WO2013007703A1 - CLASS A CpG OLIGONUCLEOTIDES FOR PREVENTION OF VIRAL INFECTION IN CATS - Google Patents

CLASS A CpG OLIGONUCLEOTIDES FOR PREVENTION OF VIRAL INFECTION IN CATS Download PDF

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WO2013007703A1
WO2013007703A1 PCT/EP2012/063437 EP2012063437W WO2013007703A1 WO 2013007703 A1 WO2013007703 A1 WO 2013007703A1 EP 2012063437 W EP2012063437 W EP 2012063437W WO 2013007703 A1 WO2013007703 A1 WO 2013007703A1
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cells
odn
cats
feline
pbmcs
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Celine ROBERT-TISSOT
Hans Lutz
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Universität Zürich
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Abstract

The invention provides for the use of class A CpG oligonucleotides for prevention and therapy of infections, particularly viral infections, in cats.

Description

Class A CpG oligonucleotides for prevention of viral infection in cats

Description

Availability of vaccination regimes has lowered the risk posed by a number of cat-specific infectious diseases, yet a large number of diseases still exist against which a vaccine is not available. Additionally, vaccination may not be a method of choice for protection in situations where the time gap before establishment of an immune response elicited by vaccination is too large, or protection is deemed economically unfeasible.

A promising alternative to vaccination is the manipulation of innate immunity. Innate pathogen recognition relies on a set of sensory molecules, the Toll-like receptors (TLRs), which enable the immediate reaction of immune cells to pathogen "danger signals", or pathogen-associated molecular patterns (PAMPs). Oligodeoxynucleotides (ODN) containing unmethylated cytosine-phosphate-guanosine (CpG) motifs are effectively recognized as PAMPs by the vertebrate innate immune system. Response to oligodeoxynucleotides containing unmethylated cytosine-phosphate-guanosine (CpG) motifs (hereinafter: CpG ODN) is conferred through TLR9, expressed mainly in the intracellular compartments of human B cells and plasmacytoid dendritic cells (pDCs). Alarmed TLR9 is the initial instigator of gene expression profiles that strongly support antiviral mechanisms: up-regulation of co- stimulatory molecules MHCII, B7.1 and B7.2 on the surface of stimulated cells provides them with a stronger antigen presenting potential and production of cytokines such as type I interferon (IFN), IL-12, IFNy, IL-6 and TNFa contributes to providing an optimal immune environment for the development of innate and adaptive responses against intracellular pathogens.

Probably the most important antiviral property of CpG ODN resides in their potential to stimulate the production of high amounts of type I IFN by pDCs. This family of cytokines, which includes IFNa, IFNoo and IFN , has been shown to considerably enhance natural killer (NK) cell cytotoxicity, promote differentiation, maturation and immunostimulatory functions of monocytes and DCs, induce B cell production of immunoglobulin and Th1 differentiation of T cells. Moreover, upon binding to their ubiquitously distributed receptor, type I IFNs effectively induce the synthesis of various intracellular proteins that interfere with the replication of a broad range of viruses. The myxovirus-resistance protein (Mx) GTPase is a well-studied example of these intracellular antiviral effectors. This enzyme is known to be directly regulated by the type I IFN, and its detection is readily used as marker for upregulation and biological activity of this cytokine family.

Distinct classes of ODN have been shown to induce differential responses in human cells. A- type CpG ODN (CpG-A; EP 1 220 684 B1 ) comprise CpG motifs in a phosphodiester core, flanked on both ends by phosphorothioate poly (G) sequences. CpG ODN of this class are characterized by their potential to both induce massive type I IFN secretion by pDCs and increase NK cytotoxicity. B-type CpG ODN (CpG-B) bear multiple CpG motifs on a phosphorothioate backbone. CpG-B promote monocyte maturation and B cell activation, thus substantially supporting the development of humoral responses. In vivo studies in outbred animals have mainly been carried out with CpG-B.

CpG ODN have been tested in human clinical trials as vaccine adjuvants and as

combinatorial or monotherapies for cancer, allergies and chronic infectious diseases.

Meanwhile, their potential as prophylactic stand-alone inducers of innate defence

mechanisms has been the subject of fewer studies. Most work in this field initially described protection of mice against bacterial and parasitic infections. More recently, induction of resistance to viral infections was shown also in mouse models for Herpes Simplex Virus, Vaccinia virus, neurotropic arenavirus and foot and mouth disease virus (Kamstrup et al., (2006) Antiviral Res 72: 42-48). With exception of the latter, all these studies were carried out with CpG-B. In an outbred species, partial antiviral protection has only been described in two studies so far, in which reduced shedding of herpes and parainfluenza was observed in lambs after administration of a type B CpG. To the knowledge of the inventors, prophylactic antiviral potential of CpG-A has not yet been described in outbred animals.

The objective of the present invention is to provide safe and efficacious means of conferring protection against infection, particularly viral infection, to cats and other felidae.

In a study of various CpG-ODN, broad immunoprotective effects of CpG-A in cats were surprisingly found. In addition to strengthening the antiviral qualities of feline immune cells, it was found that ODN 2216, the prototype of CpG-A, can stimulate the production of soluble molecules possessing prophylactic properties against the replication of at least five different families of viruses, Coronaviridae, Herpesviridae, Caliciviridae, Parvoviridae, and

Retroviridae, in feline cells.

According to a first aspect of the invention, an isolated deoxynucleic acid is provided for use in a method for prevention or therapy of infections in cats. This isolated deoxynucleic acid comprises a sequence gnGm(PAL)GpgqG, wherein g represents a guanosine deoxynucleotide linked to its 5' sequence neighbour by a phosphothioate bond,

G represents a guanosine deoxynucleotide linked to its 5' sequence neighbour by a phosphate bond, or a 3'-guanosine deoxynucleotide,

- PAL represents a deoxynucleotide sequence tract of palindromic sequence, 4 to 8 bases in length,

n is 1 -2, m is 0-4, p is 0-2 and q is 3-7.

The term guanosine deoxynucleotide is used synonymous with the term deoxyguanosine (CAS No. 961 -07-9). If the nucleoside denominated g is on the 5' end of the isolated deoxynucleic acid, the 5' hydroxyl group can be a hydroxyl group (OH), a phosphate group or a phosphothioate. In some preferred embodiments, a 5' g bears a phosphothioate.

In one embodiment, the isolated nucleic acid molecule is completely characterized by the sequence gnGm(PAL)GpgqG (SEQ ID 001 ).

In some embodiments, the isolated nucleic acid is characterized by values for n and m being 2, p being 0 and q being 5.

In some embodiments, the isolated nucleic acid is characterized by a sequence

YYCGYRCGRR, wherein YY is a purine deoxynucleotide, R is a pyrimidine deoxynucleotide, C is cytidine and G is guanosine.

In one embodiment, the isolated nucleic acid is characterized by the sequence

ggGGGACGATCGTCgggggG (SEQ ID 002; ODN 2216).

All above embodiments are specifically provided for use in a method for prevention or therapy of infections, particularly viral infections, in cats and other members of felidae.

Although the general utility of CpG nucleotides in mammals for improving immune responses was known, the utility of class A CpG in cats is surprising in light of previous findings (Satoh, Microbiol. Immunol. 201 1 , 55 184-189) reporting a lack of IFNgamma induction in feline splenocytes.

According to a second aspect of the invention, a pharmaceutical composition for use in a method for prevention or therapy of infections, particularly viral infections, in felidae, particularly in cats, is provided, said pharmaceutical composition comprising an isolated nucleic acid according to the above aspect of the invention.

Such pharmaceutical composition comprises a class A CpG ODN according to the above aspect of the invention. A preferred ODN comprises the sequence ggGGGACGATCGTCgggggG (SEQ ID 002). Pharmaceutical compositions for enteral administration, such as nasal, buccal, rectal or oral administration are contemplated. Dosage forms for parenteral administration, such as subcutaneous, intravenous, intrahepatic or intramuscular administration, are preferred. The pharmaceutical compositions comprise from approximately 0, 1 % to approximately 95% active ingredient, preferably from approximately 1 % to approximately 10% active ingredient.

For parenteral administration preference is given to the use of solutions of the active ingredient. Intravenous or subcutaneous applications are particularly preferred.

Similarly, a dosage form for use in a method for the prevention or treatment of infections, particularly viral infections, in felidae is provided, comprising a class A CpG ODN according to the above aspect of the invention. Optionally, a pharmaceutical carrier or excipient may be present.

Transdermal/intraperitoneal and intravenous applications are also considered, for example a transdermal patch, which allows administration over an extended period of time, e.g. from one to twenty days.

An isolated nucleic acid or pharmaceutical composition may be used, according to a preferred embodiment of the present invention, in a method for prevention or therapy of an infection by a member of the group comprising Coronaviridae, Herpesviridae, Caliciviridae, Parvoviridae, and Retroviridae. According to a particularly preferred embodiment, the infection is an infection by a member of the group comprising vesicular stomatitis virus, feline calicivirus, feline parvovirus, feline coronavirus and feline herpes virus.

A class A CpG-ODN according to the above aspects of the invention can be administered alone or in combination with one or more other therapeutic agents or vaccines. Possible combination therapies can take the form of fixed combinations, alternatively the administration can be staggered or the combined agents can be given independently of one another, or in the form of a fixed combination.

According to yet another aspect of the invention, a method for the prevention or treatment of viral infections in felidae is provided, comprising the administration of a class A GpG-ODN according to the above aspects of the invention to a patient in need thereof.

The treatment may be for prophylactic or therapeutic purposes. For the administration, the class A CpG-ODN is preferably in the form of a pharmaceutical preparation in chemically pure form and optionally, a pharmaceutically acceptable carrier or adjuvants. The dosage of the active ingredient depends upon the species, its age, weight, and individual condition, the individual pharmacokinetic data, the mode of administration, and whether the administration is for prophylactic or therapeutic purposes. In the case of an individual having a bodyweight of about 7 kg the daily dose administered is from approximately with 0.1 mg/kg to approximately 1000 mg, preferably from approximately 0.5 mg to approximately 100 mg/kg, of a class A CpG-ODN according to the above aspects of the invention.

Also within scope of the invention is a method for the manufacture of a medicament for the prevention or treatment of infection, particularly viral infection, in felidae, particularly in cats, comprising the use of a deoxynucleotide according to the above aspects of the invention.

Medicaments according to the invention are manufactured by methods known in the art, especially by conventional mixing, coating, granulating, dissolving or lyophilizing.

CpG-A stimulation of PBMCs from adult SPF cats not only induced proliferation of these cells, but also enhanced the presence of co-stimulatory molecules MHCII and B7.1 on their surface and influenced their gene expression profiles in a strong antiviral orientation.

Notably, transcription levels of IFNa, IL-6, IL-12, and IFNy were increased by up to 12Ό00, 40, 25, and 12-fold respectively in PBMCs pulsed for 24 hours. Incubation of the

supernatants of CpG-A stimulated PBMCs with feline cell lines of epithelial (CrFK) and fibroblastic (FEA, fcwf-4) origin strongly induced expression of the antiviral Mx gene in these target cells, with a peak of up to 2000-fold increase in transcription after 6 hours and protein levels reaching a maximum within 24 hours and remaining stable for several days thereafter. Most importantly, the supernatant-treated cells showed significantly enhanced resistance to feline viruses from 5 distinct families, namely Coronaviridae, Herpesviridae, Caliciviridae,

Parvoviridae, and Retroviridae. The rate of protection of the cells was strongly correlated with the induction of type I IFN in the PBMCs and concordantly with the level of Mx expression in the target cells to be infected. Altogether, the data presented here highlight the potential of CpG-A to induce a preventive antiviral state in outbred species and to protect populations against a broad range of viruses.

The data presented in the examples shown below demonstrate the broad immunomodulatory effects of CpG-A in feline cells, which prophylactically inhibit the replication of feline viruses from different families. This has been experimentally proven for FCV, FPV, FHV, FCoV and FeLV, but supports extension to virus infections in general. ODN 2216, the first described CpG-A, could stimulate feline PBMCs to produce soluble molecules that significantly increase resistance of various feline target cell lines to viral propagation. The observed repression of viral replication highly correlated with the expression of type I IFN genes in stimulated PBMCs as well as the induction, prior to inoculation, of antiviral mechanisms in the target cells. To the knowledge of the inventors, this is the first study reporting efficient inhibition of viral replication by a CpG-A in an outbred species. Furthermore, the results demonstrate the potent prophylactic potential of a synthetic molecule as stand alone agent against a large range of viral pathogens simultaneously.

Short description of the figures:

Fig. 1 shows (A) H3 thymidine incorporation in the feline PBMCs after stimulation with ODN.

Dots indicate the mean counts per minute (cpm) of triplicate reactions for one cat. **p<0.01 ; (B) Expression of B7.1 surface molecules 24 hours post stimulation with the indicated treatments. (B) Expression of B7.1 surface molecules 24 hours post stimulation (C) Expression of MHCII surface molecules 24 hours post stimulation. Histograms of B and C depict the fluorescence of gated PBMCs.

Fig. 2 shows mRNA expression factors of IFNa and IL-6 in (A) fcwf-4 cells. Expression data 24 hours after treatment with increasing concentrations of ODN (A left panel and B), or over time after a single stimulation with 4pg/ml ODN (A right panel and C).

Fig. 3 shows expression levels of early immune response genes in ODN stimulated PBMCs of 14 SPF cats (A, B) and TLR9 mRNA levels in feline immune cells after stimulation (C).

Fig. 4 shows (A) mRNA expression factors of the indicated genes in ODN stimulated PBMCs of individual cats from four different age groups ; (B) Mx mRNA expression factors in PBMCs after a single stimulation ; (C) Mx mRNA expression factors in fcwf-4 cells

Fig. 5 shows inhibition of viral replication by ODN ; mean OD values from spectrophotometric readings of plaque assays conducted on duplicate wells treated with supernatants from one cat. *p<0.05, **p<0.01 ; VSV = vesicular stomatitis virus, FCV = feline calicivirus, FPV = feline parvovirus, FCoV = feline coronavirus, FHV = feline herpes virus

Fig. 6 shows ODN 2216-mediated decrease of retroviral DNA and RNA loads in ODN-treated cells.

Examples

Materials and Methods

Animals: Fourteen male SPF cats of ages ranging from 6 weeks to 14 years were used in this study. The animals were purchased from Liberty Research Inc. (Waverly, NY, USA) and housed in groups in an animal-friendly environment under optimal ethological conditions. Feline PBMC isolation, cell lines and cell culture: Feline PBMCs were isolated from EDTA- supplemented whole blood by Ficoll-Hypaque density gradient centrifugation using a standard protocol (Holznagel et al. (1998) J Virol 72: 9025-9033). Purified cells were counted as described previously (Weissenbacher et al., (2010) Vet J.) prior to their utilization in the different experiments, and cultured in RPMI 1640 with Glutamax I.

Adherent Crandell-Rees feline kidney (CRFK) cells (ATCC no. CCL-94) and Feline

Embryonic Fibroblasts (FEA) were maintained in RPMI 1640 with Glutamax I, while adherent Felis catus whole fetus-4 (fcwf-4) cells (ATCC no. CRL-2787) were cultured in EMEM (ATCC 30-2003). All media were supplemented with 10% heat-inactivated fetal calf serum, 100 U/ml penicillin and 100mg/ml streptomycin.

ODN 2216 and control ODN 2243 and recombinant feline IFNa (rfelFNa) were solubilized in endotoxin-free PBS. ODN 2243 consists of the same sequence as ODN 2216, with CpG motifs inversed to GpC. In all experiments, both ODNs were diluted in RPMI 1640 with Glutamax I supplemented as described above.

Relative gene expression analysis: PBMCs were stimulated at a density of 3x106 cells/ml directly after isolation, while CrFK, FEA or fcwf-4 cells were cultured to confluency prior to stimulation. At time points relevant to each experiment, the supernatants were removed and cells were lysed with mRNA lysis buffer (mRNA isolation kit I, Roche). mRNA extractions were performed with the mRNA Isolation Kit I and MagNA Pure LC Instrument (Roche) and first strand cDNA was synthesized with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Real-time qPCR reactions consisted of 5μΙ cDNA in a total volume of 25μΙ per reaction using the TaqMan® Fast Universal PCR Master Mix (Applied Biosystems). Thermocycling conditions included an initial denaturation of 20s at 95°C followed by 45 cycles of amplification by melting at 95°C for 3s and annealing at 60°C for 45s. Primers and probes for feline cytokine genes have been previously described. mRNA expression factors of selected genes, which correspond to ratios of mRNA levels measured in ODN stimulated versus PBS stimulated cells, were calculated and normalized with GeNorm version 3.5, using either both feline β-glucuronidase (GUSB) and tryptophan 5-monooxygenase activation protein zeta polypeptide (YWHAZ) (usually) or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) alone (when specified) as reference genes, under conditions validated for the feline species. Generally depicted in the graphs are mean expression factors calculated from duplicate experiments carried out simultaneously.

Proliferation assay: PBMCs were seeded immediately after isolation at a concetration of 3x106 cells/ml in U-bottom plates. Triplicate cultures for each cat were treated with either 4ug/ml ODN or an equal volume of endotoxin-free PBS. After an initial incubation of 18 hours, the cells were pulsed for 24 hours with 3H-thymidine. Standard liquid scintillation protocols were used for harvesting of the cells and uptake of 3H was assessed with a β- counter. Proliferation rates were calculated as the mean counts per minute (c.p.m) of triplicate cultures.

Flow cytometry: PBMCs were treated at a density of 3x106 cells/ml with 4pg/ml ODN or an equivalent volume of endotoxin-free PBS. During collection of the cells, the adherent cell fraction was systematically removed with trypsin-EDTA. Harvested cells were divided into 3 fractions labelled separately with either anti feline B7.1 mouse monoclonal IgG, anti feline MHCII mouse monoclonal lgG1 or fluoresceinisothiocyanate (FITC)-conjugated mouse lgG1 isotype control. The fractions were subsequently stained with R-Phycoerythrin (RPE)- conjugated goat anti-mouse lgG1 . Fluorescence was measured using a FACSCalibur® and analyzed with the FlowJo software. Identical gates were set for all cats in such a way that they comprise the desired cell populations of each individual.

Western Blot: CrFK and fcwf-4 monolayers were stimulated with PBMC supernatants. At the time points indicated, cells were harvested, counted, and divided into two fractions. 106 cells of either cell line were resuspended in 30μΙ sample buffer (0.5M

Tris(hydroxymethyl)aminomethane, 5% SDS, 10% β-mercaptoethanol, 40% glycerol, and 0.05% bromphenol blue) and boiled at 95°C for 5 minutes. SDS-PAGE separation and submersed immunoblotting procedures were carried out as previously described. For protein visualization, membranes were first cut immediately below the 80kB marker. The top and bottom fractions were incubated with murine anti-human Mx MAb M143 (generously provided by Dr. J. Pavlovic, Institute for Virology, University of Zurich, Switzerland) and murine anti β- actin monoclonal antibody as a loading control respectively. Both fractions were

subsequently incubated with a peroxidase-labelled goat anti-mouse IgG. Bands were digitalized using the Chemigenius 2 Biolmaging System (Syngene, Cambridge, UK).

Production of supernatants: For each cat, PBMCs were resuspended in supplemented RPMI 1640 with Glutamax I at a concentration of 106 cells/ml and stimulated with 4pg/ml ODN 2216, 4pg/ml ODN 2243 or an equivalent volume of endotoxin-free PBS. After 24 hours incubation, supernatants were harvested by centrifugation of the cultures twice at 2000xg for 10 minutes and stored at -20°C.

Viruses and viral inhibition assays: Vesicular stomatitis virus Indiana strain, FCoV Wellcome strain, FPV, FHV ZH5-04 strain and FCV F9 strain were titrated on both CrFK and fcwf-4 cells. Viral stock dilutions inducing 95% cytopathic effect (CPE) after 24 hours (72 hours for FCoV and FPV) were selected for inhibition experiments in order to ensure proper measurement of inhibitory effects. Monolayers of CrFK and fcwf-4 cells were incubated for 24 hours with the supernatants produced with PBMCs from each cat. The treated cells were then inoculated with virus (VSV, FCV, FHV, FCoV) or trypsinized and allowed to settle in viral suspension (FPV), and plaque assays were carried out after 24 hours (72 hours for FCoV and FPV) according to the procedure described previously. Briefly, supernatants were discarded and cell debris was removed from the wells by 3 cycles of washing with HBSS and shaking on an orbital shaker for 15 seconds. Remaining cells were fixed with 5% formalin and stained with a crystal violet solution. For spectrophotometric measurements, 100% methanol was added to the dried out wells and absorbance was read at 595nm on a

SpectraMax Plus 384 microtiter plate reader (Molecular Devices). Viral inhibition rates were calculated with the following formula:

Mean OD values of duplicate wells treated with Sup /(divided by) Mean OD values of quadruplicate wells treated with medium alone FeLV Glasgow-1 strain was titrated on FEA cells, and the lowest stock dilution leading to productive infection of the cells after 48 hours was used for inhibition experiments. Cells were treated with supernatants as well as relevant controls immediately prior to inoculation and every second day thereafter. At appropriate time points, cells and supernatants were harvested and total nucleic acid was extracted from both the cells and supernatants using the MagNA Pure LC DNA Isolation Kit I and MagNA Pure LC Instrument (Roche

Diagnostics). Viral replication in supernatants and proviral loads in cells were measured by real-time PCR with assays previously described.

Statistical analysis: All statistical analyses were performed using GraphPad Prism for Windows, version 3.0. Due to the limited amount of cats integrated in the study, we refrained from using a parametric approach in the statistical tests. Differences between treatment groups in proliferation of PBMCs and expression of co-stimulatory molecules on the surface of these cells were analyzed with a Wilcoxon signed rank test. Relative Mx expression, OD values from plaque assays and Ct values for FeLV DNA and RNA loads were analysed with a Wilcoxon signed rank test when treatment with different supernatants were compared or a Mann-Whitney test when incubation with supernatant was opposed to treatment with medium alone or rfelFNa. Normalized and relative TLR9 expression ratios between different animal age groups were also compared with a Mann-Whitney test. Longitudinal effects on FeLV viral and proviral loads were compared with each other using a Mann-Whitney test carried out with Area Under the Curve (AUC) values. Correlations were assessed using the Spearman test. P-values <0.05 were considered statistically significant.

Example 1 :

ODN 2216 induce proliferation of primary feline immune cells and enhance their expression of costimulatory surface molecules

The potential of CpG-A to induce proliferation of feline peripheral blood mononuclear cells (PBMCs) was assessed by measurement of 3H-thymidine incorporation after stimulation. Purified PBMCs from 10 adult SPF cats proliferated significantly in presence of ODN 2216 when compared to cells either treated with ODN 2243 (p= 0.002) or PBS (p=0.0039) as inactive controls (Fig 1 A).

Another characteristic feature of stimulatory ODN is their ability to enhance interactions between various immune cell populations by upregulation of cell surface costimulatory molecules. Along these lines, the expression of B7.1 and MHCII was measured in stimulated PBMCs of 8 cats by flow cytometry. In these experiments, elemental strategies were used to circumvent the difficulties linked to both limited availability of labelled antibodies for the feline species and few possibilities for co-staining of various markers and cell populations. Thus, the expression of both co-stimulatory molecules was evaluated on a gated PBMC population as well as on gated lymphocytic (lc) and non-lymphocytic (non-lc) subpopulations. The observed effects varied considerably between the cells of individual cats, ranging from no alterations to an increase of 400% stained cells in some cellular subpopulations after ODN 2216 stimulation. Interestingly, in some animals, stimulation with the control ODN 2243 indicated similar staining patterns than PBS, whereas in other cats the induction of effects comparable to those of ODN 2216 could be observed. Altogether, a significantly increased percentage of cells expressed high fluorescence levels within each gated population after primary staining of B7.1 molecules upon a 24-hour stimulation with ODN 2216, when compared to stimulation with ODN 2243 or PBS (Fig 1 B). Histograms depict the

fluorescence of gated PBMCs of one cat selected as an example (top left panel).

Percentages of gated FL2-H hi PBMCs, lymphocytic (lc) and non lymphocytic (non-lc) subpopulations of cells stimulated with ODN 2216 and ODN 2243 were normalized to percentage of cells within the same gates after PBS stimulation (set to 100%). Results for 8 cats are shown (top right panel). Stimulation with ODN 2216, ODN 2243 and PBS was compared using Wilcoxon signed rank tests performed on logarithmic values corresponding to percentages of FL2-H hi gated cells from the indicated cell populations of 8 cats, p values are depicted in the table. ODN 2216 v ODN 2243 ODN 2216 v PBS ODN 2243 v PBS

Non-Lc 0.0039 0.0039 0.0977

Lc 0.01 17 0.01 17 0.01 17

PBMCs 0.0078 0.0039 0.0195

As, in contrast to mice, feline MHCII is constitutively expressed in most cellular

subpopulations of PBMCs, increase in expression of this molecule was assessed by differences in the mean fluorescence intensities in gated populations of differentially stimulated cells. Overall, although the expression of MHCII remained unchanged in non-lc cells after 24 hours incubation with ODN 2216, the presence of this molecule on the surface of feline PBMCs as well as in Ic cells could be significantly upregulated (Fig 1 C). Histograms depict the fluorescence of gated PBMCs of one cat selected as an example (top left panel). Mean fluorescence intensity (MFI) of gated PBMCs, Ic and non-lc subpopulations of cells stimulated with ODN 2216 and ODN 2243 were normalized to MFI of cells within the same gates after PBS stimulation (set to 100%). Results for 8 cats are shown (top right panel). Stimulation with ODN 2216, ODN 2243 and PBS was compared using Wilcoxon signed rank tests performed on logarithmic values corresponding to MFI of the indicated cell populations of 8 cats, p values are depicted in the table.

Figure imgf000012_0001

Example 2:

ODN 2216 influence type I IFN and proinflammatory gene expression in primary feline immune cells

Through interaction with TLR9, CpG-A typically induce expression of both type I IFN and proinflammatory cytokines in stimulated cells. In order to understand whether ODN 2216 exert similar effects in the cat, treated cultures of feline PBMCs, FEA, CrFK and fcwf-4 cells were systematically screened for increased expression of IFNa and IL-6 following stimulation. All tested immortalized feline cell lines failed to respond to stimulation with ODN 2216 (Fig 2A and data not shown). However, this molecule exhibited potent

immunomodulatory properties in feline PBMCs: when measured 24 hours post stimulation, a concentration of only 1 pg/ml ODN 2216 was sufficient to enhance transcription of IFNa by 40-fold and a maximum induction of this gene was observed when 4pg/ml ODN were used (Fig 2B). Although affected in a similar pattern, the expression of the proinflammatory cytokine IL-6 remained comparatively low at all concentrations tested (Fig 2B). In

experiments foreseen to determine gene expression kinetics after stimulation with the 2216 molecule in feline cells, an influence on IFNa could be measured as early as 1.5 hours after treatment of PBMCs, whereas increased levels of IL-6 were observed only as of 6 hours post stimulation. Notably, the highest induction of both genes was measured 24 hours after addition of ODN 2216 to the cultures, with transcription increasing by 9000-fold and 39-fold at this time point for IFNa and IL-6 respectively (Fig 2C). The observed effects were specifically conferred by the CpG motifs comprised in the 2216 ODN, since specific control ODN 2243 only induced slight elevations in the expression of the genes tested (Fig 2B and 2C). Importantly, trypan blue exclusion experiments indicated no evidence of cellular toxicity after treatment neither with ODN 2216 nor with ODN 2243 at all concentrations tested (data not shown).

Example 3:

ODN 2216 broadly influence the gene expression profile in primary feline immune cells of adult cats

In an effort to assess both the breadth of the effects conferred by treatment with a CpG-A and possible variability in the responses obtained in individual cats, the expression of 10 genes relevant to early immune responses was measured in the ODN 2216 stimulated PBMCs of 14 SPF cats divided in different age groups (group 1 : 2.5 months (n=4), group 2: 1.5 years (n=4), group 3: 7 years (n=4), group 4: 14 years (n=2)). Only slight individual variability in gene induction was observed after 24 hours stimulation of immune cells from adult cats ranging between 1.5 and 14 years of age (Fig 3A, groups 2-4). mRNA expression factors of the indicated genes were measured in PBMCs isolated from SPF cats of 2.5 months of age (= group 1 ), 1 .5 years of age (= group 2), 7 years of age (= group 3) and 14 years of age (= group 4) and stimulated with ODN 2216 for 24 hours.

Overall, the gene expression profile measured in stimulated PBMCs of these cats

corroborated with the induction of strong antiviral immune responses. The induction of type I IFN mRNA, including IFNa, IFNp and IFNoo was substantially higher in the immune cells from every adult cat, with minimal inductions of 490, 60 and 1600-fold respectively observed in the older animals of group 4. Moreover, all individual IFNa subtypes tested were induced at similar levels in the cells of four adult cats from group 2 (Fig 3B). mRNA expression factors of the indicated IFNa subtype genes were measured in PBMCs isolated from 4 adult SPF cats (group 2) and stimulated with ODN 2216 for 24 hours.

Increased levels of proinflammatory cytokine mRNA were also measured in most individuals of groups 2-4, with IL-6 more systematically increased than TNFa. The cells from these cats also indicated a typical Th1 orientation after stimulation, with enhanced transcription of IL-12 in all and IFNy in some individuals, together with absent or decreased induction of IL-4.

Stimulation of PBMCs with ODN 2216 also created an optimal environment for NK cell activity, as indicated by the increased expression of NK cell stimulator IL-15 by up to 20-fold and NK cell effector Granzyme B by up to 7-fold. Higher individual variability was observed after stimulation of PBMCs from kittens of group 1 , as the cells of only 2 out of 4 individuals could be stimulated with CpG 2216 to enhance expression of the tested genes and in both cats at much lower levels than observed in adult cats (Fig 3A, group 1 ).

In order to determine whether a discrepancy in expression of TLR9 between the PBMCs of adult animals and kittens could play a role in these observations, mRNA levels of this gene were measured in immune cells of each cat. Interestingly, although basal TLR9 expression was similar in the PBMCs of the cats of all age groups (Fig 3C, top panel), ODN 2216 stimulation increased TLR9 expression in the cells of kittens, but decreased expression of this receptor in the cells of adult cats so that differences in the mRNA levels of this receptor were significantly higher in young cats (group 1 ) than in adult cats (p=0.0286 with groups 2 and 3, not measurable with group 4) (Fig 3C, bottom panel). mRNA levels of TLR9 were measured by real-time qPCR in unstimulated PBMCs of all four groups of cats and normalized to the expression of a feline housekeeping gene (GAPDH) (top panel). Relative TLR9 mRNA expression factors were measured in ODN 2216 stimulated PBMCs from cats of all four groups (bottom panel). **p<0.01

Example 4:

ODN 2216 induce the production of soluble molecules that activate intracellular antiviral mechanisms in feline target cells

Protective properties of type I I FN against viruses originate from their ability to trigger the production of potent antiviral proteins in nearby cells. The expression of one of these antiviral proteins, the Mx GTPase, is known to be directly stimulated by type I I FN and can be used as marker for the induction of intracellular antiviral mechanisms by these cytokines. Thus, as detection of feline type I IFN on a protein level is rendered difficult by the lack of antibodies on the market specifically recognising these proteins, mRNA levels of Mx were measured in feline PBMCs after stimulation with ODN 2216 as indication for production of type I IFN. Transcription of Mx was significantly enhanced and remained proportional to expression of type I IFN in stimulated PBMCs of individual cats (Fig 4A). mRNA expression factors of the indicated genes were measured in ODN 2216 stimulated PBMCs of the individual cats from four different age groups.

Moreover, peak levels of Mx mRNA were measured 24 hours after stimulation of the cells, indicating the presence of optimal effects of the type I IFN present in the cell culture medium at this time point (Fig 4B). Mx mRNA expression factors were assessed at the indicated time points in PBMCs after a single stimulation with either ODN 2216 or ODN 2243.

In order to further determine the potential of the type I IFN liberated by PBMCs after ODN 2216 stimulation to induce intracellular antiviral mechanisms in non-immune target cells, cell- free supernatants of PBMCs derived from the blood of individual adult cats and stimulated in vitro for 24 hours with ODN 2216, ODN 2243 or medium alone (Sup 2216, Sup 2243 and Sup Neg respectively), were incubated with CrFK and fcwf-4 cells. Mx gene expression was significantly enhanced in cells treated with Sup 2216 compared to Sup 2243 (p=0.0078) and Sup Neg (p=0.0078), at levels comparable to those achieved by stimulation of the cells with 100U rfelFNa. Although Sup 2243 also increased Mx mRNA levels in target cells, protein levels remained barely detectable (Fig 4C). Mx mRNA expression factors were measured in fcwf-4 cells incubated for 24 hours with supernatants (Sup 2216, Sup 2243, Sup Neg) derived from PBMCs of 8 adult cats or 100U recombinant feline IFNa (rfelFNa) (left panel). Mx protein was detected by Western blot in fcwf-4 cells incubated with the indicated supernatants or with 100U recombinant feline IFNa (rfelFNa) for 24 hours (right panel).

Furthermore, peak induction of Mx transcription was observed in both cell lines already within 6 hours of incubation with Sup 2216, while protein levels achieved maximum levels within 24 hours post stimulation and remained stable thereafter at least for another 24-48 hours (Fig 4D and data not shown). Mx mRNA expression factors were measured in fcwf-4 cells at the indicated time points after a single stimulation with Sup 2216, Sup 2243 and Sup Neg respectively (left panel). Mx protein was detected by Western Blot in fcwf-4 cells at indicated time points after a single stimulation with Sup 2216 (right panel). **p<0.01 Example 5:

ODN 2216 inhibit replication of viruses belonging to 4 families in vitro

Felids are frequently affected by four viruses of different families, the feline herpesvirus (FHV), calicivirus (FCV), parvovirus (FPV) and coronavirus (FCoV). Although these viruses cannot productively infect purified PBMCs in vitro, they share the ability to induce cytopathic effects (CPE) in CrFK and fcwf-4 cells. These feline cell lines, however, do not alter their expression patterns upon direct treatment with ODN 2216 (Fig 2A and data not shown), preventing from assessing the potential of this molecule to inhibit viral replication directly in these cells. Thus, CrFK and fcwf-4 cells were incubated, prior to their inoculation, with the cell-free supernatants of PBMCs mentioned above: Sup 2216, Sup 2243 and Sup Neg. To this aim, supernatants derived from PBMCs treated for 24 hours were selected, as Sup 2216 produced by the cells several adult cats were estimated to contain optimal type I IFN amounts at this time point, according to the induction of Mx expression measured directly in these immune cells (Fig. 4A and 4B). CrFK and fcwf-4 target cells were then incubated with the supernatants for 24 hours before inoculation, as this time span had indicated highest induction of antiviral mechanisms (Fig 4D). The antiviral effects of the supernatants were initially tested on vesicular stomatitis virus (VSV) as control, as this virus is widely recognized for both its potential to induce CPE in cell lines of multiple species and for its particularly high sensitivity to the effects of type I IFN. When the Sup 2216 derived from the PBMCs of 8 adult cats that had broadly responded to in vitro ODN 2216 stimulation (Fig 3A, groups 2 and 3) were incubated with fcwf-4 cells prior to their inoculation, significant inhibition of VSV replication was observed (p=0.0039) (Fig 5A). fcwf-4 cells were incubated for 24 hours with the indicated supernatants derived from PBMCs of 8 adult cats (groups 2 and 3) or medium only as control before inoculation with the indicated viruses. Each dot represents mean OD values from spectrophotometric readings of plaque assays conducted on duplicate wells treated with supernatants from one cat.

Interestingly, VSV replication was also to some degree repressed by Sup 2243 (p=0.0039), an observation reminiscent of the slight induction of Mx in target cells incubated with these supernatants (Fig 4C). fcwf-4 cells were incubated for 24 hours with the indicated

supernatants derived from PBMCs of 4 kittens (group 1 ) or medium only as control before inoculation with the indicated viruses. Each dot represents mean OD values from

spectrophotometric readings of plaque assays conducted on duplicate wells treated with supernatants from one cat. *p<0.05, **p<0.01 In turn, the propagation of FHV, FCV, FPV and FCoV on fcwf-4 cells was also significantly decreased by the Sup 2216, however with expected lower sensitivity than VSV (Fig 5A and Table 1 ). Both Sup 2243 and Sup Neg failed to inhibit replication of this heterogeneous group of feline viruses, underlining the importance of the 2216 molecule in conferring the observed effects. Moreover, the Sup 2216 of those 2 kittens whose cells responded to ODN 2216 stimulation (Fig 3A, group 1 ) could also inhibit both VSV and FCV on fcwf-4 cells, while the supernatants derived from the PBMCs of the other 2 kittens indicated no inhibition potential on these viruses (Fig 5C). Importantly, Mx induction in target cells highly correlated with inhibition of all viruses in those cells (Fig 5B). Correlation of individual inhibition ratios of each virus with Mx mRNA expression induced in fcwf-4 cells incubated with supernatants of ODN 2216 stimulated PBMCs from 8 cats (groups 2-3).

Table 1. Means of viral inhibition rates measured in fcwf-4 cells after treatment with supernatants derived from stimulated PBMCs of 8 adult cats

Figure imgf000017_0001
VSV= Vesicular Stomatitis Virus, FCV= Feline Calicivirus, FCoV=Feline Coronavirus, FHV=Feline Herpesvirus, FPV=Feline Parvovirus

Notably, similar results were obtained when the same supernatants were incubated with CrFK cells, prior to their inoculation with all the above-mentioned viruses (data not shown). This cell line has previously indicated less sensitivity to the antiviral effects of type I IFN and concordantly, average fold viral inhibition in CrFK cells in our experiments was approximately half that observed in fcwf-4 cells.

Example 6:

ODN 2216 inhibits replication of a retrovirus in vitro

The life cycle of retroviruses is characterised by the reverse transcription of their genomic RNA into DNA and subsequent integration of this viral DNA as provirus into the genome of the host, causing permanent infection accompanied most often by persistent virus production by infected cells. The domestic cat is commonly affected by the leukemia virus (FeLV), a gammaretrovirus that can be propagated on FEA cells in vitro. As viral replication in chronically infected cats can be lowered by treatment with IFNa, the potential of Sup 2216 produced by the PBMCs of 4 adult cats (Fig 3A, group 3) to inhibit productive infection of FEA cells was analysed. In initial experiments, this cell line exhibited similar responses as fcwf-4 and CrFK cells to both direct treatment with ODN 2216 and incubation with the different supernatants (Fig 2A, 4C). Compared to incubation with medium alone, treatment of Sup 2216 with FEA cells for 24 hours prior to inoculation with FeLV followed by repetitive treatments of the cells with this supernatant every 2 days thereafter significantly reduced viral RNA (p=0.05) and DNA (p=0.05) measured in the cell culture supernatants and cells respectively as of 4 days post inoculation (Fig 6A). FEA cells were incubated for 24 hours with the respective supernatants or ODN 2216 or medium alone, before inoculation with the feline leukaemia virus (FeLV), as well as every 2 days thereafter. Viral replication was measured at the indicated time points by real time qPCR and 45 cycles - Ct values are shown in order to facilitate interpretation (left panel). FeLV DNA loads in the cells were measured at the indicated time points and Ct values were normalized to detection of a housekeeping gene (GAPDH) (right panel). Mean values from duplicate experiments carried out simultaneously with the supernatants derived from PBMCs of a selected cat and with medium alone are shown as an example. Results are indicative of those obtained with supernatants from PBMCs of 3 additional adult cats. Stars represent statistical differences in area under the curve (AUC) measurements between the curves of all 4 cats obtained in cells incubated with Sup 2216 and each of the other treatments.

Mx mRNA expression was 80 fold higher in the Sup 2216 treated than in all controls 8 days post inoculation, indicating the ability of these supernatants to sustain antiviral mechanisms when applied to the cells repeatedly (Fig 6B). Viral loads (top left panel), proviral loads (top right panel) and Mx mRNA expression factors (bottom panel) were measured in the FEA cells on day 8 post inoculation. Each dot represents the mean of duplicate measurements for an individual cat. *p<0.05, **p<0.01

Furthermore at this time point, the Sup 2216 treated cells exhibited significantly lower viral DNA loads (p=0.0313) and produced significantly less virus (p=0.0313) than cells treated with Sup 2243, Sup Neg, CpG directly or medium alone (Fig 6B).

Cells of the feline immune system were strongly influenced when cultured in the presence of ODN 2216. In contrast to other classes of ODN, CpG-A does not generally induce lymphocyte proliferation in mice (Krug A et al. (2001 ) Eur J Immunol 31 : 2154-2163). Feline PBMCs however, significantly proliferated in presence of ODN 2216 (Fig 1A), indicating a direct and or indirect stimulation of one or more immune cell subpopulations by this molecule. ODN 2216 also increased the expression of cell surface co-stimulatory molecules in PBMCs (Fig 1 B and 1 C), reinforcing possible interactions between various immune cell populations. The simultaneous transport of MHCII molecules and antigen for presentation may explain the lack of upregulation of this costimulatory molecule on the surface of non- lymphocytic cells after stimulation. Also, it should be noted that the observed effects were linked to relatively high variability among the cats, reflecting the heterogeneity of PBMC subpopulations in individuals of the same species. Differential gating adapted to the PBMC phenotypes of each animal or differentiation of various immune cell subpopulations by co- staining for appropriate markers could confer further information about intraspecies stimulatory variability. Moreover, not only the presence of CpG motifs but also the chemical structure of the ODN seemed to play an important role in the upregulation of MHCII and B7.1 co-stimulatory molecules in feline PBMCs, as control ODN 2243 also significantly increased expression of both B7.1 and MHCII in stimulated lymphocytic cells. The potential of synthetic backbones to enhance cell surface molecule expression in immune cells has indeed already been reported. Interestingly, the most astonishing effects of ODN 2216 remain the potent induction of IFNa and IFNoo, with mRNA expression of these genes increased by up to 12Ό00 and 35'000-fold respectively in PBMCs of certain animals (Fig 3B). The

measurements carried out 24 hours after stimulation of purified feline cells also supported induction of Th1 -oriented immune responses and enhanced NK cell activity, both highly desired in the contexts of viral infection. The highly similar patterns in gene expression profiles observed in the treated cells of adult cats indicate consistency in stimulatory potential of ODN 2216 among individuals. It should be noted that the SPF origin and maintenance under SPF conditions of the animals included in this study may slightly lower the variability in strength and breadth of innate immune responses, and studies with cells obtained from field cats would give further information on divergence in response to stimulation of the innate immune cells of individual animals. Interestingly, the immune cells of kittens indicated much more reticence to stimulation, with either limited or absent upregulation of both type I IFN and other genes measured after incubation with ODN 2216. Stimulated PBMCs from this group of young animals moreover indicated a tendency to develop an immunologic environment with a Th2 orientation, including higher production of IL-4 and impaired induction of IL-12 compared to cells from adult cats. Although the kittens included in this study were already of 2.5 months of age, these observations strongly corroborate with both the impaired immune cell activation via TLR9 measured in human neonatal mononuclear cells and the bias towards Th2 rather than Th1 responses in human foetuses and neonates. Furthermore, concordantly to findings in human neonatal blood, basal TLR9 expression in kittens indicated levels similar to those of adults. Interestingly however, ODN 2216 stimulation increased mRNA expression of this gene only in the young animals (Fig. 3C). The lack of specific antibodies to feline TLR9 unfortunately renders further investigations on the mechanism behind these results and their implications in CpG ODN stimulation in kittens rather challenging. However, in the endoplasmic reticulum of adult immune cells, the accumulation of TLR9 proteins as a pool awaiting transport to the endosomes upon ligand-induced stimulation substantially decreases the necessity of upregulation on the mRNA level. Young individuals hypothetical^ have yet to establish this reserve of TLR9 proteins in the ER, leading to increased transcription of this gene following stimulation with CpG ODN.

Preventive treatment of cells with CpG ODN strengthens considerably the antiviral defence mechanisms of these and surrounding cells, restricting viral infections, and thus tilting the immunological balance in favour of the host. Concordantly, in infections with rapidly dividing pathogens, optimal protection of mouse models can be observed when CpG ODN are administered prior to infection. Our data demonstrate that five viral species belonging to the Calicivirus, Herpesvirus, Parvovirus, Coronavirus and Retrovirus families were sensitive to the immunologic effects of ODN 2216 in cats.

When compared to direct initiation of antiviral mechanisms by a recombinant IFNa protein, administration of CpG ODN holds the advantage of inducing the production of all type I IFN and their subtypes (Fig 3B), which have been shown to possess differential antiviral properties. Also, CpG ODN specifically target DCs, a cell population widely recognized to link innate and adaptive immune mechanisms. Modifications in the gene expression profile of these cells enhances the expression of costimulatory molecules on their surface (Fig 1 B) as well as the secretion of a broad range of cytokines (Fig 3A), both mechanisms enabling further transfer of the stimulatory signals to cells of the innate and specific immune systems.

In view of future in vivo experiments, a 3-step in vitro system was conceptualized and utilized in this study: purified PBMCs were first stimulated with ODN 2216 to secrete soluble molecules, including type I IFN, into the culture medium; cell-free supernatants of the stimulated PBMCs were then incubated with a target cell line reminiscent of epithelial (CrFK) or fibroblastic (fcwf-4, FEA) tissue; target cells were finally inoculated with a feline virus and viral replication was monitored. This method seemed much more feasible than direct stimulation of CrFK, fcwf-4 and FEA cells, as it enabled to both circumvent the irresponsiveness of these cell lines to stimulation with ODN 2216 (Fig 2A) and profit from the interplay between the heterogenic subpopulation of immune cells comprised in PBMCs. Additionally, this system closely mimics an in vivo situation, as PBMCs not only comprise pDCs, the main players involved in recognition and response to CpG-A, but also circulate systemically, affecting many different tissues with the soluble molecules they produce.

The supernatants of PBMCs stimulated with CpG-A contain a mixture of molecules that could play a role in the total antiviral effects observed. However, the viral inhibition highly correlated with the induction of Mx transcription in the cell lines incubated with the

supernatants (Fig 5B) underlining the central role of type I IFN in protection of target cells against viral invasion. Inhibition of viral replication in the present study moreover

corroborates the sensitivity of the individual viruses to various subtypes of recombinant feline interferon reported in past studies. It is possible that Mx mRNA and protein were directly linked to inhibition of the viruses tested in this study similar to the situation known with myxoviruses, at least, Mx mRNA and proteinact as markers for induction of an antiviral state in tissue cells. Although the expression of other antiviral molecules such as

2'5'oligoadenylate synthetase (OAS) and the RNA-dependent protein kinase (PKR) has not been measured, their induction has been reported following CpG ODN stimulation of PBMCs in other species and differential interplay between several effector mechanisms most likely leads to inhibition of individual viruses. After a single 24-hour treatment with supernatants of ODN 2216 treated feline PBMCs, an increased amount of Mx protein could be detected for at least 72 hours in all target cell lines tested, corroborating the kinetics of this protein following IFNa stimulation reported in various cell types previously. Also, when fcwf-4 cells were incubated with the supernatants for 72 instead of 24 hours, similar inhibition of VSV and FCV was observed (data not shown), further supporting possible protection against viral infection by ODN 2216 for at least several days, a phenomenon described already in mouse models.

The characteristic structure of a CpG-A molecule includes poly (G) sequences on a phosphorothioate backbone at both ends and CG dinucleotides embedded in a central palindromic sequence with phosphodiester linkages. The necessity for poly (G) motifs renders CpG-A more difficult to produce than other types of CpG ODN. However, when considering the in vivo use of stimulatory molecules, CpG-A hold several considerable advantages. First, they remain the most potent inducers of type I IFN, themselves the most biologically active antiviral molecules known to date. Also, as a result of their poly (G) stretches, they can form G-tetrads, highly ordered structures with enhanced stability. Their only partial phosphorothioate structure could moreover help reduce possible long-term side effects related to synthetic backbones. Finally, the flu-like symptoms generally linked with administration of immunostimulatory molecules in vivo may remain minor in cats treated with ODN 2216, since we observed only marginal increase in expression of proinflammatory cytokines in stimulated immune cells when compared to type I IFN genes. Altogether, the data presented here reflects the strong potential for this molecule in the prevention of a large variety of viral diseases.

Claims

Claims
1. An isolated deoxynucleic acid comprising a sequence gnGm(PAL)GpgqG, wherein g represents a guanosine deoxynucleotide linked to its 5' sequence neighbour by a phosphothioate bond,
G represents a guanosine deoxynucleotide linked to its 5' sequence neighbour by a phosphate bond, or a 3'-guanosine deoxynucleotide,
PAL represents a deoxynucleotide sequence tract of palindromic sequence, 4 to 8 bases in length,
n is 1 -2, m is 0-4, p is 0-2 and q is 3-7,
for use in a method for prevention or therapy of infections in cats.
2. An isolated nucleic acid according to claim 1 , wherein n and m are 2, p is 0 and q is 5, for use in a method for prevention or therapy of infections in cats.
3. An isolated nucleic acid according to claim 1 or 2, wherein PAL is a sequence
YYCGYRCGRR (SEQ ID 001 ), wherein YY is a purine deoxynucleotide, R is a pyrimidine deoxynucleotide, C is cytidine and G is guanosine, for use in a method for prevention or therapy of infections in cats.
4. An isolated nucleic acid according to claim 1 , wherein the sequence is
ggGGGACGATCGTCgggggG (SEQ ID 002), for use in a method for prevention or therapy of infections in cats.
5. A pharmaceutical composition comprising an isolated nucleic acid according to any of the above claims, for use in a method for prevention or therapy of infections in cats.
6. An isolated nucleic acid or pharmaceutical composition according to any of the above claims, wherein the infection is a viral infection.
7. An isolated nucleic acid or pharmaceutical composition according to claim 6, wherein the infection is an infection by a member of the group comprising Coronaviridae, Herpesviridae, Caliciviridae, Parvoviridae, and Retroviridae and other virus families.
8. An isolated nucleic acid or pharmaceutical composition according to claim 6, wherein the infection is an infection by a member of the group comprising vesicular stomatitis virus, feline calicivirus, feline parvovirus, feline coronavirus and feline herpes virus and other feline viruses.
PCT/EP2012/063437 2011-07-08 2012-07-09 CLASS A CpG OLIGONUCLEOTIDES FOR PREVENTION OF VIRAL INFECTION IN CATS WO2013007703A1 (en)

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