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

The limited efficacy of current treatments against pancreatic cancer has prompted the search of new alternatives such as virotherapy. Activation of the immune response against cancer cells is emerging as one of the main mechanisms of action of oncolytic viruses (OV). Direct oncolysis releases tumor antigens, and viral replication within the tumor microenvironment is a potent danger signal. Arming OV with immunostimulatory transgenes further enhances their therapeutic effect. However, standard virotherapy protocols do not take full advantage of OV as cancer vaccines because repeated viral administrations may polarize immune responses against strong viral antigens, and the rapid onset of neutralizing antibodies limits the efficacy of redosing. An alternative paradigm based on sequential combination of antigenically distinct OV has been recently proposed

A Versatile Vector for In Vivo Monitoring of Type I Interferon Induction and Signaling.

Development of reporter systems for in vivo examination of IFN-β induction or signaling of type I interferon (IFN-I) pathways is of great interest in order to characterize biological responses to different inducers such as viral infections. Several reporter mice have been developed to monitor the induction of both pathways in response to different agonists. However, alternative strategies that do not require transgenic mice breeding have to date not been reported. In addition, detection of these pathways in vivo in animal species other than mice has not yet been addressed. Herein we describe a simple method based on the use of an adeno-associated viral vector (AAV8-3xIRF-ISRE-Luc) containing an IFN-β induction and signaling-sensitive promoter sequence controlling the expression of the reporter gene luciferase. This vector is valid for monitoring IFN-I responses in vivo elicited by diverse stimuli in different organs. Intravenous administration of the vector in C57BL/6 mice and Syrian hamsters was able to detect activation of the IFN pathway in the liver upon systemic treatment with different pro-inflammatory agents and infection with Newcastle disease virus (NDV). In addition, intranasal instillation of AAV8-3xIRF-ISRE-Luc showed a rapid and transient IFN-I response in the respiratory tract of mice infected with the influenza A/PR8/34 virus lacking the NS1 protein. In comparison, this response was delayed and exacerbated in mice infected with influenza A/PR/8 wild type virus. In conclusion, the AAV8-3xIRF-ISRE-Luc vector offers the possibility of detecting IFN-I activation in response to different stimuli and in different animal models with no need for reporter transgenic animals

<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> 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 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> 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> 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 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

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

The limited efficacy of current treatments against pancreatic cancer has prompted the search of new alternatives such as virotherapy. Activation of the immune response against cancer cells is emerging as one of the main mechanisms of action of oncolytic viruses (OV). Direct oncolysis releases tumor antigens, and viral replication within the tumor microenvironment is a potent danger signal. Arming OV with immunostimulatory transgenes further enhances their therapeutic effect. However, standard virotherapy protocols do not take full advantage of OV as cancer vaccines because repeated viral administrations may polarize immune responses against strong viral antigens, and the rapid onset of neutralizing antibodies limits the efficacy of redosing. An alternative paradigm based on sequential combination of antigenically distinct OV has been recently proposed

Recombinant in vitro assembled hepatitis C virus core particles induce strong specific immunity enhanced by formulation with an oil-based adjuvant

In the present work, immunogenicity of recombinant in vitro assembled hepatitis C virus core particles, HCcAg.120-VLPs, either alone or in combination with different adjuvants was evaluated in BALB/c mice. HCcAg.120-VLPs induced high titers of anti-HCcAg.120 antibodies and virus-specific cellular immune responses. Particularly, HCcAg.120-VLPs induced specific delayed type hypersensitivity, and generated a predominant T helper 1 cytokine pro file in immunized mice. In addition, HCcAg.120-VLPs prime splenocytes proliferate in vitro against different HCcAg.120-specific peptides, depending on either the immunization route or the adjuvant used. Remarkably, immunization with HCcAg.120-VLPs/Montanide ISA888 formulation resulted in a significant control of vaccinia virus titer in mice after challenge with a recombinant vaccinia virus expressing HCV core protein, vvCore. Animals immunized with this formulation had a marked increase in the number of IFN-&#947; producing spleen cells, after stimulation with P815 cells infected with vvCore. These results suggest the use of recombinant HCV core particles as components of therapeutic or preventive vaccine candidates against HCV