WO2012131134A1 - Use of a non-coding region of the genome of the foot-and-mouth virus for the purpose of producing an antiviral drug - Google Patents

Abstract

The present invention relates to the use of RNAs that correspond to highly structured domains of the non-coding regions of the genome of the foot-and-mouth virus (FMV) and to the use of sequences that comprise said RNAs for the production of a pharmaceutical composition for the prophylaxis and/or the treatment of diseases caused by interferon-sensitive viruses. The invention also relates to the pharmaceutical composition that comprises the non-coding regions of the invention and that furthermore comprises at least one pharmaceutically acceptable excipient and/or vehicle, and also to the use of said pharmaceutical composition.

Description

 Use of a non-coding region of the foot and mouth disease virus genome for the development of an antiviral drug

The present invention relates to the use of a non-coding region of the FMD virus genome for the preparation of a pharmaceutical composition for the prophylaxis and / or treatment of diseases caused by interferon-sensitive viruses.

STATE OF THE PREVIOUS TECHNIQUE

The innate immune response is the first line of defense against pathogen invasion based on various mechanisms and signaling pathways. In the case of a viral infection, one of the mechanisms of the innate immune response is the production of type I interferon (IFN) (IFN-α and IFN-β, or also called IFN-α / β) that has an antiviral effect. , antiproliferative and has immunomodulatory activity. The production of type I IFN occurs after the detection of pathogen-associated molecular patterns (PAMPS) present in viral products that are generated in a viral infection. PAMPS includes both single stranded ribonucleic acid (RNA) and double stranded RNA. However, interferon-sensitive viruses that evade this defense system have been described. Interferon-sensitive viruses include foot-and-mouth disease virus, vesicular stomatitis virus and West Nile virus (Randall RE et al. 2008. J Gen Virol 89: 1-47).

Foot and mouth disease virus (FMDV) is a member of the Picornaviridae family that is the cause of foot and mouth disease (glossopeda) that affects artiodactylated ungulate mammals, including ruminants and pigs . VFA is a single-stranded RNA virus approximately 8.5 kilobases (Kb) in length that contains non-coding regions (NCRs) that contain highly structured domains forming a double RNA. chain. VFA NCRs contain specific structures that participate in the control of replication and translation of the viral genome. Among the non-coding regions are the so-called S fragment, IRES ("infernal ribosome entry site") and 3'NCR (Witwer CS et al. 2001. Nucleic Acid Res 29: 5079-5089; Escarmis CM et al. 1992. Virus Res 26: 1 13-125; Belsham GJ et al. 2009. Virus Res 139: 183-192).

It has been shown that VFA is sensitive to IFN-a, IFN-β and IFN-γ but is capable of developing mechanisms to evade the immune response. The Lpro protease is the first viral protein that translates and blocks the host's innate immune response, including the INF-mediated response. Lpro is able to inhibit the induction of the messenger RNA (mRNA) of IFN-β and the expression of genes that stimulate IFN-α / β. This fact represents a problem for the control of FMD disease.

Foot and mouth disease is a disease that constitutes a serious problem in the livestock sector. It is a highly contagious disease that spreads through infected animals, livestock equipment, clothing or even shoes bearing VFA. It is currently controlled by vaccination and preventive measures, but once the animals are infected, it involves slaughter and therefore results in large economic losses in the sector.

In relation to vaccination against VFA, adaptive immunity appears several days after vaccination of animals, being approximately one week in the case of traditional vaccination. This is a problem because these animals can be exposed to the virus during the window period and be susceptible to infection during that time. Efforts have been made to cover that susceptibility window by using recombinant replication-defective adenoviruses expressing IFN-α but said strategy, in addition to involving the use of a viral vector, does not cover the VFA susceptibility window in its totality since inoculated pigs are protected in this way from 24 hours Post-infection and protection lasts between 3 and 5 days. Therefore, a tool that covers the susceptibility window against the FMD virus is necessary prior to the development of an effective adaptive immunity in which infected animals are susceptible to infection.

The vesicular stomatitis virus (VEV, "vesicular stomatitis virus", VSV) is a virus belonging to the family Rhabdoviridae that produces an acute vesicular disease in cattle, horses, deer and pigs. Serological evidence has been found that infects many other animals and occasionally infects humans as well. There are two main species, called New Jersey and Indiana. It is transmitted by insect bite vectors, mainly sand flies (Lutzomya sp.), Black flies (family Simuliidae) and insects of the Culicoides genus. Its genome consists of 1 1 - 12 Kb of single-stranded RNA and negative polarity. VEV is also able to avoid the host's immune response. In the case of VEV, the infection induces the general blockage of gene expression in the host cell, which affects the production of IFN and other antiviral molecules. Currently, disease control is performed using measures to restrict movement of infected animals, quarantine, control of vector insects and vaccination with inactivated virus. An alternative capable of activating an effective innate immune response against the virus would be very useful for the control of this disease.

The West Nile virus (WNV, "West Ni le Virus", WNV), a virus transmitted mainly by mosquitoes and whose natural host are birds, is classified within the Flaviviridae family, along with other important animal pathogens such as classical swine fever, and humans such as hepatitis C virus, dengue virus, yellow fever virus (Blitvich BJ 2008. Anim Health Res Rev 9:71-86). WNV is a virus with a lipid envelope, whose virions have icosahedral symmetry and an approximate diameter of 50 nm. As a genetic material, WNV has a single single-stranded and positive polarity RNA molecule of about 1,1000 nucleotides in length. He WNV is also able to avoid the host's immune response. WNV is a clear example of re-emerging zoonosis, which is currently a serious problem for human and animal health since it causes fatal meningoencephalitis in humans, equidae and birds, in which it generally produces subclinical infections, but in which occasionally it causes a high mortality (Granwehr BP et al. 2004. Lancet Infecí Dis 4: 547-556). In recent years, the number and severity of cases in humans and horses have increased considerably. At present, as tools for its control, only a commercial vaccine for equidae is available based on the use of inactivated virus and in the case of humans, therapies based on the administration of interferon (IFN) or compassionate antibodies (Ng et al. 2003. Dev Biol 114: 221-227; Davis et al. 2006. Ann Neurol 60: 286-300) that are not sufficient for disease control, so an effective alternative to the prophylaxis and / or treatment of West Nile disease.

EXPLANATION OF THE INVENTION

The present invention relates to the use of a non-coding region of the foot-and-mouth disease virus genome for the preparation of a pharmaceutical composition for the prophylaxis and / or treatment of diseases caused by interferon-sensitive viruses.

In order to evaluate the antiviral potential of the non-coding regions of the foot-and-mouth disease virus (VFA) genome, in the present invention, non-coding synthetic RNAs analogous to said regions were generated by in vitro transcription, the ncRNAs of the invention.

Interferon-sensitive viruses that have been shown to be sensitive to the innate immune response induced by the ncRNAs of the present invention are viruses of the families Picornaviridae, Rhabdoviridae and Flaviviridae. Within the family Picornaviridae, the FMD virus was chosen in the Rhabdoviridae family was chosen vesicular stomatitis virus, while from the family Flaviviridae West Nile virus was chosen as the most representative of said families for the performance of tests demonstrating the activity of the ncRNAs of the invention.

With the ncRNAs used in the invention, in vitro and in vivo studies were performed in which the production of messenger RNA (mRNA) of IFN-α or IFN-β and antiviral response was detected. In addition, survival trials were also conducted against viral challenge in a model of a nursing mouse. In the trials, the effect was compared with a known interferon stimulator, poly l: C (Richmond JY et al. Proc Nati Acad Sci USA 1969. 64: 81-86) and it was demonstrated that the stimulating activity of the innate immune response of the ncRNAs of the invention is superior to this control. The results obtained with the ncRNAs of the invention show that these ncRNAs stimulate the production of IFN in transfected cells. In addition, IFN mRNA induction levels correlate with antiviral activity against vesicular stomatitis virus (VEV). In viral challenge studies, the ncRNAs of the invention protect against foot-and-mouth disease virus as well as West Nile virus, with the IRES sequence of VFA being the one that offers the best protection in both cases while the S and 3 'regions NCR of said virus offer less protection. On the other hand, IFN production occurs in the first hours after transfection and antiviral activity against VEV is detectable at 12 hours post infection, which makes the ncRNAs of the invention an effective tool for the treatment of diseases. caused by IFN-sensitive viruses since the ncRNAs of the invention activate the innate immune response rapidly. The results obtained in the present invention show the potential use of the ncRNAs of the invention for the preparation of an antiviral drug against IFN-sensitive viruses. The ncRNAs of the invention can be very useful in combination with vaccines against diseases caused by IFN-sensitive viruses to cover their susceptibility window. For all that is described herein, a first aspect of the present invention relates to the use of a non-coding region of the FMD virus genome for the preparation of a pharmaceutical composition.

In the present invention it is understood as "non-coding region" (NCR, "non-coding region", "untranslated region" or UTR) that region of the foot and mouth disease virus genome located at its terminal ends (5 'and 3' ) that contains highly structured domains forming a double stranded RNA and that does not code for any protein. Among the non-coding regions are the so-called S fragment, IRES ("internal! Ribosome entry site") at the 5 'end, at the 5'NCR and at the 3' end is the 3'NCR region. RNAs obtained by in vitro transcription of the S and IRES regions of the 5'NCR and the 3'NCR region of FMD virus are used in the present invention. Hereinafter we will refer to the S and IRES regions of the 5 'end and the 3'NCR region of the VFA as the "NCRs of the invention" or "non-coding regions of the invention", while we will refer to the RNAs obtained by in vitro transcription of said regions as to the "ncRNAs of the invention" (non-coding RNAs of the invention).

The term "pharmaceutical composition" herein refers to any substance used for prevention, diagnosis, relief, treatment or cure of diseases. In the context of the present invention it refers to a composition comprising at least the ncRNAs or NCRs of the invention. The pharmaceutical composition of the invention can be used both alone and in combination with other pharmaceutical compositions, including vaccines and antivirals. The combination of said pharmaceutical composition with vaccines or antivirals could make the immune response they generate more effective, thus acting as an adjuvant. The term pharmaceutical composition and medicament are used interchangeably in that invention. In the context of the present invention the term "vaccine" refers to an antigen preparation used to elicit an immune system response to disease caused by a virus. It is a preparation of antigens that, once inside the organism, provokes the response of the immune system through the production of antibodies, and generates immunological memory producing permanent or transient immunity.

In the present invention the term "antiviral" refers to any substance that does not allow the replication, assembly or release of viruses, such as interferon, ribavirin, etc.

The ncRNAs of the invention have proven capable of inducing antiviral activity against INF-sensitive viruses, such as VFA, VEV, and WNV. For this reason, a second aspect of the present invention relates to the use of a non-coding region of the FMD virus genome for the preparation of a pharmaceutical composition for the treatment of diseases caused by interferon-sensitive viruses. "Treatment" refers to both therapeutic and prophylactic treatment or preventive measures. Those necessary for treatment include those already associated with alterations as well as those in which the alteration is prevented. An "alteration" is any condition that would benefit from treatment with the composition of the invention, as described herein.

"Interferon sensitive virus" means a virus whose replication is affected or interrupted by the interferon secreted by the host cells.

Interferon is a cytokine secreted by various cells, including cells of the immune system, which has an antiviral effect. Of the groups of interferons, it is the alpha and beta that are generally induced in response to viral infection, for which reason in the present invention "interferon" refers to IFN-α or IFN-β. Foot and mouth disease virus (FMDV) is the virus that causes foot and mouth disease. It belongs to the family Picornaviridae (genus Aphthovirus) and its genome is a single stranded RNA. Seven different serotypes with multiple subtypes and variants have been described, serotype O, A, C, Asial, SAT1, SAT2 and SAT3. The isolates used in the present invention are 01 K, belonging to serotype O (the genome of lineage isolates 01, 01 c and 01 K is found in Genbank accession numbers D10138 and X00871) and isolate C-S8c1 from serotype C (genome in Genbank accession number AJ133357). The length of the NCRs regions in the different isolates varies and the homology is 80% for the S region, 85% for the 5'NCR region comprising IRES in addition to fragments from other regions and 82% for the region 3'NCR, so the non-coding regions of the VFA would have at least 80% homology between the different serotypes (Carrillo et al. 2005. J Virol 79: 6487-6504). The ncRNAs used in the invention correspond to the S and IRES regions of the 5 'end and the 3'NCR region. The ncRNAs used in the invention constitute a non-infectious material of easy production and biotechnological manipulation. The results obtained with the ncRNAs used in the present invention demonstrate the potential use of non-coding regions of VFA for the preparation of a pharmaceutical composition for the treatment of diseases caused by interferon-sensitive viruses.

In the non - coding region 5 'end (5'NCR) of the FMDV genome is the fragment The S includes 366 nucleotides 5 S. ^fragment' -terminal of RNA and its folding is predicted in a fork - like secondary structure ( "hairpin ") long and stable (included in the access sequence in GenBank X00871). The S fragment is followed by a poly C region ("heteroplolymeric poly C tracf, Cn), several pseudo-nodes (pseudoknots, Pk), the ere-replication element ("cis-acting replication element") and the internal ribosome entry site (IRES, "internal! ribosome entry site") (Fig . one ). IRES is a 454 nucleotide region structured in multiple domains that mediates the cap-independent translation of the viral genome (included in the access sequence in GenBank D10138). On the other hand, a non-coding region called 3'NCR is also located at the 3 'end of the viral genome. The 3'NCR includes 90 nucleotides from the termination codon of the open reading phase of the viral RNA and a variable length poly A tail, where said length increases with the course of infection (the 90 nucleotides without the poly A tail are included in the access sequence in GenBank X00871). The 3'NCR of the VFA has been seen to exert an activating effect of IRES-dependent translation and that it is capable of interacting with the IRES and S regions located at the 5 'end by direct RNA-RNA interactions.

In the present invention analogs of VFA NCRs have been generated by in vitro transcription. Due to the strategies known by any person skilled in the art that derive from the biotechnological methods used in said in vitro transcription (such as the use of a complementary DNA inserted in the plasmid used in the transcription, cloning and the use of enzymes restriction), the ncRNAs of the invention contain extra nucleotides at their terminal ends. The ncRNAs of the invention are non-coding regions that are functional analogs of the NCRs of the invention.

The term "analog" refers to a region of the same function but of a different sequence. In the present invention it refers to the fact that ncRNAs have the same function as NCR regions, they comprise the NCR sequence but contain more nucleotides from the biotechnological strategy used for their synthesis. The "extra" nucleotides referred to in the present invention are ribonucleotides not present in VFA NCRs but which are the result of the biotechnological strategies used in the present invention for the generation of ncRNAs. These extra nucleotides can be incorporated into the VFA NCRs by the use, for example, of an RNA polymerase, by being nucleotides present in the plasmid in which the complementary DNA used for in vitro transcription is cloned or by entering the sequence to generate restriction targets to favor biotechnological strategies.

A "restriction target" (or restriction site) is a sequence recognized by a restriction enzyme, that is, it refers to a site or site that recognizes an endonuclease that is capable of cutting the phosphodiester bonds of the nucleotide chain by generating a cut in the chain of deoxyribonucleic acid (DNA). In the present invention it refers to a sequence recognized by a restriction enzyme that is capable of cutting the phosphodiester bonds of the complementary DNA used for in vitro transcription. For example sequences recognized by the restriction enzymes Stu I and EcoRV. The ncRNA of the 3'NCR region of the VFA (SEQ ID NO: 1) contains 188 ribonucleotides of which the first 15 and the last 16 correspond to nucleotides not present in the 3'NCR of the VFA and which are the result of biotechnological strategies used for its generation. It also contains three additional ribonucleotides that have been introduced to generate restriction sites that allow biotechnological manipulation and that do not affect the secondary structure that is generated in the 3'NCR and therefore its function. This is the case of position 36 uracil that derives from a thymine in the template DNA clone that generates a restriction target for the enzyme Stu I (AGGCCT) and the case of position 108 guanine and position uracil 10, derived from a guanine and thymine in the template clone that generate a restriction target for EcoRV (GATATC). So the 3'NCR region of the VFA referred to in the present invention is considered as the region comprised between nucleotide 23 and 11 described in SEQ ID NO: 1 (where said nucleotides are also included) in which nucleotides 36, 108 and 1 are not present 10. On the other hand, the ncRNA of the S region of the VFA (SEQ ID NO: 2) contains 404 ribonucleotides of which the first 20 and the last 18 correspond to nucleotides not present in the S sequence of the VFA and which are the result of the biotechnological strategies used for their generation. Thus, the S region of the FMD virus referred to in the present invention is the sequence between (and even) nucleotide 21 and 386 described in SEQ ID NO: 2. In the case of the ncRNA of the IRES region of the VFA (SEQ ID NO: 3), this contains 476 ribonucleotides of which the first 14 and the last 8 correspond to nucleotides not present in the IRES of the VFA and which are the result of the biotechnological strategies used for their generation. Thus, the IRES region of the FMD virus referred to in the present invention is the sequence between (and even) nucleotide 15 and 468 described in SEQ ID NO: 3.

In the present invention the term "region", "sequence" or "nucleotide sequence" is used interchangeably in memory.

From the foregoing, a preferred embodiment of the first and second aspects of the present invention relates to the use of a non-coding region of the VFA genome where said non-coding region is comprised in the nucleotide sequence selected from the list comprising: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or any combination thereof. Where SEQ ID NO: 1 comprises the 3'NCR region of the VFA, SEQ ID NO: 2 comprises the region or fragment S of the VFA, SEQ ID NO: 3 comprises the IRES region of the VFA. A more preferred embodiment refers to the use where the sequence is SEQ ID NO: 1. Another more preferred embodiment refers to the use where the sequence is SEQ ID NO: 2.

Another more preferred embodiment refers to the use where the sequence is SEQ ID NO: 3.

The present invention also relates to the use of other NCRs or ncRNAs of other virus isolates. For this reason and for the foregoing, the present invention also relates to the use of the non-coding regions of the invention and to the ncRNAs of the invention where the non-coding region refers to a region with a homology of at least 80% with that region.

The present invention also relates to the NCRs or to the ncRNAs of the invention which, by biotechnological strategies known by any person skilled in the art, have their sequence modified, for example by the generation of restriction targets, but where said modifications do not significantly affect the structure RNA secondary.

It is known to any person skilled in the art that viruses whose genome is a single stranded RNA are more sensitive to IFN than viruses with DNA. Various viruses that are sensitive to interferon have been described, including members of the Picornaviridae, Flaviviridae and Rhabdoviridae families, for example foot-and-mouth disease virus, West Nile fever virus and vesicular stomatitis virus. Despite presenting sensitivity to INF, many viruses are capable of developing mechanisms to avoid this innate immune response, as in the case of VFA, VEV and WNV (Randall RE ei al. J Gen Virol 2008. 89: 1 - 47). The present invention demonstrates that the use of the ncRNAs of the invention is useful as an antiviral even in viruses that exhibit such evasion mechanisms.

As described herein, an even more preferred embodiment refers to the use where the interferon-sensitive virus is a single-stranded RNA virus. Preferably, the virus is a virus of the Picornaviridae family, more preferably the foot and mouth disease virus. Preferably also the virus is a virus of the family Rhabdoviridae, more preferably it refers to the vesicular stomatitis virus. And also preferably the virus is a virus of the Flaviviridae family, where said virus is preferably West Nile virus.

Another preferred embodiment of the second aspect of the invention relates to the use where said diseases are diseases in non-human mammalian animals. Preferably ungulated animals, more preferably of the family Suidae (for example of the genus Sus, for example S. scrofa), of the family Bovidae or of the family Equidae. Also the present invention relates to the use in birds, preferably of the families of birds that are selected from the list comprising: Phasianidae, Corvidae, Paseridae, Fringillidae and Turdidae (belonging to the Passerine and Galliform orders), as well as families Accipitridae and Strigidae, among others.

Another preferred embodiment of the second aspect of the invention relates to the use where said diseases are diseases in humans. Diseases can be zoonotic diseases.

A third aspect of the invention relates to a pharmaceutical composition of the first or second aspects of the invention which further comprises at least one pharmaceutically acceptable excipient and / or vehicle.

The term "excipient" refers to a substance that aids in the absorption of the pharmaceutical composition comprising the NCR or the ncRNA of the invention, stabilizes it or aids in its preparation in the sense of giving it a consistency, form, taste or any Another specific functional feature. Thus, the excipients could have the function of keeping the ingredients together such as starches, sugars or cellulose, sweetening function, coloring function, drug protection function such as to isolate it from air and / or moisture, filling function of a tablet, capsule or any other form of presentation such as dibasic calcium phosphate, disintegrating function to facilitate the dissolution of the components and their absorption in the intestine, not excluding other types of excipients not mentioned in this paragraph.

A "pharmacologically acceptable vehicle" refers to those substances, or combination of substances, known in the pharmaceutical sector, used in the preparation of pharmaceutical forms of administration and includes, but are not limited to, solids, liquids, solvents or surfactants. The carrier can be an inert substance or of analogous action to any of the compounds of the present invention and whose function is to facilitate the incorporation of the drug as well as other compounds, allow a better dosage and administration or give consistency and form to the composition Pharmaceutical When the presentation form is liquid, the vehicle is the diluent. The term "pharmacologically acceptable" refers to the compound referred to being allowed and evaluated so as not to cause damage to the organisms to which it is administered. As described herein, a preferred embodiment of the third aspect of the invention relates to a pharmaceutical composition that further comprises at least one other active ingredient.

As used herein, the term "active substance"("activesubstance","pharmaceutically active substance", "active ingredient" or "pharmaceutically active ingredient") means any component that potentially provides a pharmacological activity or other different diagnostic effect , cure, mitigation, treatment, or prevention of a disease, or that affects the structure or function of the body of man or other animals. The term includes those components that promote a chemical change in the preparation of the drug and are present therein in a modified form intended to provide the specific activity or effect. The pharmaceutical composition or medicament provided by this invention may be provided by any route of administration, for which said composition will be formulated in the pharmaceutical form appropriate to the route of administration chosen. For this reason, a preferred embodiment for this aspect of the invention relates to the pharmaceutical composition wherein said pharmaceutical composition is presented in a form adapted to oral, parenteral or intradermal administration.

The present invention also relates to the use of the pharmaceutical composition of the third aspect of the invention for the preparation of a medicament for the prophylaxis and / or treatment of diseases caused by interferon-sensitive viruses.

Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following figures and examples are provided by way of illustration, and are not intended to be limiting of the present invention.

DESCRIPTION OF THE FIGURES

Fig. 1. Structural reasons in the NCRs of the VFA genome. Schematic representation of the motifs in the 5 ^' and 3 ^' NCRs of the VFA. NCR, non-coding region; S, fragment S; IRES, "ribosome entry site"; VPg, "genome linked viral protein"; Cn, poly C region; Pk, region of "pseudoknots"; ere, the cis-acting replication element; Lpro, protease "leader"; 3Dpol, 3D polymerase; SL1, "stem-loop 1"; SL2, "stem-loop 2";3'NCR, 3 '"non coding region"; ΔΑη, transcribed 3'NCR to which the poly A tail has been removed. Fig. 2. Induction of IFN-β in porcine cells. It shows the production of IFN-β by the ncRNAs of the invention of VFA in porcine cells. SK-6 cells were transfected with 20 μ9 / ηιΙ of the NCR transcripts or with 10 μ9 / ηιΙ of the poly l: C control or the negative tRNA control (transfer RNA). Induction levels of IFN-β mRNA in cells transfected with respect to cells transfected with phosphate buffered saline (PBS) at the indicated times were determined by RT-qPCR (quantitative RT-PCR) normalized to GAPDH. Error bars show the standard deviation of the average of three independent experiments performed in triplicate. IFN-β, porcine IFN-β messenger RNA; T, time; h, hours

Fig. 3. Analysis of the contribution of the 3 ^' NCR sequences of VFA in the induction of IFN-β. It shows the production of IFN-β by the 3'NCR region of VFA and fragments thereof in porcine cells. RNAs corresponding to SL1, SL2, 3 ^' NCRAA _n as well as 3 ^' NCR RNAs (20 Mg / ml) treated or not with CIP and poly l: C (10 g / ml) were transfected into SK-6 cells. Induction levels of IFN-β mRNA in cells transfected with respect to cells transfected with PBS at the indicated times were determined by standardized RT-qPCR with respect to GAPDH. Error bars show the standard deviation of the mean between triplicates. IFN-β, porcine IFN-β messenger RNA; T, time; h, hours; SL1, "stem-loop 1"; SL2, "stem-loop 2";3'NCR, 3 '"non coding region"; ΔΑη, transcribed 3'NCR in which the poly A tail has been removed; 3'NCR + CIP, 3'NCR that has been treated with phosphatases. Fig. 4. Induction of an antiviral state in cells transfected with NCR RNAs. It shows the antiviral effect against the virus of vesicular stomatitis in swine cells. After 24 h of transfection with 40 μg / ml of the transcripts corresponding to the NCRs of VFA, or 10 μg / ml of tRNA, SK-6 cells were infected with VEV (multiplicity of infection (MOI) = 1). Supernatants from infected cells were collected at 12 and 24 h post-infection and viral titers were determined in IBRS-2 cells. The bars indicate the average values of two independent experiments performed in triplicate plus the standard deviations Statistical analysis was performed using ANOVA and Bonferroni test (* p <0.05, ** p <0.01). T, post infection time; h, hours; Iog10pfu / ml, viral titration measured in Iog10 of the plaque forming units (pfu) per milliliter; mock, transfection control.

Fig. 5. VFA NCRs induce the innate response in a nursing mouse.

It shows the production of IFN-β by the ncRNAs of the invention in lactating mice. Groups of 4 to 5 Swiss lactating mice were inoculated intraperitonealemnte (ip) with 100 μg of 3 ^' NCR, S, IRES or poly l: C transcripts. The levels of IFN-α (A) and IFN-β (B) in pools of serum extracted at different post-inoculation times for each RNA were determined by ELISA. The average values of IFN-α and IFN-β for serum pools at 0 h post-inoculation were 25 and 6 pg / ml, respectively.

Figure 6. Effect of inoculation of ncRNAs on the survival of mice inoculated with WNV. The data corresponding to each group and the Kaplan-Meier survival curves analyzed by the log rank test using GraphPad Prism 5.01 (GraphPad software, San Diego, CA, USA) are shown.

Figure 7. IFN-α levels in sera of mice inoculated with the ncRNAs. The analysis was carried out by ELISA from "pools" of sera taken at 4, 8 or 24 h after inoculation of the RNAs; t, time; h, hours

Figure 8. Total specific antibody levels against VFA induced following the different vaccination guidelines. The antibody level was determined by ELISA and the means corresponding to each group are shown at the different post-vaccination times indicated. The titer was expressed as the Iog10 of the last dilution whose OD reading was greater than 2 times the value of the pre-immune serum at the minimum dilution tested (1/20); t, time; d, days; week weeks Figure 9. Levels and kinetics of neutralizing antibodies against VFA after the different vaccination guidelines. The number of animals in each group in which significant levels of neutralizing antibodies were detected is shown. The graph represents the average levels in each group at different post-vaccination times expressed as PRN70; t, time; d, days; week weeks + Pre RNA: Inoculated with 200 μg of IRES RNA + lipofectin and 24 h later inoculated with the vaccine; + RNA t = 0: Co-inoculated with the vaccine and with 200 μg of IRES RNA + lipofectin; + RNA post: Inoculated with the vaccine and 24 h later inoculated with 200 μg of IRES RNA + lipofectin.

Figure 10. Profile and levels of isotypes of antibodies against VFA induced after vaccination, in the groups that received only the vaccine (vaccine only) or vaccine co-inoculated with the IRES (vaccine + RNA t = 0). The sera of the animals, taken 9 weeks after vaccination, were analyzed individually by ELISA using a panel of mouse immunoglobulin isotyping antibodies. The results of each group are shown: A: groups that received only the vaccine (vaccine only); B: groups that received the vaccine co-inoculated with the IRES (vaccine + RNA t = 0); C: corresponding to negative (NEG) and positive (POS) control sera. M, G1, G2A, G2B, G3, A and K + L, correspond to different isotypes of antibodies against VFA.

ILLUSTRATIVE EXAMPLES OF THE INVENTION The following specific examples provided in this patent document serve to illustrate the nature of the present invention. These examples are included for illustrative purposes only and should not be construed as limitations on the invention claimed herein. Therefore, the examples described below illustrate the invention without limiting its scope of application. EXAMPLE 1: VCR genome NCRs contain PAMPs (molecular patterns associated with pathogens) that activate innate response signaling. The ability to induce the expression of IFN-β in porcine cells of highly structured elements of the 5 ^' and 3 ^' NCR regions of VFA RNA was initially analyzed. RNAs corresponding to the 5 ^' and 3 ^' NCRs and the S and IRES fragment of the foot-and-mouth disease virus were synthesized by in vitro transcription (Fig. 1). VFA ncRNAs were used to transfect SK-6 cells (porcine kidney cells) (Fig. 2). IFN-β mRNA expression induction was analyzed by real-time quantitative RT-PCR at three different times (Fig. 2) using the sense primer shown in SEQ ID NO: 4 and the antisense primer shown in SEQ ID NO: 5. Remarkable differences were observed between the different RNAs. At three times the 3 ^' NCR was the most potent IFN-β transcriptional inducer of all, reaching levels almost 200 times above the transfection control cells at 9 h posttransfection. The S fragment induced levels 10 times higher than the control followed by the full 5 ^' NCR. For the transcript corresponding to the IRES, inductions of 4.5 times were detected, slightly higher than poly l: C. These results suggest that the structures present in the 3 ^' NCR are recognized more efficiently than those of the 5 ^' NCR by viral sensors present in porcine cells and that the S fragment is the main inducing element in the 5 ^' NCR in these cells. EXAMPLE 2: Analysis of the characteristics of the 3 ^' NCR of the VFA relevant for the induction of IFN-β.

The contribution of the different structures present in the 3 ^' NCR and of the VFA poly A to the induction of the IFN-β mRNA was analyzed by transfection in SK-6 cells of the corresponding transcripts (Fig. 1 and 3). None of the two "stem-loops" present in the 3 ^' NCR, SL1 (SEQ ID NO: 6) and SL2 (SEQ ID NO: 7) were able to induce detectable levels of IFN-β, being minimally active (Fig. 3). However, the 3 ^' NCR with the poly A tail removed (3'NCR ΔΑη, SEQ ID NO: 8) was still a potent activator, reducing only between 2-10 times according to the post-transfection time analyzed (Fig. 3 ). Activation levels detected did not correlate with the size or molarity of transfected RNAs. Paper induction ^5'end triphosphate in the RNA was analyzed by treatment with alkaline phosphatase (CIP). The effect of this treatment significantly reduced induction levels at 9h post-transfection, although 20-fold inductions were still detected with respect to control cells.

EXAMPLE 3: The "PAMP" elements of the VFA genome stimulate innate immunity and antiviral response in porcine cells

To determine whether the induction of IFN-β mRNA observed in SK-6 cells transfected with the S, IRES and 3 ^' NCR elements of the VFA corresponded to the activation of an anti-viral state in the cells, the antiviral activity was analyzed. in transfection supernatants, incubating with them IBRS-2 cells (swine kidney cells) (Table 1). After 24 hours, the cells were infected with VEV. In all cases antiviral activity was detected (Table 1), except in the controls (cells transfected with poly l: C or tRNA, where the result was the same for both cases), and the levels of inhibition of VEV infection correlated well with IFN-β mRNA levels induced by each RNA. Treatment of transfection supernatants with specific monoclonal antibodies blocking IFN-α, -β confirmed that the observed antiviral activity is mainly mediated by IFNs. Table 1 . Antiviral activity in transfected porcine cells ³

 Antiviral activity

. . . Antibody Antibody Antibody _{Monoclonal Supernatant} , _untreated monoclonal clonal _mon0

 anti-IFN-a anti-IFN-β anti-IFN-α + β

VFA 3 ^' NCR 27 <2 6 <2

 VFA S 13 3.8

 VFA IRES 9

 Poly l: C or tRNA <2 NA NA NA

a SK-6 cells were transfected for 24 h with 40 μ9 / ηιΙ of the ncRNAs, or 10 μ9 / ηιΙ of poly l: C or tRNA. The antiviral activity is expressed as the reciprocal of the highest dilution of the supernatants of transfected SK-6 cells necessary to reduce the number of VEV plaques on IBRS-2 cells by 50%. The data is the average of duplicates of three independent transfection experiments. In some cases, the supernatants were previously incubated for 1 h at 37 ° C with 1 μ9 / ηπΙ of neutralizing monoclonal antibodies against IFN-α, -β or both, respectively. -, undetermined. NA, not applicable. On the other hand, the antiviral activity induced on the SK-6 cells transfected with the different RNAs was studied, infecting them with VEV at an MOI of 1. At 12 and 24 h post infection, supernatants were collected and the viral titer was determined by plating on IBRS-2 cells (in Fig. 4 the results are shown at 12 and 24 hours post infection). In the cells treated with the RNAs the viral yield had been reduced by 3 log compared to that observed in the transfected control cells (with tRNA- or with PBS, "mock"). At 12 h post infection the VEV titers showed differences of 5.5 times (although not significant) between the cells treated with the 3 ^' NCR element compared to the others, while at 24 hours the viral titres were more similar.

These results suggest that all the NCR RNAs elements used, even inducing the transcription of IFN-β at different levels, are capable of to reach the threshold level of induction necessary to trigger an effective anti-viral response, both autocrine and paracrine, in transfected porcine cells. EXAMPLE 4: Inoculation of the "PA P" elements of the VFA genome induces an innate immune response in lactating mice

To determine whether the "PAMP" elements of the VFA genome were capable of inducing an antiviral response in vivo, the transcripts corresponding to the S, IRES and 3 ^' NCR regions of the VFA were inoculated intraperitoneally to litters of Swiss mice of 5- 7 days old As a control, poly l: C was inoculated, a synthetic compound for which an effect on the induction of immune response had already been described. IFN-α and IFN-β levels in serum samples collected at 4, 8, 24 and 48 hours post-inoculation for each ncRNA group were analyzed by ELISA (Fig. 5 A and 5 B). All the elements tested proved to be potent inducers of IFN-α / β in lactating mice, even those that in pig cells in culture had induced low levels of IFN-β mRNA expression.

Serum IFN-α peaks were detected at 8 h after inoculation of the 3'NCR and IRES elements, while for S the IFN-α levels remained high for longer, with a maximum at 24 h post-inoculation (Fig. 5 A). Comparing the IFN-α levels at 8 h post-inoculation, the RNA elements of the VFA genome induced between 15 and 40 times more than the poly l: C, while at 24 h post-inoculation, the levels induced by S were 90 times higher than those induced by poly l: C. This pattern was repeated in the kinetics of IFN-β (Fig. 5 B).

The biological relevance of these IFN-α / β levels detected by serum ELISA was checked by a VEV infectivity inhibition assay. in L-929 cells (Table 2), observing a good correlation between the two trials, both in levels and kinetics.

Table 2. Antiviral activity in the serum of lactating mice.

transfected ⁹

 h post-inoculation

 RNA 4 8 24 48

 S 2 128 162 <2

 IRES 64 128 8 <2

3 ^' NCR <2 32 <2 NR

 Poly l: C 8 8 <2 <2 a Groups of 4-5 mice were inoculated with 100 μ9 of VFA ncRNAs or poly l: C. The sera were collected and mixed, at the indicated post-inoculation times, for each RNA. Antiviral activity is expressed as the reciprocal of the highest dilution of serum capable of suppressing the cytopathic effect induced by VEV in L-929 cells in 50% of the wells. No antiviral activity was detected in serum pools collected at 0 h post-inoculation (<2). NR, not done. Together these results indicate that RNA transcripts corresponding to the structural elements present in the NCRs of the 3 'and 5' ends of the VFA genome can stimulate an innate immune response and induce an antiviral state in vivo. EXAMPLE 5: Survival against the challenge with VFA or with WNV.

In order to determine which of the VFA ncRNAs is the one that most induces protection against viral challenge with VFA or with WNV, protection trials were carried out in lactating mice based on intraperitoneal (ip), and intracranial (ic) and ip inoculation in the case of WNV, of the ncRNAs and subsequent infection with different infective doses of the viruses (VFA or WNV) that cause the death of the mice at previously established doses and times. The protective effect of inoculation with ncRNAs is established by comparison with the control group of animals inoculated with buffer saline phosphate (PBS). In the case of WNV, control groups were also included that were inoculated with PBS and challenged with the same amounts of virus that had been previously incubated (1: 1 dilution) for 1 hour at room temperature with a mixture of sera from mice surviving at infection whose protective capacity against infection with WNV was known (Alonso-Padilla et al. 201 1 Vaccine 29: 1830-1835), and untreated groups that were inoculated ic with PBS as a control of good manipulation.

5.1. Protective effect of VFA ncRNAs against infection with VFA Survival results against the different dilutions tested are shown in Table 3. Briefly, the protective effect of different ncRNAs compared to that of poly l: C was gradual. The effect of 3 ^' NCR and poly l: C was similar. From 8 x 10 ³ pfu (dilution 10 ^{~ 3} ) onwards the poly l: C was better; below that dose (10 ^"4 onwards) were equivalent in protection. Inoculation of RNA S induced a greater protective effect, with 50% of the animals alive until day 1 1 post infection (pi). of dilution 10 ^{" 2} , same as poly l: C. However, at higher doses of the virus, RNA S protected 50% of the animals even against undiluted virus preparation (8 x 10 ⁶ pfu), while the poly l: C stopped protecting the 10 ^{~ 1} dilution . The best protection results were obtained by inoculating the IRES, with total protection (100%) against VFA up to 10 ^"2 dilution and 90% at higher doses until undiluted virus, indicating that even with doses of virus much higher than physiological levels, the VFA is only able to kill 10% of the animals inoculated with this RNA.No animal of the control group survived from day 1 pi with the 10 ^{~ 2} dilution. The observed "plateau" effect is interesting for RNAs with greater protection capacity (S and IRES) With these RNAs it seems to be an antiviral barrier that the infectivity of the virus is unable to overcome, around 50% for S and 10% for IRES.

The LD ₅₀ values (lethal dose-50 / ml) shown in Table 3 correspond to the dilution of the viral preparation used necessary for Kill 50% of the animals inoculated in each case. For S and IRES, since there is no dilution at which the virus kills a percentage of animals> 50%, it is only indicated that it is <10 °.

Table 3. Survival of lactating mice inoculated with the ncRNAs at 11 days post-infection with the VFA.

 RNA Dilution Percentage of

virus inoculated survivors / inoculated survival LD ₅₀ / ml

0 (5/10) 50

-1 (4/8) 50

-2 (7/12) 58

<10 ^L

-3 (10/1 1) 90

-4 (12/12) 100

-5 (1 1/1 1) 100

0 (9/10) 90

-1 (7/8) 87.5

-2 (10/10) 100

 IRES <10 °

 -3 (10/10) 100

-4 (10/10) 100

-5 (9/9) 100

-1 (0/8) 0

 -2 (1/10) 10

3'NCR -3 (6/1 1) 54 9x10 ^"

 -4 (5/5) 100

-5 (4/4) 100

0 (1/10) 10

-1 (0/10) 0

 -2 (7/1 1) 64 -2

Poly l: C 7x10

 -3 (7/8) 87.5

-4 (12/12) 100

-5 (8/8) 100

-2 (0/9) 0

 -3 (1/24) 4

PBS 5x10 ^"

 -4 (7/25) 28

-5 (18/27) 67 I -6 (9/9) 100

PBS + LP * | -4 (0/6) 0

* Lipofectin; LD, lethal dose. -1, dilution 10 ^{~ 1} . -2, dilution 10 ^{~ 2} . -3, dilution 10 ^{~ 3} . - 4, dilution 10 ^"4. -5, dilution 10 ^{" 5} .

5.2. Dose effect

An inoculation experiment was carried out with doses lower than the one commonly used (100 μ9) with the ncRNA that showed a greater protective capacity, the IRES using a high dose of virus. The animals were inoculated intraperitoneally, as described above, with the RNA and 24 h later with the 10 ^"2 dilution, equivalent to 8 x 10 ⁴ pfu (plaque forming units) of VFA. The results are shown in Table 4. Indeed, a dose effect on protection was observed, with a very slight decrease (90%) with 50 μ9 of RNA and greater, 10% with 1 μ9. This result confirms the specificity of the protection observed with ncRNAs and the suitability of the dose of 100 μ9 used in the trials.

Table 4. Survival of inoculated lactating mice with different doses of

IRES of VFA on day 1 1 post-infection with VFA

5.3. Protection kinetics

To assess the time window that covers the protection mediated by ncRNAs with respect to the time of infection, 10 ^{" 2} and 10 ^{~ 4} dilutions of VFA were inoculated in infant baits that had previously been inoculated with the IRES (intraperitoneally, 100 μ9) at different times after RNA inoculation The results are shown in Table 5. Total protection against infection with 10 ^{~ 4} dilution of foot-and-mouth disease virus was maintained at all times tested from minutes after inoculation until 72 h later. For the 10 ^{~ 2} dilution the protection was complete after 8 hours of inoculation with the IRES and 60% after 4 hours. However, almost immediately to inoculation (5-10 min, corresponding to time 0) the protection levels were 100%. On the other hand, 48 hours after RNA inoculation, protection against 10 ^"2 dilution was 50% and at 72 hours the animals were totally susceptible to infection.

Table 5. Number of survivors per day 10 post-infection / number of inoculated

5.4. Lipofectin Effect

The effect of lipofectin as a carrier agent on the protective capacity of ncRNAs was analyzed. The level of protection induced by the IRES with and without lipofectin against the 10 ^{~ 2} and 10 ^{~ 4} dilutions of the VFA was studied (Table 6). The protective effect of IRES was reduced by 30% when inoculated without lipofectin against 10 ^"2 dilution and 10% against 10 ^{~ 4} (Tables 1 and 4). No animal inoculated with PBS + lipofectin survived infection with 10 ^{~ 2} dilution of the VFA These results indicate that lipofectin per se does not induce protection although it does enhance the protective effect of ncRNAs probably increasing its stability and / or access to target cells. Table 6. Survival after infection with VFA at 24 h post-inoculation of IRES of VFA with / without lipofectin (LP)

5.5. Protective effect against infection with West Nile virus

Survival tests against WNV confirm those obtained against VFA, so that greater protection was obtained with the IRES region of VFA than with the S region of VFA, which was also greater than that obtained with control RNA (poly l: C).

Survival results 15 days post infection against the different dilutions tested are shown in Table 7. Briefly, the protective effect of the different ncRNAs (IRES and S) inoculated 24 hours before viral infection was equal to or better than that of the virus. control (poly l: C) .in all the infection conditions tested (10 pfu / mouse via ic, and 100 pfu / mouse both via ic and ip). The IRES conferred a very high protection, which became complete (100%, 100% and 46.2%). On the other hand, the survival percentages of the S region were also very notable (100%, 96% and 12.5%) compared to those obtained with poly l: C (100%, 33.3% and 0%). The survivals obtained with the ncRNAs of the invention were similar to those obtained when the virus was neutralized with murine sera, whose protective capacity was known, prior to inoculation (100%, 100% and 77%). Survival rates of the control groups inoculated with PBS were 12.5%, 40% and 0%, respectively. Table 7. Survival of lactating mice inoculated with ncRNAs at 15 days post intracranial (ic) or intraperitoneal (ip) infection with WNV.

 Ns, not meaningful

EXAMPLE 6: Inoculation of ncRNAs in adult mice significantly increases survival against infection with WNV

Groups of Swiss mice were inoculated intraperitoneally (ip) with PBS (n = 48), polyl: C (n = 12), or 200? Of RNA S (n = 24) or IRES (n = 24). The RNA inoculum was emulsified with 40 μ9 of lipofectin in PBS in a final volume of 200 μΙ. 24 hours later the mice were infected via ip with 10 ⁵ pfu of WNV. Survival was monitored daily until day 15 post-infection. The results are shown in Figure 6, and in Table 8.

Table 8. Survival of mice against infection with WNV after inoculation with ncRNAs.

While the final survival in the group of animals inoculated with PBS was 27%, the percentages were significantly higher in the groups of mice inoculated with RNA. The highest level of survival (83.4%) was observed in the group of animals inoculated with RNA S. These results support the antiviral ability of ncRNAs against WNV in adult mice, as previously observed with lactating mice.

EXAMPLE 7. Detection of IFN-α in sera of adult mice inoculated with ncRNAs

Groups of 4 Swiss adult mice with 200 μg of RNA (IRES, S or polyl: C) were inoculated with lipofectin via ip, as described above. After 4, 8 or 24 h after inoculation, serum samples were taken, grouping the pools corresponding to the 4 animals in each group. Using ELISA (PBL InterferonSource kit), IFN-α levels were determined in the sera pools of each group at the 3 times analyzed. The results are shown in Figure 7. An increase in IFN-α levels was observed at 8 h post-inoculation in the case of the groups inoculated with the S or IRES ncRNAs. The RNA that induced higher levels of IFN-α was IRES.

EXAMPLE 8. Testing of ncRNAs as vaccine adjuvants against VFA

The vaccine consisted of a chemically inactivated virus by treatment with BEI ("binary ethylenimine"), as described in (Bahnemann HG. 1975. Arch Virol 47: 47-56), in a dose equivalent to 2x10 ⁵ pfu of VFA (isolated C-S8c1) The inactivated virus was emulsified in a 1: 1 ratio with Montanide ISA50, from Seppic. Ip was inoculated as previously described (Borrego B et al. 2006. Vaccine 24: 3889-3899). The specific immune response against VFA of four groups of animals was compared:

 a) Inoculated only with the vaccine (n = 3)

 b) Co-inoculated with the vaccine and with 200 μg of IRES RNA + lipofectin.

 The inoculations were performed i.p. at different points of the abdominal cavity, right or left as RNA or "vaccine virus" is inoculated, approximately 2 cm apart (n = 5).

 c) Inoculated with 200 μg of IRES RNA + lipofectin and 24 h later inoculated with the vaccine (n = 5).

 d) Inoculated with the vaccine and 24 h later inoculated with 200 μg of IRES RNA + lipofectin (n = 5).

8.1. IRES inoculation increases the total specific antibody titer against VFA induced after vaccination

Total antibody titers against VFA in the different animals of each group were determined by ELISA of the corresponding sera taken at days 5 and 12 or 3 and 8 weeks post-vaccination, respectively, obtained by maxillary vein bleeding, inactivated and maintained. at -20 ° C until use. The ELISA used follows a C-S8c1 virus capture protocol without purification with a serotype-C rabbit anti-VFA serum (supplied by the Institute for Animal Health, Pirbright, UK), a commercial anti-mouse-HRP serum ( BioRad) and TMB (Sigma) as a substrate. The reaction was stopped with 3N sulfuric acid after 10 min incubation at room temperature, and absorbance reading was carried out at 450 nm. All incubations were performed at 37 ° C for 1 h, and between each step three washes with 0.05% PBS-Tween 20. The sera were tested (individually) in serial dilutions and the titer was defined as the Iog10 of the last dilution whose optical density reading (OD) was greater than 2 times the value of a negative (pre-immune) serum at the minimum dilution rehearsed (1/20). The figure represents the means by group.

The results are shown in Figure 8. In the three groups inoculated with RNA the antibody level was higher than the group to which the vaccine was administered exclusively. At long post-immunization times, the group that simultaneously co-administered vaccine and RNA was the one that induced the highest titer of specific antibodies against the virus.

8.2. IRES inoculation increases the titer and duration of neutralizing antibodies against VFA induced after vaccination

Serum samples from animals of the different groups, taken at different days and weeks post-vaccination and processed in the same way as in the previous section, were analyzed in a plaque number reduction test that reveals the presence of neutralizing antibodies against to the virus and allows to title them (Mateu MG et al. 1987. Virus Res 8: 261-274). IBRS-2 cells were used and neutralization titers were expressed as PRN70, the reciprocal of the maximum serum dilution (log 10) that caused a 70% reduction in plaque number. The results are shown in Figure 9. The graph represents the average values of each group, excluding negative animals. The highest level of neutralizing antibodies was observed, at all times analyzed, in the group to which the vaccine and RNA were co-inoculated. It is worth noting that after 8 weeks after vaccination, none of the animals inoculated only with the vaccine maintained levels detectable antibodies neutralizing infectivity, while the other three groups, in which the IRES RNA had been administered, a high percentage of animals (see table 9) still maintained high levels. Table 9. Number of animals that have detectable levels of neutralizing antibodies against VFA, after inoculation with IRES RNA at different times with respect to the vaccine.

8.3. IRES inoculation increases the diversity of antibody isotypes against VFA induced after vaccination

The isotype diversity of the induced antibodies was determined, after 9 weeks after vaccination, in the animals corresponding to the group of only vaccinated and that of co-inoculated with vaccine + RNA. For this, the sera were analyzed in two different dilutions (chosen according to the ELISA results described above) by means of a capture ELISA of unpurified C-S8c1 virus equivalent to that described above, although in this case the capture was performed with a serum from a pig experimentally infected with C-serotype VFA (CISA-INIA). Isotypes were determined using a mouse immunoglobulin isotyping panel (BioRad) as primary antibodies, followed by incubation with commercial HRP-conjugated rabbit anti-rabbit serum from BioRad (Borrego B et al. 2006. Vaccine 24: 3889- 3899). Figure 10 shows the results in the form of OD corresponding to the 1/60 dilution of the serum of two of the three animals that only received vaccine and of the five that were co-inoculated with vaccine and RNA. The comparison with negative and positive sera is shown. As can be seen in the figure, the immunoglobulin profile in the animals of the coinoculated group was much more diverse, similar to that observed for a “ροο of sera from experimentally infected mice (positive control). In particular, an increase in lgG1 was observed. lgG2A, appearance of lgG2B and in an animal, also of lgG3.

MATERIAL AND METHODS USED Cells and viruses.

The IBRS-2, SK-6 pig kidney cell lines (monkey kidney epithelial cells) of the Animal Health Research Center (CISA-INIA), Valdeolmos, Spain, and murine L-cells have been used 929, from the laboratory of Dr. Alcamí, from the Severo Ochoa Molecular Biology Center, Madrid, Spain. The cells were grown in DMEM supplemented with 10% fetal bovine serum, penicillin / streptomycin and glutamine.

The viruses used in the infection experiments were: the foot-and-mouth disease virus O1 K, the vesicular stomatitis virus Indiana and the West Nile virus strain NY-99.

Preparation of RNAs and transfections. The RNA transcripts corresponding to the S, 3 ^' NCR fragment and its derivatives SL1 (3 ^' NCRASL2), SL2 (3 ^' NCRASL1) and ΔΑη (with the poly A tail removed) of the VFA 01 K genome (the genome sequence of the Isolated 01 K is considered collected in Genbank accession numbers D10138 and X00871) were generated by in vitro transcription with T3 RNA polymerase from plasmids previously described and linearized with Not I (Serrano PM et al. 2006. J Gen Virol 87: 3013-3022). The RNA corresponding to the VFA IRES of the C-S8c1 isolate was obtained from a clone derived from pGEM (Ramos R et al. 1999. RNA 5: 1374-1783) assigned by E. Martínez-Salas, after linearizing with Xho \ and transcribing in vitro with T7 RNA polymerase (NEB). The RNA corresponding to the 5 ^' NCR, including the S fragment, the IRES and 212 nucleotides of the Lpro coding sequence was synthesized with the SP6 RNA polymerase, using the pO1 K clone as a template (Rodríguez-Pulido MF et al. 2009. 83 (8): 3475-3485) linearized with Acc \. Unless otherwise indicated, all transcripts contain a triphosphate group at the 5 'end and the 3'NCR elements of the VFA and their derivatives carry a 58 nucleotide poly A tail.

After transcription, the RNAs were treated with DNase RQ1 (1 U ^ g, Promega), extracted with phenol / chloroform and precipitated with ethanol. Finally they were resuspended in water and quantified by spectrophotometry. The integrity and size of the RNA were analyzed by electrophoresis in denaturing gels 6% acrylamide, 7 M urea or agarose gels.

The removal of the 5 'end phosphate groups in the 3 ^' NCR VFA transcripts was performed by incubating for 90 minutes with 0.5 U ^ g alkaline phosphatase (CIP, New England Biolabs). After this treatment, the RNA was extracted twice with phenol / chloroform, precipitated with ethanol and treated as indicated above. The integrity of these transcripts was analyzed by electrophoresis in denaturing gels 6% acrylamide, 7 M urea. This In this manner, the ncRNAS of the invention were generated SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3.

Before being transfected, the RNAs were heated for 5 minutes at 92 ° C, allowed to cool to room temperature for 10 minutes and kept on ice.

SK-6 cells were transfected using 20 μg / ml of each of the ncRNAs with Lipofectin (Invitrogen) or with 10 μg / ml of poly l: C (Sigma) using Lipofectamine (Invitrogen).

Analysis of IFN-β expression.

At different post-transfection times, the cells were lysed and the total RNA was extracted and quantified using methods known to any person skilled in the art. Retro-transcription (RT) was performed using 500 ng of total RNA and the enzyme Transcriptor RT (Roche). The quantitative polymerase chain reaction (PCR) was performed from aliquots of the back transcription reactions (RT) (1/10) and the reagents of the "LightCycler FastStart DNA Master SYBR green I" kit (Roche). reactions were performed in triplicate. The data were analyzed using the AACt method known to any person skilled in the art. In all assays the expression of the IFN-β gene was normalized with respect to the constitutive expression gene GADPH, and was expressed as an increase with respect to the transfected control cells.

The oligonucleotides used to amplify the mRNA of porcine IFN-β were the sequences referred to in SEQ ID NO: 4 (sense primer) and SEQ ID NO: 5 (antisense primer). For GAPDH, previously described oligonucleotides were used (García-Briones MM et al. 2004. Virology 322: 264-75).

IFN bioassay. The antiviral activity of the supernatants of the transfected SK-6 cells was determined by an inhibition assay of VEV infection on IBRS-2 cells. Briefly, SK-6 cells were transfected for 24 hours with 40 μ9 / ίηΙ of the NCR transcripts, or with 10 μ9 of poly l: C or tRNA (negative control since the transfer RNA is an endogenous RNA and therefore its Transfection should not induce the innate response or the induction of INF). IBRS-2 cells were incubated for 24 hours with different dilutions of transfection supernatants, washed and infected with 100 pfu of VEV. Plates were counted at 24 hours post-infection. In some cases, transfection supernatants were previously incubated for 1 hour at 37 ° C with 1 μg of porcine anti-IFN-α-specific neutralizing monoclonal antibodies (K9, from PBL InterferonSource) or anti-IFN-β previously described (Overend CR et al. 2007. J Gen Virol 88: 925-31). No inhibitory effect on VEV infectivity was observed for amounts of monoclonal antibody up to 10 μg. The antiviral activity was expressed as the highest dilution of supernatant required to reduce the number of plaques by 50%.

Mouse experiments. Baits of Swiss mice (3-7 days old) were inoculated intraperitoneally with 100 μΙ containing 100 μg of the 3 ^' NCR VFA transcripts (SEQ ID NO: 1), S (SEQ ID NO: 2), IRES (SEQ ID NO: 3) or poly l: C, in PBS buffer. For RNA transcripts, 20 μg of lipofectin (Invitrogen) was added. At 0, 4, 8, 24 and 48 hours post-inoculation serum samples were collected, and samples of 4-5 animals for each RNA were pooled and kept at -80 ° C until use. The amount of IFN-α and IFN-β in serum was determined using ELISA kits (PBL InterferonSource). In order to analyze the antiviral activity in these serum samples, a cytopathic inhibition assay was performed (Rubinstein SPC et al. 1981. J Virol 37: 755-758). Briefly, monolayers of L-929 cells seeded in M-96 multiwell plates were incubated for 24 hours with serial dilutions of the corresponding sera. Subsequently the medium was removed and replaced by fresh medium containing 100 infective doses-50 (TCID50) of VEV. At 72 hours the cytopathic effect was monitored by observation under a microscope. The antiviral activity was expressed as the reciprocal of the highest dilution of serum that protects against cytotoxicity in 50% of the wells.

The management of animals in this study has always followed the guidelines of the European Community 86/609 / EEC. The protocols were approved by the INIA Animal Experimentation Ethics Committee (Permits Number CBS 2008/016 for the VFA and 201 1/023 for the WNV).

Survival trials Groups of Swiss lactating mice approximately one week old were inoculated intraperitoneally (ip) with each RNA (ncRNA) that were subsequently inoculated with different virus dilutions: VFA (a bait of 8-12 animals per dilution) 24 h after by the same route, or WNV (a bait of 9-17 animals per dilution) 24 hours later either via ip or intracranial route (ic) (both routes of administration were used in different baits). The inoculum had a final volume of 100 μΙ and contained 100 μg of the RNA to be tested diluted in PBS and 20 μg of Lipofectin (Invitrogen). Briefly, RNAs synthesized by in vitro transcription were heated at 92 ° C for 5 min in water. The PBS was then added and allowed to renaturate at room temperature for 10 min. The lipofectin was then added and after 15 min incubation at room temperature the mixture was injected. Lipofectin is a commercial cationic liposome preparation for routine use in single-stranded RNA transfections. The methodology of inoculation of VFA RNAs in mice and pigs has been previously developed. The preparation of the inoculum of poly l: C is also performed with 100 μg of the RNA diluted in PBS but without liposomes since its ability to induce IFN in adult mice after intraperitoneal inoculation as naked RNA has been described. Infection with the virus was carried out 24 hours after inoculation with the RNAs for giving the best protection results in previous trials with the 3 ^' NCR inoculated at 4, 8 and 24 h pre-infection. The inoculum volume of VFA was 100 μΙ diluted in PBS. Be tested the dilutions between 10 ° (undiluted) and 10 ^{~ 5} of an infection supernatant in cell culture with a titer of 8x10 ⁷ plaque forming units (pfu) / ml. In the case of the challenge with WNV, the inoculation volume was 10 or 100 μΙ, depending on whether the inoculation was ic or ip, diluted in PBS. The challenge was performed with infection supernatant in WNV cell culture strain NY-99 (10 or 100 pfu / mouse via ic, and with 100 pfu / mouse via ip) 24 h after RNAs inoculation.

In the challenge test against WNV, in addition to the control group inoculated with PBS and challenged with virus (as is the case with VFA), control groups were also included that were inoculated with PBS and challenged with the same amounts of virus that had previously been incubated (1: 1 dilution) for 1 hour at room temperature with immune sera from mice whose protective capacity against WNV infection was known, and untreated groups that were inoculated ic with PBS as a good handling control.

Claims

one . Use of a non-coding region of the foot-and-mouth disease virus genome for the preparation of a pharmaceutical composition

2. Use of a non-coding region of the foot-and-mouth disease virus genome for the development of a pharmaceutical composition for the treatment of diseases caused by interferon-sensitive viruses.

3. Use according to any of claims 1 or 2 wherein said non-coding region is comprised in the nucleotide sequence selected from the list comprising: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or any of their combinations.

4. Use according to claim 3 wherein the sequence is SEQ ID NO: 1.

5. Use according to claim 3 wherein the sequence is SEQ ID NO: 2.

6. Use according to claim 3 wherein the sequence is SEQ ID NO: 3.

7. Use according to any of claims 3 to 6 wherein the non-coding region refers to a region with a homology of at least 80% with said region.

8. Use according to any of claims 2 to 7 wherein the interferon sensitive virus is a single stranded RNA virus.

9. Use according to claim 8 wherein the virus is a virus of the Picornaviridae family.

10. Use according to claim 9 wherein the virus is the foot and mouth disease virus.

eleven . Use according to claim 8 wherein the virus is a virus of the family Rhabdoviridae.

12. Use according to claim 1, wherein the virus is vesicular stomatitis virus.

13. Use according to claim 8 wherein the virus is a virus of the Flaviviridae family.

14. Use according to claim 13 wherein the virus is West Nile virus.

15. Use according to any of claims 2 to 14 wherein said diseases are diseases in non-human mammalian animals.

16. Use according to claim 15 wherein the animals belong to the Suidae family.

17. Use according to claim 15 wherein the animals belong to the Bovidae family.

18. Use according to claim 15 wherein the animals belong to the Equidae family.

19. Use according to any of claims 13 to 14 wherein said diseases are diseases in birds.

20. Use according to claim 19 wherein the birds are selected from the families of the list comprising: Phasianidae, Corvidae, Paseridae, Fringillidae, Turdidae, Accipitridae and Strigidae.

twenty-one . Use according to any of claims 2 to 14 wherein said diseases are diseases in humans.

22. Pharmaceutical composition comprising the non-coding region according to any one of claims 1 to 21 which further comprises at least one pharmaceutically acceptable excipient and / or carrier.

23. Pharmaceutical composition according to claim 22 further comprising at least one other active ingredient.

24. Pharmaceutical composition according to any of claims 22 or 23, wherein said pharmaceutical composition is presented in a form adapted to oral, parenteral or intradermal administration.

25. Use of the pharmaceutical composition according to any of claims 22 to 24 for the preparation of a medicament for the treatment of diseases caused by interferon-sensitive viruses.