WO2012131134A1 - Utilisation d'une région non codante du génome du virus de la fièvre aphteuse en vue de l'élaboration d'un médicament antiviral - Google Patents

Utilisation d'une région non codante du génome du virus de la fièvre aphteuse en vue de l'élaboration d'un médicament antiviral Download PDF

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WO2012131134A1
WO2012131134A1 PCT/ES2012/070198 ES2012070198W WO2012131134A1 WO 2012131134 A1 WO2012131134 A1 WO 2012131134A1 ES 2012070198 W ES2012070198 W ES 2012070198W WO 2012131134 A1 WO2012131134 A1 WO 2012131134A1
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virus
use according
vfa
rna
ifn
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PCT/ES2012/070198
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Margarita SÁIZ ZALABARDO
Francisco SOBRINO CASTELLÓ
Belén BORREGO RIVERO
Miguel Ramón RODRÍGUEZ PULIDO
Juan Carlos SÁIZ CALAHORRA
Miguel Ángel MARTÍN ACEBES
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Consejo Superior De Investigaciones Científicas (Csic)
Fundació Centre De Recerca En Sanitat Animal (Cresa)
Instituto Nacional De Investigación Y Tecnologían Agraria Y Alimentaria (Inia)
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Publication of WO2012131134A1 publication Critical patent/WO2012131134A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/17—Immunomodulatory nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/18—Type of nucleic acid acting by a non-sequence specific mechanism

Definitions

  • 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.
  • the innate immune response is the first line of defense against pathogen invasion based on various mechanisms and signaling pathways.
  • one of the mechanisms of the innate immune response is the production of type I interferon (IFN) (IFN- ⁇ and IFN- ⁇ , or also called IFN- ⁇ / ⁇ ) that has an antiviral effect. , antiproliferative and has immunomodulatory activity.
  • IFN type I interferon
  • 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.
  • 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 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.
  • NCRs non-coding regions
  • VFA NCRs contain specific structures that participate in the control of replication and translation of the viral genome.
  • 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).
  • 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- ⁇ and the expression of genes that stimulate IFN- ⁇ / ⁇ . This fact represents a problem for the control of FMD disease.
  • mRNA messenger RNA
  • 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.
  • VSV vesicular stomatitis virus
  • sand flies Lutzomya sp.
  • Black flies family Simuliidae
  • 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.
  • the infection induces the general blockage of gene expression in the host cell, which affects the production of IFN and other antiviral molecules.
  • 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.
  • WNV West Nile virus
  • WNV West Ni le Virus
  • WNV West Ni le Virus
  • 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.
  • 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 (Granburg BP et al. 2004. Lancet Infec ⁇ Dis 4: 547-556).
  • the number and severity of cases in humans and horses have increased considerably.
  • 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.
  • 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.
  • 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.
  • ncRNAs used in the invention in vitro and in vivo studies were performed in which the production of messenger RNA (mRNA) of IFN- ⁇ or IFN- ⁇ and antiviral response was detected.
  • survival trials were also conducted against viral challenge in a model of a nursing mouse.
  • 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.
  • IFN mRNA induction levels correlate with antiviral activity against vesicular stomatitis virus (VEV).
  • VVE vesicular stomatitis virus
  • 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.
  • 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.
  • ncRNAs of the 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.
  • 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.
  • non-coding region 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.
  • 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.
  • pharmaceutical composition 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.
  • pharmaceutical composition and medicament are used interchangeably in that invention.
  • 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.
  • antiviral refers to any substance that does not allow the replication, assembly or release of viruses, such as interferon, ribavirin, etc.
  • ncRNAs of the invention have proven capable of inducing antiviral activity against INF-sensitive viruses, such as VFA, VEV, and WNV.
  • 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- ⁇ or IFN- ⁇ .
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • restriction target 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).
  • restriction enzyme 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.
  • sequences recognized by the restriction enzymes Stu I and EcoRV are sequence recognized by the restriction enzymes Stu I and EcoRV.
  • the ncRNA of the 3'NCR region of the VFA 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.
  • the ncRNA of the S region of the VFA 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.
  • 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.
  • 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.
  • region In the present invention the term "region”, “sequence” or “nucleotide sequence” is used interchangeably in memory.
  • 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.
  • 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.
  • 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.
  • 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.
  • the interferon-sensitive virus is a single-stranded RNA virus.
  • the virus is a virus of the Picornaviridae family, more preferably the foot and mouth disease virus.
  • the virus is a virus of the family Rhabdoviridae, more preferably it refers to the vesicular stomatitis virus.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • the presentation form is liquid, the vehicle is the diluent.
  • pharmaceutically 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.
  • a preferred embodiment of the third aspect of the invention relates to a pharmaceutical composition that further comprises at least one other active ingredient.
  • 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.
  • 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
  • ⁇ transcribed 3'NCR to which the poly A tail has been removed.
  • Fig. 2 Induction of IFN- ⁇ in porcine cells. It shows the production of IFN- ⁇ by the ncRNAs of the invention of VFA in porcine cells.
  • SK-6 cells were transfected with 20 ⁇ 9 / ⁇ of the NCR transcripts or with 10 ⁇ 9 / ⁇ of the poly l: C control or the negative tRNA control (transfer RNA).
  • Induction levels of IFN- ⁇ 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.
  • Fig. 3 Analysis of the contribution of the 3 ' NCR sequences of VFA in the induction of IFN- ⁇ . It shows the production of IFN- ⁇ 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- ⁇ 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- ⁇ porcine IFN- ⁇ messenger RNA
  • T time; h, hours
  • ⁇ 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.
  • MOI multiplicity of infection
  • VFA NCRs induce the innate response in a nursing mouse.
  • mice show the production of IFN- ⁇ by the ncRNAs of the invention in lactating mice.
  • Groups of 4 to 5 Swiss lactating mice were inoculated intraperitonealemnte (ip) with 100 ⁇ g of 3 ' NCR, S, IRES or poly l: C transcripts.
  • the levels of IFN- ⁇ (A) and IFN- ⁇ (B) in pools of serum extracted at different post-inoculation times for each RNA were determined by ELISA.
  • the average values of IFN- ⁇ and IFN- ⁇ for serum pools at 0 h post-inoculation were 25 and 6 pg / ml, respectively.
  • FIG. 6 Effect of inoculation of ncRNAs on the survival of mice inoculated with WNV.
  • FIG. 7 IFN- ⁇ 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
  • FIG. 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 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.
  • VCR genome NCRs contain PAMPs (molecular patterns associated with pathogens) that activate innate response signaling.
  • PAMPs molecular patterns associated with pathogens
  • 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- ⁇ 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.
  • the 3 ' NCR was the most potent IFN- ⁇ transcriptional inducer of all, reaching levels almost 200 times above the transfection control cells at 9 h posttransfection.
  • 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
  • a SK-6 cells were transfected for 24 h with 40 ⁇ 9 / ⁇ of the ncRNAs, or 10 ⁇ 9 / ⁇ 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 ⁇ 9 / ⁇ of neutralizing monoclonal antibodies against IFN- ⁇ , - ⁇ or both, respectively. -, undetermined. NA, not applicable.
  • 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.
  • 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).
  • the viral yield had been reduced by 3 log compared to that observed in the transfected control cells (with tRNA- or with PBS, "mock").
  • 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.
  • 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
  • poly l: C was inoculated, a synthetic compound for which an effect on the induction of immune response had already been described.
  • IFN- ⁇ and IFN- ⁇ 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- ⁇ / ⁇ in lactating mice, even those that in pig cells in culture had induced low levels of IFN- ⁇ mRNA expression.
  • Serum IFN- ⁇ peaks were detected at 8 h after inoculation of the 3'NCR and IRES elements, while for S the IFN- ⁇ levels remained high for longer, with a maximum at 24 h post-inoculation (Fig. 5 A). Comparing the IFN- ⁇ 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- ⁇ (Fig. 5 B).
  • 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.
  • VFA ncRNAs 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).
  • PBS buffer saline phosphate
  • 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.
  • 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.
  • 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 °.
  • 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.
  • EXAMPLE 6 Inoculation of ncRNAs in adult mice significantly increases survival against infection with WNV
  • RNA inoculum was emulsified with 40 ⁇ 9 of lipofectin in PBS in a final volume of 200 ⁇ . 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.
  • RNA RNA
  • S or polyl: C RNA
  • IFN- ⁇ 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- ⁇ 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- ⁇ was IRES.
  • the vaccine consisted of a chemically inactivated virus by treatment with BEI ("binary ethylenimine”), as described in (Bruemann HG. 1975. Arch Virol 47: 47-56), in a dose equivalent to 2x10 5 pfu of VFA (isolated C-S8c1)
  • BEI binary ethylenimine
  • 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:
  • 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.
  • IRES inoculation increases the titer and duration of neutralizing antibodies against VFA induced after vaccination
  • 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.
  • 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.
  • the immunoglobulin profile in the animals of the coinoculated group was much more diverse, similar to that observed for a “ ⁇ 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.
  • 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. Alcam ⁇ , 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.
  • RNA transcripts corresponding to the S, 3 ' NCR fragment and its derivatives SL1 (3 ' NCRASL2), SL2 (3 ' NCRASL1) and ⁇ (with the poly A tail removed) of the VFA 01 K genome 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).
  • 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. Mart ⁇ nez-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 (Rodr ⁇ guez-Pulido MF et al. 2009.
  • transcripts 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.
  • 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.
  • ncRNAS of the invention were generated SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3.
  • RNAs 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 ⁇ g / ml of each of the ncRNAs with Lipofectin (Invitrogen) or with 10 ⁇ g / ml of poly l: C (Sigma) using Lipofectamine (Invitrogen).
  • Retro-transcription was performed using 500 ng of total RNA and the enzyme Transcriptor RT (Roche).
  • PCR quantitative polymerase chain reaction
  • PCR quantitative polymerase chain reaction
  • the oligonucleotides used to amplify the mRNA of porcine IFN- ⁇ were the sequences referred to in SEQ ID NO: 4 (sense primer) and SEQ ID NO: 5 (antisense primer).
  • SEQ ID NO: 4 sense primer
  • SEQ ID NO: 5 antisense primer
  • GAPDH previously described oligonucleotides were used (Garc ⁇ a-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 ⁇ 9 / ⁇ of the NCR transcripts, or with 10 ⁇ 9 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.
  • transfection supernatants were previously incubated for 1 hour at 37 ° C with 1 ⁇ g of porcine anti-IFN- ⁇ -specific neutralizing monoclonal antibodies (K9, from PBL InterferonSource) or anti-IFN- ⁇ 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 ⁇ g. The antiviral activity was expressed as the highest dilution of supernatant required to reduce the number of plaques by 50%.
  • 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.
  • 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 ⁇ and contained 100 ⁇ g of the RNA to be tested diluted in PBS and 20 ⁇ g of Lipofectin (Invitrogen).
  • 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 ⁇ g 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 ⁇ 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.
  • the inoculation volume was 10 or 100 ⁇ , 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.
  • 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.

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Abstract

L'invention concerne l'utilisation d'ARN correspondants à des domaines hautement structurés des régions non codantes du génome du virus de la fièvre aphteuse (VFA) et l'utilisation de séquences qui les comprennent pour l'élaboration d'une composition pharmaceutique de prophylaxie et/ou de traitement de maladies causées par des virus sensibles à l'interféron. L'invention concerne également une composition pharmaceutique qui comprend les régions non codantes de l'invention et qui, en outre, comprend au moins un excipient et/ou un véhicule pharmaceutiquement acceptable, ainsi que l'utilisation de cette composition pharmaceutique.
PCT/ES2012/070198 2011-03-25 2012-03-23 Utilisation d'une région non codante du génome du virus de la fièvre aphteuse en vue de l'élaboration d'un médicament antiviral WO2012131134A1 (fr)

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Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
BIGERIEGO, P. ROSAS ET AL.: "Heterotypic inhibition of foot-and-mouth disease virus infection by combinations of RNA transcripts corresponding to the 5' and 3' regions.", ANTIVIRAL RESEARCH., vol. 44, no. 2, December 1999 (1999-12-01), pages 133 - 141 *
GUTIERREZ, A. ET AL.: "Specific inhibition of aphthovirus infection by RNAs transcribed from both the 5' and the 3' noncoding regions.", JOURNAL OF VIROLOGY., vol. 68, no. 11, November 1994 (1994-11-01), pages 7426 - 7432 *
MEIER, A. ET AL.: "MyD88-dependent immune activation mediated by human immunodeficiency virus type 1-encoded toll-like receptor ligands.", JOURNAL OF VIROLOGY., vol. 81, no. 15, August 2007 (2007-08-01), pages 8180 - 8191 *
RODRIGUEZ-PULIDO, M. ET AL.: "Inoculation of newborn mice with non-coding regions of foot-and-mouth disease virus RNA can induce a rapid, solid and wide-range protection against viral infection.", ANTIVIRAL RESEARCH., vol. 92, no. 3, December 2011 (2011-12-01), pages 500 - 504. *
RODRIGUEZ-PULIDO, M. ET AL.: "structural domains in noncoding regions of the foot-and-mouth disease virus genome trigger innate immunity in porcine cells and mice.", JOURNAL OF VIROLOGY., vol. 85, no. 13, July 2011 (2011-07-01), pages 6492 - 6501. *
ZHANG, Y. ET AL.: "Molecular cloning and functional characterization of porcine toll-like receptor 7 involved in recognition of single-stranded RNA virus/ssRNA.", MOLECULAR IMMUNOLOGY., vol. 45, no. 4, February 2008 (2008-02-01), pages 1184 - 1190, XP022322747, DOI: doi:10.1016/j.molimm.2007.07.014 *
ZHANG, Y.-L. ET AL.: "Hepatitis C virus single-stranded RNA induces innate immunity via Toll-like receptor 7.", JOURNAL OF HEPATOLOGY., vol. 51, no. 1, July 2009 (2009-07-01), pages 29 - 38, XP026209776, DOI: doi:10.1016/j.jhep.2009.03.012 *

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