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. 2015 Jun 11;7(6):2980-98.
doi: 10.3390/v7062756.

Recombinant Immunomodulating Lentogenic or Mesogenic Oncolytic Newcastle Disease Virus for Treatment of Pancreatic Adenocarcinoma

Pascal Buijs  1 , Stefan van Nieuwkoop  2 , Vincent Vaes  3 , Ron Fouchier  4 , Casper van Eijck  5 , Bernadette van den Hoogen  6
Affiliations

Recombinant Immunomodulating Lentogenic or Mesogenic Oncolytic Newcastle Disease Virus for Treatment of Pancreatic Adenocarcinoma

Pascal Buijs et al. Viruses. .

Abstract

Oncolytic Newcastle disease virus (NDV) might be a promising new therapeutic agent for the treatment of pancreatic cancer. We evaluated recombinant NDVs (rNDVs) expressing interferon (rNDV-hIFNβ-F₀) or an IFN antagonistic protein (rNDV-NS1-F₀), as well as rNDV with increased virulence (rNDV-F₃aa) for oncolytic efficacy in human pancreatic adenocarcinoma cells. Expression of additional proteins did not hamper virus replication or cytotoxic effects on itself. However, expression of interferon, but not NS1, resulted in loss of multicycle replication. Conversely, increasing the virulence (rNDV-F₃aa) resulted in enhanced replication of the virus. Type I interferon was produced in high amounts by all tumor cells inoculated with rNDV-hIFNβ -F₀, while inoculation with rNDV-NS1-F₀ resulted in a complete block of interferon production in most cells. Inoculation of human pancreatic adenocarcinoma cells with rNDV-F₃aa caused markedly more cytotoxicity compared to rNDV-F₀, while inoculation with rNDVβ-hIFN -F₀ and rNDV-NS1-F₀ induced cytotoxic effects comparable to those induced by the parental rNDV-F₀. Evaluation in vivo using mice bearing subcutaneous pancreatic cancer xenografts revealed that only intratumoral injection with rNDV-F₃aa resulted in regression of tumors. We conclude that although lentogenic rNDVs harboring proteins that modulate the type I interferon pathway proteins do have an oncolytic effect, a more virulent mesogenic rNDV might be needed to improve oncolytic efficacy.

Keywords: Newcastle disease virus; immunotherapy; innate immunity; oncolytic virotherapy; oncolytic virus; pancreatic adenocarcinoma.

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Figures

Figure 1
Figure 1
Generation and characterization of rNDVs. (a) Cloning strategy to obtain full length NDV plasmids. The nucleotide length of the NDV genome is noted below the plasmid names and all full-length plasmids and pGEM-T-PM-cassette are drawn to scale. The 3′-terminal leader, intergenic regions and 5′-terminal trailer are shown as horizontal lines. Gene-end and gene-start sequences in the PM intergenic region are depicted as vertical black and white rectangles, respectively. fwd: forward; rev: reverse; nt: nucleotides; pGEM-T: plasmid backbone of pGEM-T-PM-cassette; NP: nucleoprotein gene; P: phosphoprotein gene; V: accessory V gene; M: matrix gene; F: fusion gene; HN: hemagglutinin-neuraminidase gene; L: large protein gene; (b) PCR product of PM intergenic region. RNA of indicated rNDV stocks was reverse transcribed into cDNA and the PM intergenic region was amplified with RT-PCR using flanking primers; (c) Western blot for expression of NS1 protein. Whole cell lysates of Vero-118 cells (mock-) inoculated with indicated viruses were assayed for expression of the NS1 protein or tubulin protein.
Figure 2
Figure 2
Replication kinetics of rNDVs in 6 different human pancreatic adenocarcinoma cell lines (HPACs). Cells were inoculated in triplo with m.o.i. 0.1 and samples were taken at indicated time points and titrated by end-point dilution assay in Vero-118 cells. Means and standard deviations of duplicate titrations are plotted. H.p.i.: hours post inoculation. * = p < 0.05 vs. rNDV-F0 (one-way ANOVA + Bonferroni post-test), tested for time points 24, 48 and 96 h.p.i.
Figure 3
Figure 3
IFN responses upon inoculation with rNDVs. Cells were either mock inoculated or inoculated with the indicated rNDV at m.o.i. 3. After 24 h: (a) RNA was isolated and quantitative real-time polymerase chain reaction (qRT-PCR) was performed for endogenous hIFNβ mRNA. Results are presented as fold change gene induction of treated versus mock treated cells calculated using the 2−ΔΔCT method [31]. Means and ranges of triplicate experiments are plotted; (b) Supernatants were tested for functional IFN protein content using an ISRE-luc bioassay [14]. Results are presented as fold change in luminescence compared to mock inoculated cells. Means and standard deviations of triplicate experiments are plotted. x = p < 0.05 vs. MRC-5 (one-way ANOVA + Bonferroni post-test), * = p < 0.05 vs. rNDV-F0 (one-way ANOVA + Bonferroni post-test).
Figure 4
Figure 4
Median LD50 upon inoculation with rNDVs. Cells were either mock inoculated (not shown; set as 100% viable) or inoculated with rNDV at different m.o.i. (range 0.0001–100). Cytotoxicity was measured after 5 days by LDH assay and LD50 values were calculated. Means and ranges of LD50 calculations are plotted. * = p < 0.05 as compared rNDV-GFP-F0, rNDV-hIFNβ-F0 and rNDV-F3aa compared to rNDV-F0. # = p < 0.05, rNDV-F3aa compared to rNDV-hIFNβ-F0. ^ = p < 0.05 rNDV-F3aa compared to rNDV-NS1-F0.
Figure 5
Figure 5
Efficacy of rNDV treatment in a mouse model using SU.86.86, BxPC-3 or MIA PaCa-2 subcutaneous tumor xenografts. Animals were treated and evaluated as described in the material and methods section. Graphs with specific xenograft-treatment combinations depict individual mouse tumor volumes with black lines and dots. Last observed tumor volumes were carried forward to calculate median volumes per group in the lowest plots. *: p < 0.05 as compared to PBS group. D.p.i.: days post first injection.
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