Characterization of Morreton virus as an oncolytic virotherapy platform for liver cancers

Abstract

Background: Morreton virus (MORV) is an oncolytic Vesiculovirus , genetically distinct from vesicular stomatitis virus (VSV).

Aim: To report that MORV induced potent cytopathic effects (CPEs) in cholangiocarcinoma (CCA) and hepatocellular carcinoma (HCC) in vitro models.

Approach and results: In preliminary safety analyses, high intranasal doses (up to 10 10 50% tissue culture infectious dose [TCID 50 ]) of MORV were not associated with significant adverse effects in immune competent, non-tumor-bearing mice. MORV was shown to be efficacious in a Hep3B hepatocellular cancer xenograft model but not in a CCA xenograft HuCCT1 model. In an immune competent, syngeneic murine CCA model, single intratumoral treatments with MORV (1 × 10 7 TCID 50 ) triggered a robust antitumor immune response leading to substantial tumor regression and disease control at a dose 10-fold lower than VSV (1 × 10 8 TCID 50 ). MORV led to increased CD8 + cytotoxic T cells without compensatory increases in tumor-associated macrophages and granulocytic or monocytic myeloid-derived suppressor cells.

Conclusions: Our findings indicate that wild-type MORV is safe and can induce potent tumor regression via immune-mediated and immune-independent mechanisms in HCC and CCA animal models without dose limiting adverse events. These data warrant further development and clinical translation of MORV as an oncolytic virotherapy platform.

Trial registration: ClinicalTrials.gov NCT01628640.

Figure 1.

A, Genome Organization and Ultrastructure of MORV. MORV, a negative-sense RNA Vesiculovirus comprising five major genes (MORV-N, MORV-P, MORV-M, MORV-G, and MORV-L) and 11,181 nucleotides in genome. B, Transmission electron microscopy image of MORV virus, a bullet-shaped virus of 200 nm (length) and 75 nm (width). Defective interfering (DI) particles designated as incomplete particles (IP) were found after several plaque purifications. VP (viral particle) and G (glycoprotein).

Figure 2.

Characterization of MORV. A, Left-Viral titers of MORV and vesiculovirus stomatitis virus (VSV). Recombinant MORV and laboratory-adapted VSV were used to infect BHK-21 cells. B, Supernatants from infected cells were collected at different time points, and viral titer was determined using a TCID₅₀ (50% tissue culture infective dose) method on Vero cells (1.5 ×10⁴). Right- Serial dilutions of MORV and VSV were used to infect a monolayer of Vero cells (5 ×10⁵), then stained 48 hours later with crystal violet to reveal viral plaques. C, A549 (2 ×10⁴), interferon responsive lung cancer cells were pretreated with various concentrations of universal type I interferon-alpha (IFN-α), infected with viruses at a multiplicity of infection (MOI) of 0.01. Cell viability was measured using a colorimetric assay (MTS, Promega USA) after 48-hours post-infection. D and E, Normal human serum, anti-VSV glycoprotein (anti-VSV-G) antibody, serum from patients treated with VSV (PT13-D2, PT14–22) expressing human IFN-beta (VSV-hIFN-β) were evaluated for their ability to neutralize 400 TCID₅₀ units of MORV and VSV in Vero cells (2 ×10⁴). Data are expressed as means of triplicate from three independent experiments.

Figure 3.

Cytopathic Effect of MORV and VSV In a Panel of Liver Cancer Cells. A, Monolayers (1.5×10⁵) of a transformed cholangiocyte cell line (H69), human CCA ( EGI-1, PAX-42, HuCCT-1, SNU-1079, RBE, GBD-1, CAK-1, SNU-245, SNU-308, SNU869), murine CCA (SB), human hepatocellular carcinoma (Hep3B, HepG2,Huh7, Sk-Hep1) and murine HCC ( R1LWT, R2LWT, HCA-1 and Hepa 1–6) cells were mock-infected or infected with MORV (A) and VSV (B) at an MOI of 0.01, 0.1 and 1. The percentage of cell viability was determined 72 hours post-infection by MTS assays (Promega, USA). The average of three independent experiments are plotted.

Figure 4.

Assessment of MORV and VSV Infectivity and Cytotoxicity In Low Density Lipoprotein Receptor (LDLR) Knockout Cell Line (HAP-1). A, Expression of low-density lipoprotein receptor (LDLR) was measured by flow cytometry using fluorescein (FITC) anti-human LDLR antibody (solid line) or an isotype control antibody (dashed line). B, we infected HAP1 WT or LDLR KO cells (1×10⁴) cells with different MOIs (10,1, 0.1, 0.01, 0.001, and 0.0001) of MORV or VSV, and 72 hours post-infection, cell viability was measured by MTS assay. C, Representative images of infected or mock-infected HAP1 WT and LDLR KO cells fixed at 48 hours, stained with crystal violet (scale bar=200 μm). Data are shown as mean ± SEM from three independent experiments.

Figure 5.

Intranasal Administration of MORV and VSV in Immunocompetent Mice. A and B, Measurement of body weight and C, Pictures of Hematoxylin and eosin (H&E) stained brain sections from mice treated with high dose of MORV and VSV (1×10¹⁰ TCID₅₀/kg). D and E, complete blood count (white blood cells (WBC) and lymphocytes) values following intranasal administration of increasing doses (1×10^7, 1×10⁸, 1×10⁹ and 1×10¹⁰ TCID₅₀/kg) of MORV and VSV.

Figure 6.

In Vivo Anti-tumor Efficacy of MORV and VSV in Xenograft Mouse Models of Cholangiocarcinoma (CCA) and Hepatocellular Carcinoma (HCC). Single or multiple doses (2) of 1 × 10⁷ TCID₅₀ of MORV or VSV were injected intratumorally into HuCCT-1 (CCA) and Hep3B (HCC) tumor-bearing mice. Analysis of the effect of MORV and VSV on tumor growth and tumor inhibition in HuCCT-1 (A) and (B), and Hep3B (C) and (D) xenografts.

Figure 7.

MORV Reduced Tumor Burden At a Lower Dose Compared to VSV In Murine CCA. A, Overview of the in vivo experiment. B, Tumor weight of mice treated with phosphate-buffered saline (PBS), MORV, and VSV at 1 ×10⁷ TCID₅₀ or 1×10⁸ TCID₅₀. C, Frequency of tumor-infiltrating CD3+ CD8+ CTLs. D, E, F and G, graphs showing changes in serum ALP, ALT, IgM and IgG2b.