WO2014199166A1 - Method of increasing viral growth rate and/or titre in cells - Google Patents

Abstract

Methods of increasing viral growth rate and/or titre in cell culture. A method of increasing viral growth rate and/or titre in cells for virus production, through the use of inhibitors of the interferon induction and/or signalling cascade.

Description

METHOD OF INCREASING VIRAL GROWTH RATE AND/OR TITRE IN CELLS Field of the Invention

The present invention relates to a method of increasing viral growth rate and/or titre in cells and the use of inhibitors of the interferon induction and/or signalling cascades in cells to increase viral growth rate and/or titre. The present invention also relates to a method of screening to identify cell line/interferon induction and/or signalling cascade inhibitor combinations providing increased viral growth rate and/or titre in cells. Background of the Invention

Interferons (IFNs) are a group of cytokines whose expression is induced in host cells in response to viral infections. This is known as the IFN induction cascade (Figure 1 A, left panel). This induction leads to the initiation of signalling cascades (the IFN signalling cascade; Figure 1A, right panel) in neighbouring non-infected cells, resulting in an antiviral response capable of controlling most viral infections.

There are three main classes of IFNs (type I, II and III), characterised based on the receptors through which they signal. Type I IFNs (including IFN-a and IFN-β) bring about antiviral changes in the cell by binding to the IFN-a receptor (IFNAR).

The IFN Induction Cascade

In IFN-α/β secreting cells, pattern-recognition receptors (PRRs) such as MDA-5, RIG-I and TLR3 detect molecules associated with viral infection such as double-stranded RNA (dsRNA) and viral proteins. This recognition leads to the initiation of a signalling cascade, which results in the activation of transcription factors such as NF-κΒ and IRF3/IRF7. These transcription factors lead to the activation of the IFN-α/β promoters resulting in IFN-α/β transcription and expression.

The IFN Signalling Cascade

IFN-α/β is secreted from infected host cells and this results in the induction of an antiviral state in neighbouring non-infected cells. This is achieved through binding of secreted IFN-α/β to IFNAR and resulting activation of the Janus-kinase Signal Transducer and Activator of Transcription (JAK-STAT) pathway. JAK proteins associate with IFN receptors, and following its activation, JAK phosphorylates STAT transcription factors. Phosphorylated STAT-1 and STAT-2 recruit IRF-9 to form a transcription complex known as IFN-stimulated gene factor 3 (ISGF-3). ISGF-3 then translocates to the nucleus and binds to the IFN-stimulated response element (ISRE) in the promoter region of IFN-stimulated genes (ISGs). This signalling pathway leads to the upregulation of hundreds of ISGs, which are involved in the antiviral response [1 ].

The I FN system constitutes an extremely powerful antiviral response that is capable of controlling most if not all virus infections in the absence of adaptive immunity. However, it rarely works to full capacity because almost all viruses have evolved I FN antagonists that use a wide variety of mechanisms to circumvent the I FN response by either directly or indirectly targeting the IFN-induction or IFN-signalling cascades, or both [1 ].

Activation of the I FN response also results in a variety of complementary antiviral effects on the cell, for example (i) activation of the I FN induction cascade through IRF3 can lead to the induction of certain ISGs, such as ISG56, which have an antiviral activity in the absence of IFN and (ii) IRF3 possess a distinct activity that induces apoptosis in virus infected cells [1 , 2]. Counteracting the IFN induction/signalling cascades by using viral IFN antagonists or inhibitors may have beneficial effects on viral growth by counteracting such complementary antiviral mechanisms. Growing viruses in cells has applications in vaccine manufacture, gene therapy, oncolytic virotherapy, virus diagnostics and isolation, amongst other things.

Vaccines have proved extremely successful in controlling many viral infections. However, vaccines still have to be developed against many viruses, e.g. among the negative-strand RNA viruses, respiratory syncytial virus (RSV), the parainfluenza viruses, Ebola virus, and members of the Bunyaviridae family, including Hantavirus. It would also be desirable to develop improved vaccines for a number of negative-strand RNA viruses, including the measles, mumps and influenza viruses. One of the most successful approaches in producing virus vaccines has been the generation of attenuated viruses, which are administered to mimic natural infection and induce protective immunity without causing disease. In the past, the generation of attenuated viruses has been empirical. However, with the advent of recombinant technology, the possibility of designing attenuated viruses that have been engineered to possess specific phenotypes has become a reality. One general approach to producing attenuated viruses would be to engineer viruses so as to disable their capacity to circumvent the IFN response [3-6]. This is feasible because viral anti-IFN proteins are usually dispensable for virus replication in cell culture and viruses in which the genes encoding these proteins have been knocked out are attenuated in vivo. However, it can be difficult to grow such viruses to high-titre in cells that produce and respond to IFN [7]. In this regard, the vaccine industry is largely restricted to a very limited selection of cell lines (e.g. vera cells) that have lost their ability to produce IFN [8, 9]. Some vaccines, for example the influenza vaccine, are manufactured using shell eggs. The viruses are injected into the eggs which are then incubated for a number of days, allowing the virus to replicate. The virus is then harvested from the allantoic fluid and the virus purified. An improvement in virus yield would be beneficial in the production of egg-derived vaccines, as millions of eggs are currently used in such vaccine manufacture.

Gene therapy utilizes DNA as a pharmaceutical agent to treat disease. The term encompasses a wide variety of different therapeutic applications. For example, the DNA may correct a genetic disorder by replacing a missing or defective gene or the DNA may encode a protein that modulates cell behaviour, e.g. blocking cancer cell proliferation by expression of a protein that interferes with cell cycle regulation. Irrespective of the application, the DNA must be introduced to the cell and the properties of viruses make them excellent gene delivery vectors. Choice of viral vector is based on the specific attributes of the viral vector for the specific application. Many viral vectors are being developed, although retro-, adeno-, adeno-associated and herpes viral vectors have been the most extensively used in clinical trials to date [10]. Viral gene delivery vectors are often generated from disabled viruses that have been engineered such that the viral genome only contains viral cis-acting elements that are required for viral genome function and the therapeutic gene of interest. Trans-acting elements, such as viral structural proteins, are expressed from a different genetic element such as a helper virus or in a packaging cell line. High titre stocks of some of these viral vectors can be generated (e.g. adenoviral vectors - 10¹⁰ plaque forming units/ml) but this is difficult to achieve for other classes of viral vectors (e.g. adeno- associated viral vectors) [10]. Any improvement in overall viral titres or the time it takes to generate equivalent titres would be advantageous in the manufacture of viral vectors for therapeutic gene delivery purposes. Replication-competent oncolytic viruses that selectively infect and damage cancerous tissues without causing harm to normal tissues are being developed for use in oncolytic virotherapy to destroy cancers [1 1 ]. A wide range of viruses are being developed for this application, including adenovirus, coxackie virus, herpes simplex virus, measles virus, Newcastle disease virus, parvovirus, poliovirus, reovirus, Seneca valley virus, retrovirus, vaccinia virus and vesicular stomatitis virus [1 1 ]. The choice of virus is directed by the properties of the virus, for example, some viruses have a natural predilection for certain types of cancer cells whereas others can be adapted or engineered to make them target cancer specific cells. One way of engineering viruses to mediate tumour specificity is to knockout their anti-l FN proteins [1 1 , 12]. The rational for this approach is to exploit the fact that tumourigensis can result in impairment of innate immune responses. Therefore viruses that can no longer counteract the IFN response are often able to propagate in tumour cells but not normal cells and are thus proposed to mediate tumour-specific killing with minimal off-target toxicity. As previously stated, it can be difficult to grow such attenuated viruses to high-titre in cells that produce and respond to IFN. Generating high-titre stocks of viruses for oncolytic virotherapy is crucial because high doses are required to be administered for clinical efficacy. Virus manufacture is recognised as a limiting factor for many oncolytic viruses and is required at orders-of-magnitude higher yields than is currently possible [1 1 ].

Viral growth is also essential for virus isolation purposes in diagnostic laboratories. Human cell lines (e.g. MRC5 and Hep2) are often used for isolating known and/or unknown viruses; however the range of viruses that will efficiently replicate in these cell-lines is limited due to the presence of the IFN system, amongst other things.

The present inventors have previously engineered a variety of cell lines, including MRC5, which are particularly relevant to vaccine manufacture and/or viral diagnostics, to express the parainfluenza type 5 virus (PIV5) V protein, which is a powerful IFN antagonist [7]. The growth of a variety of viruses, including slow-growing wildtype viruses and vaccine candidate viruses, was analysed and the inventors demonstrated the formation of larger plaques and increased viral titres (10- to 4000-fold) in these IFN non-responsive cells [7]. However, genetically engineering cell lines is time consuming and their use creates regulatory problems for vaccine manufacturers. Thus, further methods of increasing viral growth rates and overall viral titre in cells would be desirable. It is an object of the present invention to obviate and/or mitigate at least one of the abovementioned disadvantages.

Summary of the Invention

The present invention is based in part on studies by the inventors into inhibitors of the I FN induction and/or signalling cascade and the discovery that supplementing tissue culture media with inhibitors of the I FN induction or signalling cascade can result in increased viral growth rate and/or titre.

According to a first aspect of the invention, there is provided a method of increasing viral growth rate and/or titre in cells, the method comprising adding an inhibitor of the IFN induction and/or signalling cascade to the cells.

There is also provided the use of an inhibitor of the IFN induction and/or signalling cascade to increase viral growth rate and/or titre in cells.

The cells of the present invention may include, for example, cells grown in cell culture, in egg culture, or any other suitable form of maintaining or propogating cells. By "egg culture" we refer to the culture of shell eggs, for example chicken or duck eggs. The cells of the present invention include any cells suitable for growing a virus therein, although excludes human embryonic stem cells obtained through destruction of an human embryo.

As used herein, the phrase "cell culture" encompasses the maintenance and/or propogation of cells and could be in vitro or ex vivo. As used herein, the phrase "ex vivo" refers to cells taken from an organism and subsequently maintained or propogated outside of that organism. In particular, this may refer to cells taken from an adult human for gene therapy purposes, but excluding embryonic stem cells.

If the cells are grown in cell culture, the method of the present invention may comprise adding an inhibitor of the IFN induction and/or signalling cascade to the tissue culture medium.

Alternatively, if the cells are grown in egg culture, the method of the present invention may comprise adding an inhibitor of the IFN induction and/or signalling cascade to the egg in which a virus is cultured. The skilled person would be well aware of the methods for introducing such inhibitors into eggs, for example injection of an inhibitor suspension.

In embodiments of the invention, cells grown in cell culture and inhibitors added to the tissue culture medium of such cultured cells are excluded.

The inventors have observed that addition of I FN induction or signalling cascade inhibitors to tissue culture media results in increased viral growth rate and/or titre. The use of the inhibitors of the present invention can result in at least a 10, 20, 50, 100, 200 or 400-fold increase in viral growth rate. It may be possible to obtain at least a 1000- 50,000-fold increase, such as at least a 2000, 4000, 10000 or 40000-fold increase in viral growth rate compared to viral growth in untreated cells. The use of the inhibitors of the present invention can result in at least a 10, 20, 50, 100, 200 or 400-fold increase in viral titre. It may be possible to obtain at least a 1000-50,000-fold increase, such as at least a 2000, 4000, 10000 or 40000-fold or more increase in viral titre compared to viral titres in untreated cells.

The inhibitors for use in the present invention are intended to be used to target the I FN induction and/or signalling cascades. The activation of multiple signalling pathways by the engagement of I FN with its receptors is critical for the generation of IFN-mediated biological function. As used herein, the phrase "IFN induction cascade" encompasses any virus-induced pathway that leads to the activation of IFN-α/β transcription and expression. The molecules in the IFN induction cascade that may be targeted by the inhibitors may include, but are not limited to TLR3, MDA5, RIG-I, Cardif, ΤΒΚ1/ΙΚΚε, ΙΚΚα/β, IRF3, NFKB or ATF-2/C-JUN. As used herein, the phrase "IFN signalling cascade" encompasses any I FN-activated signalling pathway whose activation leads to antiviral changes in the cell. The signalling pathway targeted may include, but is not limited to, the JAK-STAT pathway. The inhibitors for use in the present invention may target one or more components of the IFN induction and/or signalling cascades shown in Table 1 . The inhibitors used in the present invention may be used in isolation or in combination. Table 1 : Possible targets for inhibition in the IFN induction and signalling cascades.

In one embodiment of the invention, the inhibitors for use in the present invention target the JAK/STAT signalling pathway. The inhibitors may target any component of the JAK/STAT pathway. The targeted components of the JAK/STAT pathway may include, but are not limited to, Tyk2, Jak1 , Jak2, STAT-1 , STAT-2 and IRF-9. In one embodiment, the inhibitor targets Jak1 . In a further embodiment of the invention, the inhibitors for use in the present invention target the ΤΒΚ-1/ΙΚΚε/ΙΚΚ2 induction pathway.

To achieve the desired inhibition of the IFN signalling or induction cascade and increase in viral growth rate and/or titre, whilst limiting cellular toxicity it is preferred that the final concentration of inhibitor in the tissue culture medium is in the range of 0.1 -20 μΜ. In one embodiment, the final concentration of inhibitor may be in the range of 0.25- 15 μΜ.

The skilled person will appreciate that the concentration of inhibitor used in tissue culture medium would also be suitable for egg culture, where the "tissue culture medium" could be considered as the fluid inside the egg, for example the allantoic fluid. The inhibitors for use in the present invention can be any molecule which decreases the activity of the IFN induction and/or signalling cascades. Non-limiting examples of inhibitors which could be used in accordance with the present invention include small molecule inhibitors, siRNAs, imiRNAs, lipocalins, plastic antibodies, antibodies or antibody fragments.

If the inhibitor was designed to target Jak1 , one could envisage an antibody, plastic antibody or antibody fragment raised against Jak1 such that in use, the antibody, plastic antibody or antibody fragment binds to Jak1 and prevents it phosphorylating STAT transcription factors, for example.

Alternatively, if the inhibitor were an miRNA or siRNA, one could envisage the miRNA or siRNA having a sequence complementary to a portion of the Jak1 imRNA sequence, such that the miRNA or siRNA inhibitor would bind Jak1 imRNA, thereby reducing translation and reducing the abundance of Jak1 in the cell.

Preferably the inhibitor is a small molecule. As used herein the phrase "small molecule" refers to a low molecular weight (less than approximately 800 Da) organic or inorganic compound.

The small molecule inhibitor for use in the present invention can function through competitive, uncompetitive, mixed or non-competitive inhibition. In one embodiment, the inhibitor is a competitive inhibitor. The small molecule inhibitor for use in the present invention may be N-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4- yl)benzamide (Cyt387), an ATP-competitive inhibitor of the Jak1 mediated step of the IFN signalling cascade. To achieve the desired inhibition of the IFN signalling cascade and increase in viral growth rate and/or titre, whilst limiting cellular toxicity, it is preferred that the final concentration of Cyt387 in the tissue culture medium is in the range of 2.5 -10 μΜ.

In a further embodiment of the invention, the small molecule inhibitor may be AZD1480, an ATP-competitive Jak2 inhibitor which also displays selectivity against Jak3, Tyk2 and Jak1 . To achieve the desired inhibition of the IFN signalling cascade and increase in viral growth rate and/or titre, whilst limiting cellular toxicity, it is preferred that the final concentration of AZD1480 in the tissue culture medium is in the range of 0.5 - 4 μΜ, preferably 2 - 4 μΜ.

In a further embodiment of the invention, the small molecule inhibitor may be Ruxolitinib, an inhibitor of the Jak1 mediated step of the I FN signalling cascade. To achieve the desired inhibition of the IFN signalling cascade and increase in viral growth rate and/or titre, whilst limiting cellular toxicity, it is preferred that the final concentration of Ruxolitinib in the tissue culture medium is in the range of 0.25 - 4 μΜ. In a further embodiment of the invention, the small molecule inhibitor may be Tofacitinib, an inhibitor of Jak3, which is also displays selectivity against Jak1 and Jak2. To achieve the desired inhibition of the IFN signalling cascade and increase in viral growth rate and/or titre, whilst limiting cellular toxicity, it is preferred that the final concentration of Tofacitinib in the tissue culture medium is in the range of 0.25 - 4 μΜ, preferably 1 - 4 μΜ.

In a further embodiment of the invention, the small molecule inhibitor may be BX795 (C₂3H₂₆IN₇0₂S) an inhibitor of the TBK-1/ΙΚΚε mediated step of the IRF-3 arm of the IFN induction cascade. To achieve the desired inhibition of the IFN induction cascade and increase viral growth rate and/or titre, whilst limiting cellular toxicity, it is preferred that the final concentration of BX795 in the tissue culture medium is in the range of 1 - 10 μΜ.

In a further embodiment of the invention, the small molecule inhibitor may be TPCA-1 , an inhibitor of the IKK2 mediated step of the IFN induction cascade. To achieve the desired inhibition of the IFN induction cascade and increase viral growth rate and/or titre, whilst limiting cellular toxicity, it is preferred that the final concentration of TPCA-1 in the tissue culture medium is in the range of 0.5 - 4 μΜ, preferably 2 - 4 μΜ.

As mentioned above, the concentrations preferred when growing cells in cell culture may equally apply when growing cells in egg culture, where the "tissue culture medium" could be considered as the fluid inside the egg, for example the allantoic fluid.

The term "antibody fragment" as used herein refers to a fragment of an antibody that immunospecifically binds to a molecule in the IFN induction or signalling cascade. Antibody fragments may be generated by any technique known to one of skill in the art and by proteolytic or non-proteolytic cleavage. For example, Fab and F(ab')2 fragments may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments). F(ab')2 fragments contain the complete light chain, and the variable region, the CH1 region and the hinge region of the heavy chain. Antibody fragments can be also produced by recombinant DNA technologies. Antibody fragments may be one or more complementarity determining regions (CDRs) of antibodies. The term "plastic antibody" as used herein refers to synthetic polymer nanoparticles with antibody-like functions that have the ability to sequester molecules in the I FN induction and/or signalling cascades.

The term "Npocalin" as used herein refers to a protein which can bind primarily hydrophobic molecules, in particular molecules in the I FN induction and/or signalling cascades. Lipocalins have been linked to many biological processes including the innate immune response.

The viruses for use in the present invention may be wildtype, genetically modified or recombinant viruses. The modified viruses may be substantially complementary to wildtype viruses. However, in an embodiment of the invention, a modification will be introduced that results in an attenuated virus for use in vaccine production or oncolytic virotherapy. In one embodiment, the virus will be genetically modified so as to disable its capacity to overcome the I FN response. Techniques used for genetic modification will be known to a person skilled in the art, but for reference see Sambrook & Russell, Molecular Cloning: A Laboratory Manual (3^rd edition).

The present invention is applicable to all types of viruses: DNA, RNA and DNA-RNA viruses, single-stranded (positive and negative sense) and double-stranded viruses, and those with a circularised or linear genome. In one embodiment of the present invention, the virus is a negative strand RNA virus. Non-limiting examples of the viruses used in the present invention are parainfluenza virus 5, respiratory syncytial virus, Influenza virus, mumps virus, measles virus and members of the Bunyaviridae family. According to a further aspect of the present invention, there is provided a method of screening to identify a cell line/IFN induction and/or signalling cascade inhibitor combination providing increased viral growth rate and/or titre compared to the growth rate and/or titre found in uninhibited cell lines.

The viruses analysed in the method of screening of the present invention may be wildtype, genetically modified or recombinant viruses. The modified viruses may be substantially complementary to wildtype viruses. However, in an embodiment of the invention a modification will be introduced that results in an attenuated virus for use in vaccine production or oncolytic virotherapy. In one embodiment, the virus will be genetically modified so as to disable its capacity to overcome the IFN response. Techniques used for genetic modification will be known to a person skilled in the art, but for reference see Sambrook & Russell, Molecular Cloning: A Laboratory Manual (3^rd edition).

The method of screening according to the present invention could be carried out in a plurality of conical flasks, test tubes or cell culture plates or alternatively in multiwell plates. Preferably the method of screening comprises performing multiwell-plate based analysis of viral growth rate and/or titre in a range of cell line/inhibitor combinations. The use of multiwell plates would allow high-throughput analysis of the effect of cell line/inhibitor combinations on viral growth rate and/or titre in cells.

Non-limiting examples of techniques which can be used to assess viral growth rate and/or titre include analysing the number of plaque forming units or size of plaques; detecting fluorescence from a suitable reporter construct using a fluorescent plate reader or FACS; utilising an immunoassay through use of a suitable antibody-linked reporter. In one embodiment, viral growth rate and/or titre would be assessed by detecting fluorescence from a suitable reporter construct using a fluorescent plate reader or FACS, to allow high-throughput analysis of the effect of cell line/inhibitor combinations on viral growth rate and/or titre in cell culture. In a further embodiment, viral growth rate and/or titre would be assessed using an immunoassay, for example an enzyme-linked immunosorbent assay (ELISA), to allow high-throughput analysis of the effect of cell line/inhibitor combinations on viral growth rate and/or titre in cells. As used herein, the term "cell line" encompasses any type of cell in which viruses may be grown, but excludes embryonic stem cells. The cell lines screened may include, but are not limited to vera, MRC5, PERC6, MDCK and Hep2. The inhibitors screened in the present invention are intended to target the I FN induction and/or signalling cascades. The inhibitors screened in the present invention may target one or more components of the IFN induction and/or signalling cascades shown in Table 1 . The molecules in the IFN induction cascade that may be targeted by the inhibitors may include, but are not limited to TLR3, MDA5, RIG-I, Cardif, ΤΒΚ1/ΙΚΚε, ΙΚΚα/β, IRF3, NFKB or ATF-2/C-JUN. In one embodiment of the invention, the inhibitors screened in the present invention target the ΤΒΚ-1/ΙΚΚε/ΙΚΚ2 induction pathway. The signalling pathway targeted by the inhibitors may include, but is not limited to, the JAK-STAT pathway. In one embodiment, the inhibitors screened in the present invention target the JAK/STAT signalling pathway. The inhibitors may target any component of the JAK/STAT pathway. The targeted components of the JAK/STAT pathway may include, but are not limited to, Tyk2, Jak1 , Jak2, STAT-1 , STAT-2 and IRF-9. In one embodiment, the inhibitor targets Jak1 .

The inhibitors screened in the present invention may be used in isolation or in combination.

The inhibitors screened in the present invention can be any molecule which decreases the activity of the IFN induction and/or signalling cascades. Non-limiting examples of inhibitors which could be screened in accordance with the present invention include small molecule inhibitors, siRNAs, miRNAs, lipocalins, plastic antibodies, antibodies or antibody fragments. Preferably the inhibitors screened are small molecules. As used herein the phrase "small molecule" refers to a low molecular weight (less than approximately 800 Da) organic or inorganic compound. The small molecule inhibitors screened in the present invention can function through competitive, uncompetitive, mixed or non-competitive inhibition. Preferably, the inhibitors screened are competitive inhibitors. Detailed Description

The present invention will now be described with reference to the following non-limiting examples and figures, which show: Figure 1 : Schematic of the cellular interferon (IFN) system and GFP reporter construct in A549 pr(IFN-p).GFP and A549 pr(ISRE).GFP cell lines.

(A) Left panel: IFN's are a group of cytokines expressed in response to viral infection. Pattern-recognition receptors (PRRs) such as MDA-5, RIG-I and TLR3 recognise molecules associated with viral infection such as viral double-stranded RNA. This recognition leads to a signalling cascade that results in the activation of transcription factors such as NF-κΒ and IRF3. These transcription factors activate the IFN-β promoter inducing expression of IFN-β. Right panel: Secreted IFN binds to the IFN receptor (IFNAR) on neighbouring non-infected cells and activates the JAK-STAT pathway. The STAT proteins are latent transcription factors that become phosphorylated by JAK-1 and TYK-2. Phosphorylated STAT-1 and STAT-2 recruit IRF- 9 to form a complex known as IFN-stimulated gene factor 3 (ISGF-3). ISGF-3 translocates to the nucleus and binds to the IFN-stimulated response element (ISRE) in the promoter region of IFN-stimulated genes (ISGs). This pathway leads to the upregulation of several hundred ISGs, many of which have direct antiviral activities. (B) Schematic representation of two reporter cell lines utilised to facilitate detection of IFN induction (left panel) or signalling (right panel). Left panel: In the A549/pr(IFN- P).GFP cell line the gene encoding green fluorescent protein (eGFP) is placed under the control of the IFN-β promoter. Right panel: In the A549/pr(ISRE).GFP cell line the eGFP gene is placed under the control of an ISRE within an ISG promoter region. Modified from [13].

Figure 2: The effect of Cyt387, a Jak1 inhibitor, on IFN signalling using a A549/pr(ISRE)GFP reporter cell line. The A549/pr(ISRE)GFP cell line contains an eGFP reporter gene under control of IFN-stimulated response element (ISRE) within the promoter region of the Mx1 IFN-stimulated gene. Purified IFN is used to induce activation of the IFN signalling cascade two hours post Cyt387 treatment.

Figure 3: The effect of Cyt387, an inhibitor of IFN signalling, on viral plaque formation. (A) A549 cells infected with PIV5-VAC virus, in which the V protein has been truncated to render the virus sensitive to the I FN response. (B) Effect of the V protein IFN antagonist on plaque formation. A549 cells constitutively expressing the V protein infected with PIV5-VAC. Figure 4: The effect of Cyt387, an inhibitor of IFN signaling, on viral plaque formation. A549 and MRC5 na^'ive cells were infected with recombinant Bunyamwera virus (BUNANSs). BUNANSs is a recombinant virus in which expression of the NSs protein has been inactivated by point mutations to render the virus sensitive to the IFN response. Addition of 5 μΜ Cyt387 results in an increase in plaque size for BUNANSs virus in both A549 cells and MRC5 na^'ive cells, which are approved for vaccine manufacture.

Figure 5: The effect of Cyt387, an inhibitor of IFN signaling, on virus growth kinetics and titre. A549 na^'ive cells or recombinant A549 cells expressing the PIV5 IFN antagonist V were infected with BUNANSs at an MOI of 0.001 and grown either in the presence or absence of 5 μΜ Cyt387 for four days. Virus titre was measured at 24, 48, 72 and 96 hours post-infection by plaque assay serial dilution on Vero cells and presented as (A) plaque forming units per ml (pfu/ml) at each post-infection time point and (B) as the actual plaques formed on vero cells at the 48 hour post-infection time point.

Figure 6: The effect of the ΤΒΚ1/ΙΚΚε inhibitor BX795, on the IFN induction cascade, using a A549/pr(IFN-p).GFP reporter cell line. The A549/pr(IFN-p).GFP reporter cell line contains an eGFP reporter gene under the control of the IFN-β promoter. The recombinant PIV5 virus PIV5-VAC induces efficient GFP expression in the absence of BX795. Increasing concentrations of BX795 result in a dose-dependent reduction in GFP expression.

Figure 7: The effect of BX795, an inhibitor of IFN induction, on viral plaque formation. A549 cells were infected with recombinant Bunyamwera virus BUNANSs virus in which expression of the NSs protein has been inactivated by point mutations to render the virus sensitive to the IFN response. Addition of 2 μΜ BX795 results in a dramatic increase in plaque size in A549 cells, which is equivalent to the effect of 5 μΜ Cyt387, an inhibitor of IFN signalling. Figure 8: The effect of various inhibitors of IFN induction and signaling using A549/pr(IFNp).GFP and A549/pr(ISRE).GFP reporter cell lines. (A) The

A549/pr(IFN-p).GFP cell-line contains eGFP reporter gene under control of the IFN-β promoter. The recombinant PIV5 virus PIV5-VAC induces efficient GFP expression in the absence of TPCA-1 . Increasing concentrations of an IKK-2 inhibitor (TPCA-1 ) was tested. (B) The A549/pr(ISRE).GFP cell-line contains eGFP reporter gene under control of an ISRE within an ISG promoter region (Mx1 ). Purified IFN is used to induce activation of the IFN signalling cascade. Increasing concentrations of four JAK inhibitors (Cyt387, AZD1480, Ruxolitinib, Tofacitinib) were tested.

Figure 9: The effect of a panel of IFN inhibitors on viral plaque formation. The inhibitor panel consists of small molecules that target the IKK2 (TPCA-1 ) and TBK1 (BX795) components of the IFN induction pathway and Jak1 (Cyt387, AZD1480, Ruxolitinib, Tofacitinib) a component of the IFN signaling pathway. The effect of the inhibitors on Bunyamwera virus BUNANSs plaque formation was assessed in A549 cells and compared with A549 cells constitutively expressing viral IFN antagonists that block IFN production (BVDV-Npro) or signaling (PIV5-V). The effect of various inhibitor concentrations on viral plaque size was observed. Plaques were visualized 2 days post-infection.

Figure 10: The effect of a panel of IFN inhibitors on viral growth kinetics and titre.

The inhibitor panel consists of small molecules that target the IKK2 (TPCA-1 ) and TBK1 (BX795) components of the IFN induction pathway and Jak1 (Cyt387, AZD1480, Ruxolitinib, Tofacitinib) a component of the IFN signaling pathway. The effect of the inhibitors on Bunyamwera virus BUNANSs growth was assessed in A549 cells and compared with A549 cells constitutively expressing viral IFN antagonists that block IFN production (BVDV-Npro) or signaling (PIV5-V). Virus titre was measured at 24, 48, 48 and 72 hours post-infection by plaque assay serial dilution on Vero cells in the presence of 2 μΜ inhibitor or the equivalent volume of DMSO and presented as plaque forming units per ml (pfu/ml) at each post-infection time point.

Figure 11 : The effect of Ruxolitinib (RUX) on viral plaque formation in cell lines derived from different mammalian species. (A) The effect of RUX on Bunyamwera virus BUNANSs plaque formation in human cell lines A549 and MRC5 and their derivatives constitutively expressing the viral IFN antagonist PIV5-V. (B) The effect of RUX on Bunyamwera virus BUNANSs and BUN WT (wildtype) plaque formation in cell lines derived from mouse (BalB/C), rabbit (RK13), cow (MDBK), dog (MDCK) and pig (PK1 BRS2) was investigated. In both (A) and (B), 4 μΜ RUX or an equivalent volume of DMSO was added to the cells and the plaques were fixed on the day indicated and visualized by immunostaining with an anti-Bunyamwera N protein antibody.

Figure 12: The effect of Ruxolitinib (RUX) on plaque formation of a selection of viruses. (A) Respiratory Syncytial Virus (RSV) wildtype (WT) and derivatives with deleted IFN antagonists NS1 (ANSI ) and NS2 (ANS2). RSV plaques were grown in the MRC5 cell-line or derivative constitutively expressing PIV5-V, fixed on the days indicated and visualized by immunostaining with an anti-RSV F protein antibody. (B) Measles Edmonston (MeV Edm) and Mumps Enders (MuV End) live-attenuated vaccine strains. MeV and MuV plaques were grown in MRC5 cell-line or derivative constitutively expressing PIV5-V fixed on the day indicated and visualized by immunostaining with an anti-MeV NP or anti-MuV NP antibodies respectively. RUX was added at 4 μΜ and compared with the equivalent volume of DMSO.

Figure 13: The effect of IFN inhibitors in the DF1 chicken fibroblast cell-line. (A)

DF1 chicken fibroblast cells transfected with the reporter construct ChlFN-p-luc in which lucif erase (Luc) is placed under control of the IFN-β promoter. Fourty-eight hours post transfection, cells were treated with IFN inhibitors for two hours and then the IFN induction response induced with polyl:C overnight and Luc activity determined. (B) DF1 chicken fibroblast cells transfected with the reporter construct ChiMx(ISRE)-luc in which Luc is placed under thje control of the Mx ISRE promoter. Fourty-eight hours post transfection, cells were treated with IFN inhibitors for two hours and then the IFN signalling response induced with purified IFN (1000 U/ml) for 6 hours, and Luc activity determined.

Abbreviations:

CDR: Complementarity determining regions

Cyt387: (N-(cyanomethyl)-4-(2-(4-morpholinophenylamino)pyrimidin-4-yl)benzamide)

DMEM: Dulbecco's modified eagles medium

FBS: Fetal bovine serum

GFP: Green fluorescent protein

IFN: Interferon IFNAR: Interferon a receptor

ISG: Interferon-stimulated gene

ISGF: Interferon-stimulated gene factor

ISRE: Interferon-stimulated response element

JAK-STAT: Janus-kinase Signal Transducer and Activator of Transcription

MEM: Minimum essential media

MeV: Measles virus

MOI: Multiplicity of infection

MuV: Mumps virus

NCS: Newborn calf serum

PBS: Phosphate buffered saline

PFU: Plaque forming unit

PIV5: parainfluenza type 5 virus

PRR: Pattern-recognition receptor

RSV: Respiratory Syncytial virus

RUX: Ruxolitinib

WT: Wildtype

Materials and Methods

Cells, Viruses and Inhibitors

A549 (human) cells and their derivatives, Vera (monkey) , MRC5 (human), mouse (BalB/C), rabbit (RK13), cow (MDBK), dog (MDCK), pig (PK1 BRS2) and chicken (DF1 ) cells were maintained in Dulbecco's modified eagles medium (DMEM) supplemented with 10% v/v fetal bovine serum (FBS) at 37°C, 5% C0₂. The A549 cell derivatives were A549/PIV5-V cells expressing the V protein of PIV5 [7], A549/Pr(IFNp).GFP reporter cells in which green fluorescent protein (GFP) is placed under the control of the IFN-β promoter [14] and A549/Pr(ISRE).GFP reporter cells in which GFP is placed under the control of the ISRE within the promoter region of the Mx1 ISG (Randall et al., unpublished). The viruses utilised were wildtype Bunyamwera virus (BUNWT) and recombinant PIV5 and Bunyamwera viruses in which expression of their respective I FN antagonists (V and NSs) has been inactivated by point mutations: PIV-VAC [15] and BUNANSs [16], Respiratory Syncytial Virus (RSV) and ANSI and ANS2 derivatives, measles (MeV) Edmonson and Mumps (MuV) Enders vaccine strains (NIBSC). All inhibitors [the Jak1 inhibitors Cyt387 (N-(cyanomethyl)-4- (2(4morpholinophenylamino)pyrimidin-4-yl)benzamide) (Selleck Chemicals, UK), AZD1480, Ruxolitinib, Tofacitinib, the ΤΒΚ1/ΙΚΚε inhibitor BX795 ((C₂₃H₂₆IN₇0₂S) ) (Calbiochem, UK), and IKK2 inhibitor TPCA-1 ] were stored as a 10 mM stock in DMSO at -20°C and used at the indicated concentrations. Inhibition of IFN signalling by Cyt387, AZD1480, Tofacitinib and Ruxolitinib

Inhibition of IFN signalling by Cyt387, AZD1480, Tofacitinib and Ruxolitinib was determined using the A549/Pr(ISRE).GFP reporter cells. The A549/Pr(ISRE).GFP reporter cells were plated in 96 well plates at 3x10⁴ cells/well such that a confluent cell monolayer was obtained. The monolayer was treated with varying concentrations of drug. Two hours post drug treatment the IFN signalling cascade was induced by treatment with 10⁴ units/ml of purified a-IFN (Roferon, NHS) and the cells incubated at 37°C. At 48 hours post IFN treatment, GFP expression was measured as fluorescence using a Tecan Infinite plate reader (Tecan) at excitation/emission 488 nm/518 nm. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma) and crystal violet assays were conducted in parallel to measure the cellular cytotoxicity of the drug. In addition, the effect of Ruxolitinib on IFN signalling in DF1 chicken fibroblast cells was determined by using DF1 cells transfected with the reporter construct ChlMx(ISRE)-luc in which Luc is placed under the control of the Mx ISRE promoter. Fourty-eight hours post transduction, cells were treated with IFN inhibitors for two hours and then the IFN signalling repsonse induced with purified IFN (1000 U/ml) for 6 hours and Luc activity determined.

Inhibition of IFN induction by BX795 and TPCA-1

Inhibition of IFN induction by BX795 and TPCA-1 was determined using the A549/Pr(IFN-p).GFP reporter cells. The A549/Pr(IFN-p).GFP reporter cells were plated in 96 well plates at 3x10⁴ cells/well such that a confluent cell monolayer was obtained. The monolayer was treated with varying concentrations of drug. Two hours post drug treatment the IFN induction cascade was induced by treatment with 1 μΙ of PIV5 VAC virus stock rich in IFN inducing defective particles and the cells incubated at 37°C. At 16 hours post IFN treatment, GFP expression was measured as fluorescence using a Tecan Infinite plate reader (Tecan) at excitation/emission 488 nm/518 nm. MTT (3-(4,5- Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma) and crystal violet assays were conducted in parallel to measure the cellular cytotoxicity of the drug. In addition, the effect of BX795 and TPCA-1 on IFN induction in DF1 chicken fibroblast cells was determined by using DF1 cells transfected with the reporter construct ChlFN-p-luc in which lucif erase (Luc) is placed under the control of the IFN-β promoter. Fourty-eight hours post transfection, cells were treated with IFN inhibitors for two hours and then the I FN induction response induced with polyl:C overnight, and Luc acitivity determined. Plaque Assay

Plaque assays were used to titre virus stocks and to determine the effect of Cyt387, BX795, TPCA-1 , AZD1480, Ruxolitinib and Tofacitinib on viral plaque formation. Plaque assay were performed in Vera (monkey), A549 (human), A549/PIV5-V (human) MRC5 (human), mouse (BalB/C), rabbit (RK13), cow (MDBK), dog (MDCK) and pig (PK1 BRS2) cells, which were seeded into 6 or 12 well plates such that they formed confluent monolayers. Virus was diluted to a predetermined multiplicity of infection (MOI) to allow visualization of plaque formation in phosphate buffered saline (PBS), supplemented with 2% v/v newborn calf serum (NCS) and 200 μΙ of this inoculum used to infect the cell monolayer. Virus infection was performed for one hour at 37°C with intermittent shaking. Following infection the virus inoculum was removed and replaced with Minimum Essential Media (MEM) supplemented with 2 mM L-glutamine, 25% v/v NCS, 1 % v/v sodium bicarbonate and 0.6% v/v avicell. When required the MEM/Avicell mix was supplemented with Cyt387, BX795, TPCA-1 , AZD1480, Ruxolitinib or Tofacitinib at the indicated concentration. Cells were incubated for 3-5 days and then fixed with 5% v/v formaldehyde in PBS. Plaques were visualised by crystal violet staining (0.15% v/v) or immunostaining using the following primary antibodies: anti- Bunyamwera N protein (kind gift of Richard Elliot, University of Glasgow); anti-RSV F protein (Serotech), anti-MeV NP (Abeam) and anti-MuV NP (Abeam) followed by the appropriate alkaline phosphatase conjugated secondary antibody and SIGMA FAST BCIP/NBT substrate.

Viral Growth Kinetics

A549, A549/PIV5-V, A549/BVDV-Npro cells were seeded as a confluent monolayer and then infected with BUNANSs at a MOI of 0.001 . The infection was performed for 1 hour at 37°C with intermittent shaking. Following infection, the virus inoculum was removed and cells washed with PBS. The cells were then incubated in DMEM/10% FBS either without drug or supplemented with Cyt387, BX795, TPCA-1 , AZD1480, Ruxolitinib or Tofacitinib at the indicated concentration. At various time points postinfection (24, 36, 48, 72, 96 hours) 0.3 ml of supernatant was removed and stored at - 70°C. The collected supernatant samples were subsequently used in plaques assays performed in Vero cells to determine viral titres as plaque forming units (pfu)/ml.

Results

The present inventors have conducted experiments to investigate the effect of inhibitors of the IFN signalling cascade on viral titre in cell culture. The small molecule Cyt387, an ATP-competitive inhibitor of Jak1 and Jak2 (Selleck Chemicals) was selected to investigate this. To test inhibition of IFN signalling by Cyt387 the A549/pr(ISRE).GFP reporter cell line was utilized. The A549/pr(ISRE).GFP cell line contains an eGFP reporter gene under the control of IFN-stimulated response element (ISRE) within the promoter region of Mx1 , an IFN-stimulated gene (Figure 1 B, right panel). Cells were grown in the presence of varying concentrations of Cyt387 or DMSO and purified IFN was used to stimulate the IFN signalling cascade and induce expression of GFP under the control of the ISRE promoter. In the presence of increasing concentrations of Cyt387, expression of GFP decreased; the greatest reduction in GFP expression was seen at 5 μΜ Cyt387 (Figure 2). MTT and crystal violet cellular cytotoxicity assays were conducted in parallel (data not shown). These data indicate that Cyt387 efficiently inhibits the IFN signalling cascade at an optimal concentration of 5 μΜ without detectable cellular toxicity.

The inventors next investigated the effect of Cyt387 on plaque formation of recombinant parainfluenza type 5 virus (PIV5) PIV5-VAC in A549 cells. PIV5-VAC has been modified so that the V protein is truncated. The V protein acts as a powerful IFN antagonist; therefore when the virus contains truncated V protein it is sensitive to IFN [15]. A dose-dependent increase in plaque size was observed with increasing concentrations of Cyt387 (Figure 3A). The increase in plaque size when the media was supplemented with 5 μΜ Cyt387 was equivalent to that seen in A549 cells which have been engineered to constitutively express the PIV5 V protein (Figure 3B). These data support the hypothesis that inhibition of the IFN signalling cascade can lead to increased viral growth.

These studies were extended to a derivative of Bunyamwera virus (BUNANSs) in which the IFN antagonist NSs is inactivated by the introduction of point mutations that disrupt the ATG start codon and introduce a stop codon [16]. The effect of Cyt387 on plaque formation of BUNANSs was tested in A549 cells and also MRC5 cells, which are suitable for vaccine manufacture. A significant increase in viral plaque formation was observed in both cell lines in the presence of 5 μΜ Cyt387 (Figure 4). This increase was most dramatic in the BUNANSs infected MRC5 cells supplemented with 5 μΜ Cyt387. These data support the observation that inhibition of the IFN signalling cascade can lead to increased viral growth and extends the observation to a different virus and a different cell line (MRC5), which is approved for vaccine manufacture.

Next the effect of Cyt387 on viral growth kinetics and titre in A549 na^'ive and A549/PIV5-V cells was investigated. In A549 na^'ive cells at 48 hours post infection BUNANSs titre was 1 .4 x 10⁸ pfu/ml in the presence of 5 μΜ Cyt387 compared to 3.0 x 10⁵ pfu/ml on the absence of Cyt387 (Figure 5). Therefore growth of BUNANSs in the presence of Cyt387 was significantly increased by 459-fold compared to viral growth in the absence of the IFN signalling inhibitor. Fold-increase in viral growth was approximately equivalent in the presence of Cyt387 or stably expressed PIV5 IFN antagonist V protein or a combination of both Cyt387 and V protein. This demonstrates that addition of a single small molecule inhibitor is as effective at increasing viral growth and yield as previously reported data demonstrating increased viral growth due to stable expression of an IFN antagonist in which the maximum fold-increase in viral growth achieved was 4000-fold [7]. These data support the hypothesis that addition of small molecules inhibitors of the IFN signalling cascade can significantly increase viral growth and titre by an order of magnitude of at least 2-3 log increases.

The present inventors have also conducted experiments to investigate the effect of inhibitors of the IFN induction cascade on viral titre in cell culture. The small molecule BX795, an inhibitor of ΤΒΚ1/ΙΚΚε (Calbiochem) was selected to investigate this.

To test inhibition of IFN induction by BX795 the A549/pr(IFN-p).GFP reporter cell line was utilized. The A549/pr(IFN-p).GFP cell line contains an eGFP reporter gene under the control of IFN-β promoter (Figure 1A, left panel). Cells were grown in the presence of varying concentrations of BX795 or DMSO and PIV5 VAC virus stock rich in IFN inducing defective particles was used to stimulate the IFN induction cascade and induce expression of GFP under the control of the IFN-β promoter. In the presence of increasing concentrations of BX795, expression of GFP decreased; the greatest reduction in GFP expression was seen at 4 μΜ BX795 (Figure 6). MTT and crystal violet cellular cytotoxicity assays were conducted in parallel (data not shown). These data indicate that BX795 efficiently inhibits the IFN signalling cascade at an optimal concentration of 2-4 μΜ with no detectable cell toxicity. The inventors next investigated the effect of BX795 on plaque formation of recombinant Bunyamwera virus BUNANSs in A549 cells. An increase in plaque size was observed in the presence of 2 μΜ BX795 (Figure 7). The increase in plaque size when the media was supplemented with 2 μΜ BX795 was equivalent to that seen in the presence of 5 μΜ of the IFN signalling inhibitor Cyt387 (Figure 7), which we have demonstrated to increase viral growth by at least 400-fold. These data support the hypothesis that inhibition of the IFN induction cascade can lead to increased viral growth and titre.

Taken together, these data provide evidence that small molecule inhibitors of the IFN induction and/or signalling cascade can be used as a supplement in tissue culture medium to increase the rate of virus growth and/or viral titre by at least 400-4000-fold.

A number of other inhibitors of the IFN induction and signalling cascades are available commercially for testing; the present inventors therefore investigated the effect of the following inhibitors on viral growth:

• TPCA-1 (an IKK-2 inhibitor);

• BX795 (discussed above);

• Cyt387 (discussed above);

• AZD1480 (a Jak1 inhibitor);

· Ruxolitinib (a Jak1 inhibitor); and

• Tofacitinib (a Jak1 inhibitor).

To test inhibition of IFN induction by TPCA-1 the A549/pr(IFN-p).GFP reporter cell line was utilized. The A549/pr(IFN-p).GFP cell line contains an eGFP reporter gene under the control of IFN-β promoter (Figure 1A, left panel). Cells were grown in the presence of varying concentrations of TPCA-1 or DMSO and PIV5 VAC virus stock rich in IFN inducing defective particles was used to stimulate the IFN induction cascade and induce expression of GFP under the control of the IFN-β promoter. In the presence of increasing concentrations of TPCA-1 , expression of GFP decreased; the greatest reduction in GFP expression was seen at 4 μΜ TPCA-1 (Figure 8A). These data indicate that TPCA-1 efficiently inhibits the I FN signalling cascade at an optimal concentration of 2-4 μΜ.

To test inhibition of IFN signalling by AZD1480, Ruxolitinib and Tofacitinib the A549/pr(ISRE).GFP reporter cell line was utilized. The A549/pr(ISRE).GFP cell line contains an eGFP reporter gene under the control of IFN-stimulated response element (ISRE) within the promoter region of Mx1 , an IFN-stimulated gene (Figure 1 B, right panel). Cells were grown in the presence of varying concentrations of AZD1480, Ruxolitinib, Tofacitinib or DMSO and purified IFN was used to stimulate the IFN signalling cascade and induce expression of GFP under the control of the ISRE promoter. In the presence of increasing concentrations of AZD1480, Ruxolitinib or Tofacitinib, expression of GFP decreased; the greatest reduction in GFP expression was seen at 4 μΜ (Figure 8B). These data indicate that AZD1480, Ruxolitinib and Tofacitinib efficiently inhibit the IFN signalling cascade at an optimal concentration of 4 μΜ.

The present inventors went on to test the effect of these inhibitors on viral growth. The effect of the inhibitors on viral plaque formation was examined using A549 cells infected with recombinant Bunyamwera virus (BUNANSs), in which the NSs IFN antagonist has been inactivated rendering the virus IFN sensitive. Standard plaque assays (as described above) were performed and fixed two days post-infection. A dose-dependent increase in plaque size was observed for all the inhibitors (Figure 9). The Jak1/2 inhibitor Ruxolitinib (RUX) had the most substantial effect; at >1 μΜ RUX, plaque formation was equivalent to that observed in A549 PIV5-V expressing cells. The effect of these six inhibitors on promoting viral growth was investigated further by examining their effect on BUNANSs growth kinetics. As shown in Figure 10, at 48 hours post-infection titres of greater than ~ 5 logs were obtained for RUX, Tofacitinib, AZD1480 and TPCA-1 compared to treatment with DMSO. The maximum titre achieved was equivalent to that reached in A549/PIV5-V cells. These results further strengthen the finding that supplementing cell culture medium with a variety of IFN inhibitors that target different components of the IFN response significantly boost viral growth and titre.

The present inventors also extended their study to demonstrate the effect of IFN inhibitors on viral growth in cell lines from a variety of mammalian species (human, mouse, rabbit, cow, dog and pig), using BUNANSs and/or WT Bunyamwera virus (BUN-WT) as test viruses. The IFN inhibitor RUX was utilised for this purpose. RUX increased plaque size in all cell lines tested (Figure 1 1 A and B). In MRC5 and A549 cells, BUNANSs plaque size was increased to the size seem in MRC5/PIV5-V and A549/PIV5-V cells. These results indicate that the use of IFN inhibitors offers a general approach to increasing viral growth in a variety of species. These results also support the concept that supplementing cell-culture medium with IFN inhibitors provides a flexible method to improve techniques to isolate emerging viruses by aiding virus growth in a range of cell-lines derived from different species.

Respiratory Syncytial Virus (RSV) is an example of a virus currently being developed as IFN-sensitive attenuated vaccine candidate. The present inventors therefore investigated the effect of the IFN inhibitor RUX on the growth of RSV, along with the measles and mumps viruses. Deletion of RSV IFN antagonists NS1 and NS2 impairs viral growth in MRC5 cells (Figure 12A). However, addition of RUX increased plaque size formation in both viruses to that equivalent of MRC5/PIV5-V cells (Figure 12A). Therefore, IFN inhibitors could be useful in the industrial production of IFN-sensitive attenuated vaccine candidates. Plaque size of wildtype RSV also increased in the presence of RUX (Figure 12A). The inventors also tested two traditional vaccine strains, measles (MeV) Edmonson and the mumps (MuV) Enders, which have been generated empirically using nonsystematic attenuation methods. Plaque size of the MeV and MuV vaccine strains were significantly increased in the presence of RUX (Figure 12B). MeV vaccine strains contain attenuating mutations in the P, V and C proteins that contribute to IFN antagonism. However, MuV Enders contains a functional V protein IFN antagonist, providing evidence that IFN inhibitors can boost the yield of viruses with reduced replication rates due to attenuating mutations that do not affect viral IFN antagonists, presumably due to the balance between kinetics of virus replication and induction of the IFN response. Shell eggs are routinely used for production of vaccines, in particular influenza vaccines, and therefore improvement of vaccine yield in eggs would be beneficial, as millions of eggs have to be used during vaccine production. The present inventors therefore went on to test the effect of IFN inhibitors on the IFN induction and signalling cascades in DF1 chicken fibroblast cells. To test the inhibition of IFN induction in DF1 chicken fibroblast cells by TPCA-1 and BX795, DF1 cells were transfected with the reporter construct ChlFN-p-luc in which lucif erase (Luc) is placed under the control of the IFN-β promoter. Forty-eight hours post transfection, cells were treated with IFN inhibitors for two hours and then the IFN induction response induced with polyl :C overnight and Luc activity determined. In the presence of both TPCA-1 and BX795 a dose-dependent decrease in Luc activity was observed (Figure 13A) indicating that TPCA-1 and BX795 efficiently inhibit the IFN induction cascade in chicken fibroblast cells. To test the inhibition of IFN signalling in DF1 chicken fibroblast cells by Ruxolitinib, DF1 cells were transfected with the reporter construct ChlMx(ISRE)-luc in which Luc is placed under the control of the Mx ISRE promoter. Forty-eight hours post transfection, cells were treated with IFN inhibitors for two hours and then the IFN signaling response induced with purified IFN (1000 U/ml) for 6 hours and Luc activity determined. In the presence of both 2 μΜ and 4 μΜ RUX, Luc activity was decreased (Figure 13B) indicating that RUX efficiently inhibits the IFN signalling cascade in chicken fibroblast cells.

Given this data, the previous data performed in cell culture and the conservation of the innate immune response at the evolutionary level (e.g. the Jak1 protein shares 80% homology between human and chicken) the inventors hypothesise that the addition of IFN inhibitors to chicken cells (in eggs or in cell culture) will improve the yield of a variety of vaccines. Such viruses may include, for example: (i) IFN sensitive viruses (e.g. designated live attenuated vaccines with deletion of IFN antagonists, e.g. NS1 ); (ii) mutated viral strains, for example influenza strains in quadrivalent FluMist vaccine (Medimmune) which are cold-adapted, temperature sensitive and attenuated strains; (iii) wildtype viruses used to generate 'killed' vaccines in egg culture. The present inventors propose to test the inhibitors demonstrated as effective in inhibiting the IFN induction and signalling cascades in cell culture for their ability to increase viral growth rate in egg culture. Increasing viral growth in this way would be of significant benefit to the vaccine industry. References

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Claims

CLAIMS:

I . A method of increasing viral growth rate and/or titre in cells, the method comprising adding an inhibitor of the I FN induction and/or signalling cascade to the cells.

2. The use of an inhibitor of the I FN induction and/or signalling cascade to increase viral growth rate and/or titre in cells.

3. The method or use according to any preceding claim, wherein the cells are in cell culture or egg culture.

4. The method or use according to claim 3 wherein the cells are in cell culture. 5. The method or use according to claim 3 wherein the cells are in egg culture.

6. The method according to claim 4, wherein the inhibitor of the I FN induction and/or signalling cascade is added to the tissue culture medium.

7. The method according to claim 5, wherein the inhibitor of the I FN induction and/or signalling cascade is added to the egg in which a virus is cultured.

8. The method or use according to any preceding claim, wherein the virus is genetically modified.

9. The method or use according to any preceding claim, wherein the virus is genetically modified so as to disable its capacity to overcome the I FN response.

1 0. The method or use according to any preceding claim, wherein the inhibitor targets one or more of the following molecules: Cardif/VISA/MAVS/I PS-1 ,

CBP/p300, c-jun/ATF-2, DAI/DLM-1 /ZBP1 , FADD, I FNAR1 , I FNAR2, ΙκΒ, ΙΚΚα, ΙΚΚβ, ΙΚΚε, I RAK-1 , I RAK-4, I RF-1 , I RF-3, I RF-5, I RF-7, I RF-9, JAK1 , LGP2, Mda-5, MyD88, NAP1 , NEMO, NFKB, Osteopontin, RIG-1 , RI P1 , SI NTBAD, SMAD3, STAT1 , STAT2, TAB2, TAB3, TAK1 , TANK, TBK-1 , TLR3, TLR4, TLR7, TLR8, TLR9, TRAF3, TRAF6, TRI F, TRI M25 and Tyk2.

I I . The method or use according to any preceding claim, wherein the inhibitor is a small molecule.

1 2. The method or use according to any preceding claim, wherein the inhibitor is a competitive inhibitor.

13. The method or use according to any preceding claim, wherein the inhibitor targets the I FN signalling cascade.

14. The method or use according to any claim 13, wherein the inhibitor targets the JAK/STAT signalling pathway.

1 5. The method or use according to claim 14, wherein the inhibitor targets Jak1 .

16. The method or use according to claim 15, wherein the inhibitor is Cyt387, AZD1480, Ruxolitinib or Tofacitinib.

17. The method or use according to any one of claims 1 -12, wherein the inhibitor targets the I FN induction cascade.

18. The method or use according to claim 17, wherein the inhibitor targets the TBK-

1/ΙΚΚε/ΙΚΚ2 induction pathway.

19. The method or use according to claim 18, wherein the inhibitor is BX795 or TPCA-1 .

20. A method of screening to identify a cell line/IFN induction and/or signalling cascade inhibitor combination providing increased viral growth rate and/or titre compared to the growth rate and/or titre found in uninhibited cell lines.

21 . A method according to claim 20, wherein the method comprises performing multiwell-plate based analysis of viral growth rate and/or titre in a range of cell line/inhibitor combinations.

22. A method according to claim 20 or 21 , wherein viral growth rate and/or titre is assessed by detecting fluorescence from a suitable reporter construct by fluorescent plate reader or FACS.

23. A method according to claim 20 or 21 , wherein viral growth rate and/or titre is assessed using an immunoassay, for example an enzyme-linked immunosorbent assay.

24. A method according to any one of claims 20-23, wherein the cell lines are selected from vera, MRC5, PERC6, MDCK and Hep2.

25. The method according to any one of claims 20-24, wherein the inhibitor targets one or more of the following molecules: Cardif/VISA/MAVS/IPS-1 , CBP/p300, c- jun/ATF-2, DAI/DLM-1/ZBP1 , FADD, I FNAR1 , I FNAR2, ΙκΒ, ΙΚΚα, ΙΚΚβ, ΙΚΚε,

I RAK-1 , I RAK-4, I RF-1 , I RF-3, I RF-5, I RF-7, I RF-9, JAK1 , LGP2, Mda-5, MyD88, NAP1 , NEMO, NFKB, Osteopontin, RIG-1 , RI P1 , SI NTBAD, SMAD3, STAT1 , STAT2, TAB2, TAB3, TAK1 , TANK, TBK-1 , TLR3, TLR4, TLR7, TLR8, TLR9, TRAF3, TRAF6, TRI F, TRIM25 and Tyk2.

26. A method according to any one of claims 20-25, wherein the inhibitors are small molecule inhibitors.

27. A method according to any one of claims 20-26, wherein the inhibitors are competitive inhibitors.

28. A method according to any one of claims 20-27, wherein the inhibitors target the I FN signalling cascade.

29. The method according to claim 28, wherein the inhibitors are small molecule inhibitors of the JAK/STAT signalling pathway.

30. The method according to any one of claims 20-27, wherein the inhibitors target the I FN induction cascade.

31 . The method according to claim 30, wherein the inhibitors are small molecule inhibitors of the ΤΒΚ-1/ΙΚΚε/ΙΚΚ2 induction pathway.