulated splenocytes. Stimulated splenocytes described in b were cocultured with fresh B16-F10 cells for 4 hours at the indicated ratios and specific cytotoxic activity was determined by measurements of lactate dehydrogenase release (^*P < 0.015, ^**P < 0.0007).