<i>In vivo</i> activity of AAV8-3xIRF-ISRE-Luc in hamster in response to intravenous NDV-F3AA-GFP LaSota administration.

<p>Syrian hamsters were inoculated iv with 1x10¹¹ vg of the AAV8-3xIRF-ISRE-Luc vector. Three weeks later, animals received an iv administration of 1x10⁹ iu NDV-F3AA-GFP LaSota. A) <i>In vivo</i> luciferase activity was monitored at 10, 24, 48 and 72 hours after iv administration of the NDV virus. Each line represents an individual hamster. B) Image of a representative hamster before (basal) and 10 hours after the first NDV administration. C) Luciferase activity stimulation in hamsters receiving two doses of NDV-F3AA-GFP LaSota 6 weeks apart one from the other. D) Type I IFN activity in serum of hamsters before and 24 hours after second NDV administration, measured by bio-assay. E) NDV neutralizing antibodies in serum of hamsters before and 6 weeks after the first NDV administration. These data are from one experiment representative of two.</p

<i>In vivo</i> reporter activity of AAV8-3xIRF-ISRE-Luc in the respiratory tract.

<p>Mice received an intranasal instillation of AAV8-3xIRF-ISRE-Luc (1x10¹¹ viral genomes/mouse) and were then divided in 4 groups, according to the following intranasal stimuli: Saline solution (Mock); 2x107 iu NDV-F3AA-GFP LaSota; 2x10⁷ iu influenza A/PR8/34-ΔNS1 or 2x10² iu Wt A/PR8/34. A) Luciferase activity was measured by BLI in the upper and lower respiratory tract at the indicated times. B) Representative images of mice before (baseline) and 120 hours after Wt PR8 infection. Each line represents an individual mouse. These data are from one experiment representative of two.</p

<i>In vitro</i> characterization of IFN-I reporters.

<p>A) Schematic representation of reporter plasmid constructs. B) Dual luciferase reporter assay in HuH-7 cells transfected with the indicated plasmids in response to 500 units/ml of human IFN-α for 24 hours. C) Luciferase activity (fold induction) in HuH-7 cells transfected with the indicated reporter plasmids and treated with 500 units/ml of human IFN-α for 24 hours. D) Dual luciferase reporter assay in Hepa 1.6 cells transfected with the indicated plasmids in response to Sendai Cantell virus (20 hemagglutination units, 24 hours). E) Fold induction of luciferase activity in cells transfected with the 3xIRF-ISRE reporter plasmids in the indicated cell lines treated during 24 hours with 500 units/ml of the corresponding species-specific IFN-α. F) Luciferase activity fold induction in murine cell lines transfected with the 3xIRF-ISRE reporter plasmid and treated with 500 units/ml murine IFN-α, 500 units/ml murine IFN-β, 50 μg/ml poly I:C or 1 μg/ml poly I:C mixed with 250 μg/ml DEAE-Dextran for 24 hours. G) Time course of luciferase activity in HuH-7 cells transfected with the 3xIRF-ISRE-luc reporter plasmids and treated with 500 units/ml of human IFN-α. These data are from one experiment representative of four. *** p<0.001, ns: not significant. TK: Thymidine kinase.</p

<i>In vivo</i> characterization of 3xIRF-ISRE-Luc reporter plasmid delivered to mouse liver by hydrodynamic injection.

<p>A) Mice received a hydrodynamic injection with 20 μg of 3xIRF-ISRE-Luc reporter plasmid through the tail vein. Once luciferase activity stabilized (one month after injection), mice were treated intraperitoneally with the indicated doses of murine IFN-β. Light emission was quantified by BLI 10 and 24 hours after treatment. Values correspond to fold luciferase activity, using baseline (pre-induction) activity as a reference. B) Quantitative RT-PCR of <i>OAS</i> and <i>Mx1</i> genes in peripheral blood lymphocytes of animals treated for 24 hours with different doses of recombinant murine IFN-β. C) Reporter activity re-induction in mice determined every week by intraperitoneally administration of 10,000 units of IFN-β. Each line represents an individual mouse. D) Representative BLI images of mice before and 10 hours after administration of 10,000 U of murine IFN-β. These data are from one experiment representative of three.** p<0.01 <i>vs</i> 3,000, 1,000 and 0 IFN-β units.</p

<i>In vivo</i> activity of AAV8-3xIRF-ISRE-Luc in mice in response to intravenous NDV-F3AA-GFP LaSota administration.

<p>Mice were inoculated iv with 3x10¹⁰ vg of the AAV8-3xIRF-ISRE-Luc vector or a control vector AAV8-AAT-Luc, in which luciferase expression is controlled by a constitutive liver-specific promoter. Two weeks later, animals started to receive iv administrations of NDV-F3AA-GFP LaSota virus at different doses and schedules A) <i>In vivo</i> luciferase activity stimulation in the AAV8-3xIRF-ISRE-Luc transduced animals at 10, 24, 48 and 72 hours after iv administration of the NDV virus at 2x10⁷ iu. Each line represents an individual mouse. B) Mice transduced with AAV8-3xIRF-ISRE-Luc received the indicated doses of NDV-F3AA-GFP LaSota virus or UV-inactivated virus (UV), and the stimulation of luciferase activity was determined 10 hours later (indicated as average fold induction for each group). C) Mice transduced with AAV8-3xIRF-ISRE-Luc (upper panel) or AAV8-AAT-Luc (lower panel) received repeated iv inoculations of 2x10⁷ iu NDV one week apart. Stimulation of luciferase activity was determined 10 hours after every NDV injection. Each line represents an individual mouse. D) Concentration of IFN-α in the serum of mice, determined by ELISA 24 hours after each NDV administration E) NDV neutralizing antibodies in serum of mice, determined one day after each round of NDV administration. F) A sub-group of mice was sacrificed after the first or fourth NDV-GFP administration, and expression of virally encoded GFP was determined by qRT-PCR in liver samples. These data are from one experiment representative of three.</p

<i>In vivo</i> activity of AAV8-3xIRF-ISRE-Luc in response to IFN-agonist.

<p>A) Schematic representation of the AAV8-3xIRF-ISRE-Luc vector (not drawn to scale). B) Mice were inoculated iv with 3x10¹⁰ vg of the vector. Two weeks later, animals were divided in different groups and received the following iv stimuli: poly I:C, CpG DNA, Imiquimod (R848) or murine IFN-β. Treatments were repeated weekly for 3 weeks. Luciferase activity was quantified by BLI. Each line represents the fold luciferase activity of each individual mouse. C) Representative BLI images of AAV8-3xIRF-ISRE-Luciferase transduced mice before and 10 hours after murine IFN-β administration. These data are from one experiment representative of two.</p

Additional file 1: of Enhanced therapeutic effect using sequential administration of antigenically distinct oncolytic viruses expressing oncostatin M in a Syrian hamster orthotopic pancreatic cancer model

Additional Figure S1 Measurement of type I IFN in conditioned media from HaP-T1 cells infected with of Ad or NDV. H2T cells were exposed for 8 h to conditioned media from HaP-T1 cells infected during 24 h with the indicated viruses. mRNA levels of the type I IFN stimulated genes OAS (a) and GBP1 (b) were determined by qRT-PCR. H2T cells treated with conditioned media from uninfected cells (control) or cells treated with 500 units/ml recombinant human IFNα are included as negative and positive controls, respectively (representative results of at least 2 experiments performed in triplicate). *** p < 0.001. Additional Figure S2 Rapid onset of NAbs reduces the efficacy of Ad redosing. Hamsters bearing orthotopic pancreatic cancer tumors received a local administration of Ad-WT-Luc (1 × 109 iu/hamster) at day 0 and were then separated into two groups (n = 4). One of them received a second administration of the same dose of virus at day 3 (a) and the other one received it at day 7 (b). Expression of luciferase was quantified by in vivo bioluminescence imaging at the indicated times. The graphs show total light emission (in photons/s) measured from the tumor area. (c) Representative image of a hamster expressing luciferase at day 1, showing the region of interest for quantification. Note the predominant light emission in the anatomical region corresponding to the injected pancreatic tumor (left panel, ventral view; right panel, lateral view). In the artificial color code, red and blue represent maximal and minimal light emissions, respectively. Additional Figure S3 Elevation of OSM in serum causes severe lung inflammation in Syrian hamsters. Animals treated with 1 × 109 iu Ad-OSM show signs of toxicity and most of them die or require euthanasia during the first week after virus administration. The microphotographs show histopathological examination of lungs from representative animals from the untreated (control) and Ad-OSM-treated groups (Hematoxylin-Eosin, x200). Note the presence of edema, inflammatory infiltrate and deposition of extracellular matrix. Additional Figure S4 Ad-OSM replicates in PDAC tumors in vivo. Hamsters bearing orthotopic pancreatic cancer tumors received a local administration of Ad-OSM at 2.5 × 108 iu/hamster (n = 5) or a first-generation adenoviral vector encoding luciferase (AdCMVLuc) at 2 × 109 iu. Hamsters were sacrificed 4 days later for tumor collection. (a) Quantification of infectious units in tumor lysates (expressed as iu/mg tissue). (b) Detection of hexon expression in tumor cells by immunohistochemistry. A cluster of positive cells is marked by a black arrow (200x magnification). (PDF 1298 kb

Usage of Adenovirus Expressing Thymidine Kinase Mediated Hepatocellular Damage for Enabling Mouse Liver Repopulation with Allogenic or Xenogenic Hepatocytes

<div><p>It has been shown that the liver of immunodeficient mice can be efficiently repopulated with human hepatocytes when subjected to chronic hepatocellular damage. Mice with such chimeric livers represent useful reagents for medical and clinical studies. However all previously reported models of humanized livers are difficult to implement as they involve cross-breeding of immunodeficient mice with mice exhibiting genetic alterations causing sustained hepatic injury. In this paper we attempted to create chimeric livers by inducing persistent hepatocellular damage in immunodeficient Rag2^-/- γc^-/- mice using an adenovirus encoding herpes virus thymidine kinase (AdTk) and two consecutive doses of ganciclovir (GCV). We found that this treatment resulted in hepatocellular damage persisting for at least 10 weeks and enabled efficient engraftment and proliferation within the liver of either human or allogenic hepatocytes. Interestingly, while the nodules generated from the transplanted mouse hepatocytes were well vascularized, the human hepatocytes experienced progressive depolarization and exhibited reduced numbers of murine endothelial cells inside the nodules. In conclusion, AdTk/GCV-induced liver damage licenses the liver of immunodeficient mice for allogenic and xenogenic hepatocyte repopulation. This approach represents a simple alternative strategy for chimeric liver generation using immunodeficient mice without additional genetic manipulation of the germ line.</p> </div

Analysis of liver damage in Rag2^-/- IL2γc^-/^- mice treated with AdTk and GCV.

<p>A) Animals were bled weekly and aminotransferases (aspartate aminotransferase, AST and alanine aminotransferase, ALT) were measured in 11 AdTk +GCV (25 mg/ml) treated animals represented by independent lines. Control, GCV and AdTk Rag2^-/- IL2γc^-/^- animals are represented with black circles and lines, presenting AST values between 50 and 120 IU/ml and ALT values between 20 and 70 IU/ml through all the experiment. Each line represents values in one animal throughout the experimental period. X axis represents the weeks post GCV administration while Y axis represents units per ml. B) Histology of control, AdTk infected and AdTk/GCV treated mice livers at week 4 after GCV administration. Original magnification x200.</p

Repopulation of AdTk/GCV treated mice livers with human hepatocytes and analysis of lipid accumulation.

<p>A) Mice infected with AdTk and treated with GCV were transplanted with human hepatocytes, animals sacrificed at diverse time points after transplantation and liver sections were immunostained with hCK18 antibody. Liver samples were obtained from 9 different independent experiments and at least two stained liver sections from 6 mice belonging to three independent studies were analyzed at each time point. Obtained images were examined, quantified and represented as the percentage of hepatic section replaced by human hepatocytes (** <i>p</i>< 0.01, *** <i>p</i>< 0.001, two-tailed Student’s <i>t</i>-test). Presented images show human mouse chimeric livers collected at week 14 after transplantation. B) Frozen section of a chimeric liver containing human hepatocytes stained with fat red for detection of lipids (pink colour). C) A serial section of the same liver sample immunostained for hCK18 identifying human hepatocytes. Original magnification x20.</p