WO2012107614A1 - Vaccin contre le virus de la peste porcine africaine basé sur des virus recombinants pauvres en réplication - Google Patents

Vaccin contre le virus de la peste porcine africaine basé sur des virus recombinants pauvres en réplication Download PDF

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WO2012107614A1
WO2012107614A1 PCT/ES2012/070058 ES2012070058W WO2012107614A1 WO 2012107614 A1 WO2012107614 A1 WO 2012107614A1 ES 2012070058 W ES2012070058 W ES 2012070058W WO 2012107614 A1 WO2012107614 A1 WO 2012107614A1
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virus
gene
recombinant virus
vaccine
swine fever
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María Luisa SALAS FALGUERAS
Javier María RODRÍGUEZ MARTÍNEZ
Linda Kathleen DIXON
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Consejo Superior de Investigaciones Cientificas CSIC
Pirbright Institute
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Pirbright Institute
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • A61P31/20—Antivirals for DNA 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/12011—Asfarviridae
    • C12N2710/12022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/12011—Asfarviridae
    • C12N2710/12034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/12011—Asfarviridae
    • C12N2710/12051—Methods of production or purification of viral material
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/12011—Asfarviridae
    • C12N2710/12061—Methods of inactivation or attenuation
    • C12N2710/12062—Methods of inactivation or attenuation by genetic engineering
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor

Definitions

  • the present invention relates to a recombinant virus of the virulent strain BA71 of the African swine fever virus, deficient in replication, to the empty viral particles that are produced in isolated cells after infection with said virus and to the use of said material for the development of a vaccine against the African swine fever virus.
  • the African swine fever virus is a double-stranded DNA virus of the Asfarviridae family that causes a very serious hemorrhagic disease in the domestic pig.
  • This disease produces great economic losses in the African continent, where it is endemic, and from where it has spread since 2007 to countries in the Caucasus region and Russia, which poses a danger of re-entry into Western Europe and, likewise, a threat to China and other Asian countries (Rowlands et al. 2008. Emerg Infect Dis 14: 1870-1874).
  • the development of an effective vaccine is, therefore, essential to control the spread of this disease.
  • viruses derived from the attenuated strain BA71 V (adapted to grow in Vero cells) have been produced but no immunological study has been possible with said viruses or with empty particles derived from these, because they are not pathogenic (Zsak et al. 2001. J Virol 75: 3066-3076). Therefore, it is impossible to use viruses derived from strain BA71 V as components of a vaccine against VPPA that produces an effective immune response in vivo.
  • replication-deficient VPPA strains have been produced by conditional expression of certain viral genes required for morphogenesis and genome encapsidation.
  • the viral genes were placed under the control of an IPTG-inducible promoter ( ⁇ sopropyl-BD-1-thiogalactopyranoside), through the introduction into the viral genome of the lac operon repressor / operator system of Escherichia coli (Garc ⁇ a-Escudero et al. 1998. J Virol 72: 3185-3195).
  • IPTG-inducible promoter ⁇ sopropyl-BD-1-thiogalactopyranoside
  • BA71 V viruses have been constructed with repression of pp220 polyproteins (Andres et al. 2002. J Virol 76: 2654-2666), pp62 (Suárez et al. 2010. J Virol 84: 176-187) and of the pB438L protein (Epifano et al. 2006.
  • viruses that do not express genes available for replication but important for virulence and for inhibiting the host's immune response This is the case of viruses that do not express the thymidine kinase (TK) gene generated by the introduction of a repressor + selection cassette in the locus of this gene (Andrés et al. 2002. J Virol 76: 2654-2666) . By introducing the cassette the expression of the TK is eliminated and the recombinants are selected.
  • TK thymidine kinase
  • This system has been used to generate TK- virus through the introduction into the TK locus of the Lac I gene of the Lac repressor, under the control of the viral promoter pU104 and, as a selection marker, the gusA gene ( ⁇ -glucuronidase gene ) under the control of the p72 viral promoter.
  • Viruses have also been created that do not express the homologous protein of the adhesion receptor CD2, a structural protein present in the external domains of the virus and that acts as an immunosuppressant (hinders the immune response of the infected organism), introducing the LacZ gene through low homologous recombination the control of p72, thus breaking the reading frame of the EP402R gene coding for CD2 (Rodr ⁇ guez et al. 1993. J Virol 67: 5312-5320; Ruiz-Gonzalvo et al. 1996. Virology 218: 285-298). Again, in none of these cases immunological studies could be performed in vivo since these viruses deficient in genes available for replication derived from the non-pathogenic strain BA7 V.
  • the tools for generating them were the following: repression of TK expression by the introduction of the repressor + selection cassette composed of the gene / acl under the control of the viral promoter pU104 and the gusA marker gene under the control of the viral promoter p72 and the replacement of the original promoters of the genes of pp220, pp62 and pB438L by the IPTG-inducible promoter p72.l composed of the late promoter p72.4 and the lac operon operator sequence of E. co /; and in addition to the lacZ marker gene for the selection of recombinants.
  • the attenuated strain BA71 V derived from the pathogenic strain BA71 (the strain used in the invention and which is freely accessible to the public), was generated after adaptation to Vero cells, and its genome is sequenced (Yá ⁇ ez et al. 1995. Virology 208: 249-278). The two strains differ at the ends of the DNA, since BA71 V contains two deletions 2.5 and 7 kb in length in said terminal regions (Blasco et al. 1989. Virology 1 68: 330-338). In addition, significant deletions in the left terminal region (LVR) containing genes of the members of the multigenic families MGF360 and MGF530 have been described in strain BA71 V.
  • LVR left terminal region
  • the present invention relates to replication-deficient recombinant viruses derived from the virulent or pathogenic strain BA71, to the empty particles generated in cultures of isolated cells infected by these recombinant viruses under repression conditions and to the use of both for the preparation of a medicine, preferably a vaccine against the African swine fever virus.
  • This invention offers a novel alternative to current treatments against African swine fever by being able to generate an immune response in the animal capable of inducing effective protection against the virus.
  • the replication-deficient recombinant viruses of the present invention derived from the pathogenic strain BA71 possess certain viral genes located under the control of an inducible promoter that allows their conditional expression. Thus, under repression conditions, defective viruses are generated without DNA, but with all the external domains of the viral particle and which can effectively leave the infected cell. Thus, infection with these inducible viruses mimics a normal infection, synthesizing all viral proteins, except those whose expression is repressed in the recombinant virus, and can therefore induce a strong immune response. In this sense, they have a clear advantage over the use as antigens of individual viral proteins or in certain combinations.
  • these inducible recombinant viruses have greater safety, since the viral particles produced are not infective, and there is only one replication cycle.
  • the system of the invention is compatible with the deletion of other viral genes, such as those involved in the control of the immune response by the virus, in the virulence or DNA repair, which would increase the efficiency and safety of the vaccine.
  • strain BA71 can grow and form lysis plaques in COS cells (ATCC, CRL-1650 TM), a cell line that can be transfected very effectively, which is very useful for the construction of recombinant viruses by homologous recombination.
  • the virus produced in these cells retains its virulence, even after the high number of passes required for the construction and purification of the recombinant virus.
  • a vaccine comprising replication-deficient recombinant viruses derived from the pathogenic strain BA71 capable of infecting in vivo and generating an effective immune response, which is impossible in viruses derived from the attenuated strain.
  • BA71 V replication-deficient recombinant viruses derived from the pathogenic strain BA71 capable of infecting in vivo and generating an effective immune response, which is impossible in viruses derived from the attenuated strain.
  • empty viral particles of said viruses have been generated for use in vaccination against virulent isolates of VPPA which, being derived from the pathogenic virus, mimic the viral antigens of the virus and would be more effective than recombinant protein vaccines previously developed, by including many of the structural proteins of the virus.
  • a first aspect of the invention relates to a recombinant virus characterized in that it is obtained from the virulent strain BA71 of the African swine fever virus and because it is deficient in virus replication (hereinafter, "virus of the invention ").
  • African Swine Fever Virus also called in the literature "VPPA”, “ASFV”, “African Swine Fever Virus”
  • VPPA African swine fever virus isolated in Badajoz in 1971 which is highly virulent and whose DNA contains 178 kbp ⁇ Enjuanes et al. 1976. J Gen Virol 32: 471-477).
  • the BA71 strain is open to the public.
  • viruses of the invention have been generated by modifying the viral genome to allow inducible expression of one or more of the genes encoding the pp220, pp62 or pB438L proteins, respectively.
  • a preferred embodiment of the invention refers to the virus of the invention that has repressed the expression of at least one gene that codes for the protein selected from the list comprising polyprotein pp220, polyprotein pp62 and protein pB438L.
  • the polyprotein pp220 in the present invention refers to the polyprotein encoded by strain BA71 which is homologous to pp220 encoded by strain BA71 V or to functional variations thereof.
  • the pp220 of strain BA71 V refers to SEQ ID NO: 1 (accession number NP_042785.1)
  • the term "homologous protein” refers to that protein that has a similar sequence and exerts the same function in the African swine fever virus, regardless of the virus strain. In the present invention, it refers to proteins of strain BA71 that have an amino acid sequence similar to those of strain BA71 V, where similar sequence refers to a sequence with a similarity greater than 95%, which perform the same function in the virus
  • a more preferred embodiment refers to the viruses of the invention where the repressed gene is the one that codes for pp220 polyprotein.
  • Polyprotein pp62 in the present invention refers to the polyprotein encoded by strain BA71 which is homologous to pp62 encoded by strain BA71 V or to functional variations thereof.
  • the pp62 of strain BA71 V refers to SEQ ID NO: 2 (accession number NP_042787).
  • pB438L protein in the present invention refers to the protein encoded by strain BA71 that is homologous to pB438L encoded by strain BA71 V or to functional variations thereof.
  • PB438L of strain BA71 V refers to SEQ ID NO: 3 (accession number NP_042769.1).
  • another more preferred embodiment relates to the viruses of the invention where the repressed gene is the one that codes for the pB438L protein.
  • the inducible expression of the genes encoding the pp220 or pp62 polyproteins or the pB438L protein has been performed by replacing the original promoter of these genes with the inducible promoter p72.l formed by the late viral promoter p72.4 and the operator sequence Or ? of the Lac de E. coli operon.
  • the inducible promoter ⁇ 74 can also be used. That difference of p74.l in the distance from the operating sequence to the beginning of the transduction of the inducible gene (2 and 8 bases, respectively). Both promoters are inducible by IPTG.
  • the present invention also relates to viruses of the invention where genes are induced by other inducers, for example tetracycline, and which contain promoters inducible by them.
  • an even more preferred embodiment of the invention refers to the virus of the invention where said virus comprises a genetic construct comprising an inducible promoter capable of directing said repression.
  • a genetic construct is understood as a nucleotide sequence comprising the coding sequence of the virus of the invention operably linked with at least one promoter that directs the transcription of said sequence, and with other sequences necessary or appropriate to regulate its transcription, by for example, start and end signals, cut sites, polyadenylation signal, origin of replication, transcriptional activators ⁇ enhancers), transcriptional silencers ⁇ silencers), etc.
  • a promoter is a nucleotide sequence that controls the transcription of a given gene.
  • the term "inducible”, as used in this description, refers to the possibility that the promoter has a control element that allows activating or deactivating (repressing) the transcription of the gene that regulates, in the presence of a factor external to the promoter, for example, the addition of a substance to the culture medium in which the organism containing the virus of the invention grows.
  • An IPTG inducible promoter is one that has the necessary elements for transcription to occur only in the presence of this substance.
  • Recombinant viruses also contain at least one selection gene (lacZ that codes for ⁇ -Galactosidase or another selection gene) under the control of the p72 viral promoter.
  • the viruses of the invention further comprise at least one selection gene.
  • selection gene refers to a gene that encodes a protein that confers a characteristic to the organism in which said gene is expressed and which is not found in said organism of In a natural way, such as surviving the presence of an antibiotic, producing a colored substance in the presence of a specific reagent or emitting light.
  • An antibiotic resistance gene codes for a protein that confers on the cell that expresses it, which would normally be sensitive to the antibiotic, the ability to overcome the antibiotic's effect. This protein is usually an enzyme that inactivates the antibiotic in question.
  • an even more preferred embodiment of the invention relates to the virus of the invention, wherein said virus comprises a genetic construct comprising at least one selection gene.
  • viruses of the invention wherein said virus comprises a genetic construct comprising an inducible promoter capable of directing said repression where the promoter is inducible by IPTG.
  • the mechanism to achieve this is based on the constitutive repression either of genes involved in virulence (particularly, the thymidine kinase gene), or of genes that participate in immunosuppression (particularly, the EP402R gene that codes for CD2) or of genes involved in DNA repair (such as the 0174L gene that codes for DNA polymerase X). In all of them it has been possible to use the tools described above for the generation of the replication-deficient viruses derived from BA71 V since it is not described that there are significant differences between this strain and the strain of the invention in these genes available for replication.
  • the repressor + selection cassette that contains (i) the Lac I repressor gene of the E. coli Lac operon under the control of the early / late promoter of the U104L gene (pU104) of the VPPA and (ii) the p72GUS marker, which consists of the ⁇ -glucuronidase ( ⁇ -Gus or gusA) gene under the control of the p72 promoter of the p72 protein gene.
  • the present invention also supports the deletion of the expression of genes available for replication by insertion of a selection gene (eg GFP or EGFP, encoding the green fluorescent protein and the enhanced green fluorescent protein) "or" enhanced green fluorescent protein ", respectively) that interrupts the reading frame of the gene available for replication.
  • a selection gene eg GFP or EGFP, encoding the green fluorescent protein and the enhanced green fluorescent protein
  • enhanced green fluorescent protein or enhanced green fluorescent protein ", respectively
  • TK thymidine kinase
  • strain BA71 that is homologous to thymidine kinase encoded by strain BA71 V or to functional variations thereof.
  • Thymidine kinase of strain BA71 V refers to SEQ ID NO: 4 (accession number NP_042744.1).
  • the CD2 (also called “adhesion receptor homologue CD2" or “hemagglutinin”) in the present invention refers to the protein encoded by the EP402R gene by strain BA71 which is homologous to the CD2 encoded by strain BA71 V or to variations Functional of it.
  • the CD2 of strain BA71 V refers to SEQ ID NO: 5 (accession number NP__042752.1).
  • Polymerase X in the present invention refers to the protein encoded by the 0174L gene by strain BA71 which is homologous to pol X encoded by strain BA71 V or to functional variations Of the same.
  • Pol X of strain BA71 V refers to SEQ ID NO: 6 (accession number NP_042790.1).
  • the genes available for repiration comprise at least one gene that is selected from the list comprising: gene that participates in the virulence of the virus, gene that participates in immunosuppression, and gene that participates in DNA repair.
  • the gene available for repiration is selected from the list comprising: the thymidine kinase gene, the EP402R gene and the 0174L gene, or any combination thereof.
  • a more preferred embodiment relates to the virus of the invention where genes available for repiration are repressed because they have varied reading frames and do not express a viable protein.
  • Also part of the invention are genetic DNA constructs that encode sequences of the recombinant virus of the invention.
  • Said genetic construction of DNA would direct the intracellular transcription of the virus sequence or fragment thereof, and comprises at least one of the following types of sequences: a) DNA sequence comprising at least the coding sequence of the virus the invention for its intracellular transcription, b) an expression cassette comprising the DNA sequence defined in a) operably linked to transcription control elements and optionally translation; c) DNA sequence corresponding to a gene expression system or vector comprising the coding sequence of the virus of the invention, operably linked with at least one promoter that directs the transcription of said sequence, and with other sequences to regulate its transcription such as, for example, start and end signals, cut sites, or polyadenylation signal, where preferably the vector is a plasmid.
  • Another aspect of the invention relates to the host cell comprising the nucleic acid, the expression cassette or the vector encoding sequences of the virus of the invention, where the cell is preferably a mammalian cell (not belonging to the group of cells embryonic or germinal human), of insect, yeast or bacteria.
  • the introduction of said gene constructs can be carried out with the methods known in the state of the art. It also refers to the method of production of the virus of the invention comprising culturing said host cells.
  • Another preferred embodiment relates to the use of the viruses of the invention for the preparation of a pharmaceutical composition.
  • 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 viruses of the invention.
  • a more preferred embodiment relates to the use of the virus of the invention for the preparation of a pharmaceutical composition for the treatment of African swine fever, preferably for the prophylaxis of African swine fever.
  • composition of the invention can be used both alone and in combination with other compositions for the treatment or prevention of African swine fever.
  • 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.
  • An even more preferred embodiment of the invention relates to the use of the virus of the invention for the preparation of a pharmaceutical composition for the prophylaxis of African swine fever where said pharmaceutical composition is a vaccine.
  • the term "vaccine” refers to an antigen preparation used to elicit a response of the immune system to the disease caused by an African swine fever 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.
  • Another aspect of the invention relates to a vaccine against the African swine fever virus comprising a therapeutically effective amount of the virus of the invention.
  • therapeutically effective amount refers to the amount of modulating agents calculated to produce the desired effect and, in general, will be determined, among other causes, by the characteristics of said agents (and constructions) and the therapeutic effect to be achieved.
  • Pharmaceutically acceptable adjuvants and vehicles that can be used in said compositions are the vehicles known to those skilled in the art.
  • the vaccine comprising the virus of the invention further comprises at least one excipient and / or at least one pharmacologically acceptable carrier.
  • excipient refers to a substance that aids in the absorption of the vaccine containing the virus of the invention, stabilizes said vaccine or aids in the preparation of the pharmaceutical composition in the sense of giving it a consistency, form, taste or any other specific functional characteristic.
  • the excipients could have the function of keeping the ingredients together such as starches, sugars or cellulose, sweetening function, dye function, drug protection function such as to isolate it from air and / or moisture, function of filling a tablet, capsule or any other form of presentation such as dibasic calcium phosphate, a disintegrating function to facilitate the dissolution of the components and their absorption in the intestine, without 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.
  • compositions or vaccine provided by this invention can be facilitated by any route of administration, for which said composition will be formulated in the pharmaceutical form appropriate to the route of administration chosen.
  • the vaccine containing the viruses of the invention comprises at least one other active ingredient.
  • the present invention also relates to the use of the vaccine containing the virus of the invention for the manufacture of a pharmaceutical composition for the prophylaxis of mammalian animals, preferably of the Suidae family, for example pigs.
  • the viruses of the invention under repression conditions (that is, in the absence of the promoter inducer under whose control certain viral genes have been placed) the viruses of the invention are capable of infecting the host cell but not producing complete viruses, and the viral particles that leave the cell are empty, since they lack the DNA. Therefore, in an infection in vivo with the viruses of the invention, said empty viral particles naturally occur inside the infected animal as it is a system that lacks the inductor.
  • a preferred embodiment is the process for obtaining empty viral particles (hereafter referred to as "viral particles of the invention") comprising the infection of isolated cells with the recombinant virus of the invention in the absence of the promoter inducing agent which controls the viral genes whose transcription you want to repress.
  • “Infection” in the present invention is understood as that pathology generated by the invasion or colonization of any host tissue by the African swine fever virus. "Infection of isolated cells” is understood to be an in vitro infection in which isolated cells infected by the virus of the invention are deprived of the inducer for the correct generation of empty viral particles.
  • isolated cell is understood herein to be that cell grown in vitro for the production of empty viral particles when they are not cultured with the inducing agent or for the production of the recombinant viruses of the invention when they are cultured with the inducer.
  • the isolated cells are preferably cell lines.
  • An even more preferred embodiment of the invention refers to the empty viral particle generated by infection of the isolated cells with the viruses of the invention, hereinafter "viral particle of the invention”.
  • Another preferred embodiment of the invention relates to the use of the viral particle of the invention that is generated by infection by the virus of the invention for the preparation of a pharmaceutical composition, preferably for the treatment of African swine fever, more preferably for the African swine fever prophylaxis. More preferably said pharmaceutical composition is a vaccine.
  • Said vaccine further comprises a therapeutically effective amount of the viral particles of the invention, and preferably also comprises at least one pharmaceutically acceptable adjuvant, excipient and / or vehicle, where in another even more preferred embodiment the adjuvant is Freund's adjuvant, and that it can also comprise at least one other active ingredient.
  • Another aspect of the invention relates to the use of the vaccine containing the viral particles of the invention against the African swine fever virus for the preparation of a pharmaceutical composition for the prophylaxis of mammalian animals, preferably of the Suidae family, for example pigs
  • polynucleotide and the term “nucleic acid” refer to a polymeric form of nucleotides of any length, both ribonucieotides and deoxyribonucleotides.
  • peptide oligopeptide
  • polypeptide polypeptide
  • polyprotein protein
  • FIG. 1 Virulence assay of strain BA71 grown in COS cells. Virulence of the BA71 strain virus grown in COS cells was tested to verify that its virulence was not affected by its growth in said cell line.
  • A determination of the rectal temperature of pigs infected with different doses (10 4 Dl 50 and 100 Dl 50j being Dl 50 the 50% infectious dose) of BA71 virus before (BA71) and after (BA71 Cos) of 10 proliferation cycles in COS cells.
  • dO day in which the virus was inoculated
  • d1 -10 days post inoculation.
  • B survival test of infected pigs as described in A; "n" is the number of animals in each group.
  • FIG. 1 Construction of the replication-deficient recombinant virus, BA71.v220i.TK-.
  • the BA71 .v220i.TK- virus was constructed that inducibly expresses pp220 polyprotein and is not virulent because it does not express thymidine kinase (TK), using the molecular tools previously described starting from the non-pathogenic strain BA71 V.
  • A scheme of the BA71 .v220i.TK- virus. It is appreciated that the Lac I repressor gene and the ⁇ -glucuronidase (B-gus) marker gene are inserted into the TK locus to generate the intermediate virus BA71 .vGUSREP.TK-.
  • the BA71 .v220i.TK- virus is obtained by placing the pp220 polyprotein gene under the control of the inducible promoter p72.L
  • a marker gene, lac Z, which codes for the B-galactosidase allows the selection of recombinant B, Western blot to detect the expression of pp220 in COS cells after infection with the BA71 .v220i.TK- virus in the presence (+) or absence (-) of the IPTG inducer.
  • the absence of pp220 is shown in two clones of infected and grown COS cells in the absence of IPTG (1 and 2) and how the presence of IPTG in the culture medium results in the expression of pp220.
  • the p72 capsid protein was used as load control.
  • repressor + selection cassette containing the lac I gene under the control of the constitutive promoter pU104 of the virus, the B-gus marker gene under the control of the late viral promoter p72 and the flanking regions of the EP402R (viral CD2) gene for recombination homologous with BA71, where 10T are transcription termination signals.
  • the restriction targets used are indicated: Spe ⁇ and Af B, scheme of the BA71 virus .v220i.CD2-
  • the pp220 polyprotein gene is placed under the control of the inducible promoter p72.i.
  • a marker gene, lac Z which codes for B-galactosidase allows the selection of recombinants.
  • Lac I repressor gene and the B-glucuronidase marker gene ⁇ B-gus are inserted into the locus of the CD2 gene under the control of the promoters pU104 and p72, respectively.
  • the BA71 .v220i.CD2-.PolX- virus was constructed using the tools previously described for the replication-deficient recombinant viruses derived from the non-pathogenic strain BA71 V.
  • the intermediate virus BA71 .pp220 was generated with the gene of pp220 under the control of the p72.fy promoter, as a marker gene, lac Z, which codes for ⁇ -galactosidase allowing the selection of recombinants as described above. Subsequently, the Lac I repressor gene and the ⁇ -gus or gusA marker gene are inserted into the locus of the CD2 gene, thus generating the BA71 .v220i.CD2- virus. Starting from said virus, the expression of the pol X gene is deleted by insertion of the EGFP ("enhanced green fluorescent protein") gene under the p72 promoter.
  • EGFP enhanced green fluorescent protein
  • 061R is a gene used in this construct for homologous recombination.
  • Figure 5. Immunological tests with the BA71.v220i.TK- virus.
  • A ELISA showing the levels of anti-VPPA antibodies in pigs vaccinated with 10 5 Dl 50 (P4 to P9), 10 7 Dl 50 ⁇ P10 to P15) of the inducible virus BA71 .v220.TK- or inoculated with PBS (buffer saline phosphate) as a control (P1 to P3), and subsequently infected with 10 3 Dl 50 of virulent parental virus BA71.
  • PBS buffer saline phosphate
  • EXAMPLE 1 The BA71 virus retains virulence after COS cell growth.
  • COS cells (ATCC, CRL-1650 TM) are used for the generation of recombinant viruses and empty viral particles, to verify that the growth in said cells does not attenuate the virulent strain BA71, it was analyzed whether the virulence of the strain BA71 was affected by its proliferation in said cells.
  • BA71 Cos (pigs 7-9) (pigs 10-12)
  • inoculation with strain BA71 virus was very pathogenic, causing severe clinical symptoms, including high fever from day 3 post-innocuation until the end of the experiment, and regardless of the dose used.
  • a dose of 10 4 Dl 50 of the BA71 virus passed by COS (Ba71 Cos)
  • the disease followed the same course as with the BA71 virus without that pass (BA71)
  • lower doses (10 2 Dl 50 ) some pigs they suffered a slight delay in the onset of clinical symptoms, including fever (Fig. 1 A), and in the decrease of their survival (Fig. 1 B).
  • EXAMPLE 2 Obtaining the recombinant viruses that do not express the thymidine kinase gene BA71.v220i.TK-, BA71.v62i.TK- and BA71.vB438Li.TK- and in which the expression of pp220, pp62 and proteins pB438, respectively, is inducible.
  • thymidine kinase negative (TK-) from strain BA71, the molecular tools described above were used and in Andrés et al. 2002. J Virol 76: 2654-2666; Suárez et al. 2010. J Virol 84: 176-187; and Epifano et al. 2006. J Virol 80: 1 1456-1 1466; with some modification that we will detail.
  • a virus of strain BA71 To generate the BA71 .v220i.TK- virus, an attenuated recombinant virus, deficient in replication and in which the expression of pp220 is inducible by IPTG, was started from a virus of strain BA71.
  • the expression of the TK was deleted (Fig. 2A) using a repressor cassette + homologous recombination selection that is inserted into the TK locus.
  • This cassette contained (i) the lactose repressor gene ⁇ Lac I) under the control of the early / late promoter of the VP10 U104L gene (pU104) and (i ⁇ ) the p72GUS marker, which consists of the ⁇ - gene glucuronidase under the control of the late promoter p72 (Fig. 2A).
  • the intermediate virus, BA71 .GUSREP.TK- was purified by successive rounds of proliferation and isolation of lysis plaques in COS cells to complete purity.
  • the inducible BA71.v220i.TK- virus in the pp220 polyprotein gene was constructed from the intermediate virus BA71 .GUSREP.TK-, by introducing the pp220 gene under the control of the IPTG-inducible promoter (Z2./), which consists of the p72 promoter, the E. coli lac operator and the tac Z gene (which encodes ⁇ -galactosidase used as a marker to select recombinant viruses), following the procedure described for the construction of a similar recombinant virus starting from strain BA71 V described in Andrés et al. J Virol 2002. 76: 2654-2666.
  • Z2./ IPTG-inducible promoter
  • the resulting BA71 .v220i.TK- virus was purified by successive rounds of lysis plate isolation in COS cells.
  • the expression of pp220 in COS cells infected with this virus and grown in media with and without IPTG was determined.
  • Expression of pp220 polyprotein was examined by Western blot using specific antibodies (Andrés et al. J Virol 202. 76: 2654-2666). As shown in Fig. 2B, the expression of pp220 only occurs when culturing infected cells in a medium supplemented with IPTG.
  • the BA71 .v62i.TK- and BA71 .vB438Li.TK- viruses were generated with the same tools as for the BA71 .v220i.TK- but the genes that are regulated by the inducible promoter code for pp62 and pB438L, respectively.
  • EXAMPLE 3 Obtaining the inducible virus BA71.v220i.CD2-.
  • a virus was first constructed with the expression of inducible pp220, starting from a virus of strain BA71 and placing the pp220 polyprotein gene under the control of the IPTG-inducible promoter (p72.f) (Fig. 3B).
  • a repressor + selection cassette (essentially identical to that used for the construction of the TK- viruses) was inserted by homologous recombination into the CD2 gene, the EP402R (Fig. 3A), thus deleting his expression.
  • EXAMPLE 4 Obtaining the inducible virus BA71.v220i.CD2- PolX-. To obtain this replication-deficient recombinant virus, with expression of DNA polymerase X and deletioned CD2, and inducible expression of pp220 as explained above, the tools for the elaboration of replication-deficient viruses derived from strain BA71 were used. V described in Garc ⁇ a-Escudero et al. 1998. J Virol 72: 3185-3195; Rodr ⁇ gez et al. 1993. J Virol 67: 5312-5320; and Andrés et al. 2002. J Virol 76: 2654-2666; with some modification that we will detail.
  • TK + and CD2- that did not express the 0174L gene, which codes for the reparative DNA polymerase, pol X ..
  • the methodology used is that described above. The procedures previously described in the present invention were followed starting from the BA71 .v220i.CD2- virus which has the repressor + selection cassette inserted in the CD2 gene.
  • the pol X- virus was generated by introducing the EGFP gene into the pol X gene, which serves as a selection marker, under the control of the p72 promoter, as shown in the scheme of Figure 4.
  • EXAMPLE 5 Immunization and survival assays with the inducible virus BA71.v220i.TK-.
  • groups of pigs were immunized with 10 7 pfu or 10 5 pfu (plaque forming units) of BA71 .v220i.TK- (six pigs per group, twice in an interval of two weeks) and the results were compared with control pigs in which PBS (three animals) were inoculated.
  • PBS three animals
  • Serum and blood samples were collected after each dose of vaccine at different days after infection to study the humoral and cellular response induced by a specific ELISA test to detect VPPA (Fig. 5A) and an ELISPOT test (Fig. 5B).
  • a specific stimulus in the ELISPOT assay 10 5 pfu of BA71 .v220i.TK- per million peripheral blood mononuclear cells (PBMCs) was used in a 20 hr trial.
  • PBMCs peripheral blood mononuclear cells
  • the survival trial revealed that pigs inoculated with PBS died before day 7 after infection, while 50% of pigs vaccinated with the maximum dose of BA71 .v220i.TK- survived until day 9 after infection (Fig . 5C).
  • EXAMPLE 6 Obtaining empty icosahedral particles and tubular viral structures.
  • empty icosahedral particles are produced that contain the external domains of the virus (internal envelope, capsid and external envelope), but not internal ones (intermediate and nucleoid domain) and therefore They also lack the DNA of the virus.
  • COS cells were infected with 0.1 pfu of the inducible virus BA71 .v220i.TK-, BA71 .v62i.TK- or BA71 .vB438Li.TK- per cell and cultured in the absence of the inducer.
  • Viral particles were purified from the medium by Percoll gradient centrifugation and Sephacryll column chromatography (Carrascosa et al. J Virol 1985. 54: 337-344). Empty viral particles generated by repression of pp220, pp62 and pB438L were obtained, respectively.
  • Preconfluent monolayers of COS cells were infected with BA71 .v220i.TK- virus, BA71 .v62i.TK- virus or BA71 .vB438Li.TK- virus, at a multiplicity of infection of 0.1 pfu per cell
  • the cells were maintained in DMEM medium with 2% fetal calf serum and 1 mM IPTG. After 2 hours of adsorption of the virus at 37 9 C, in an incubator with 5% CO 2 , the inoculum was removed and fresh medium with IPTG was added at the same concentration, incubating as before at 37 S C until it was observed a total cytopathic effect (usually after 48-72 hours).
  • the culture medium containing the extracellular virus produced was collected and centrifuged at low speed (Sorvall GS3 rotor at 2500 rpm for 15 min at 4 Q C) to remove cellular debris and then centrifuged at high speed (rotor Sorvall GS3 at 6500 rpm for 8.5 h at 4 ⁇ C) to sediment the virus.
  • the sediment was resuspended in 1/100 of the initial volume and the virus was purified by centrifugation for 1 h at 4 9 C on a 25% sucrose mattress in PBS, using a Sorvall SW41. T ⁇ rotor at 100,000g.
  • the sediment obtained was resuspended in 1/500 of the initial volume and titrated in COS cells in the presence of it IPTG concentration by the lysis plate formation method (Enjuanes et al. 1976. J Gen V ⁇ rol 32: 471-477).
  • the virus concentration is expressed as plaque forming units (pfu) by me.
  • COS cells infected by BA71 .v220i.TK- were lysed in the presence or absence of the IPTG inducer and the pp220 levels were analyzed by immunoblot or western biot with specific antibodies described in Andrés et al. J Virol 202. 76: 2654-2666.
  • the antibody against p72 was used as a loading control.
  • Each well of a 96-well ELISA plate is upholstered for 12 hours at 4 s with 100 ⁇ of the corresponding protein diluted ⁇ 5 Mg / ml) in carbonate-bicarbonate buffer pH 9.6, and incubated for 12 hours at 4 9 C. After washing the plates 3 times with PBS-Tween 20 0.05% (v / v), they are blocked with 100 ⁇ PBS-1% bovine serum albumin (BSA) for 1 hour at 37 S C Serum samples are added to the appropriate dilutions to each well and incubated for 1 hour at 37 9 C, after which the plates are washed and the peroxidase-conjugated secondary antibody (pig anti-lgG at 1: 20000 is added).
  • BSA bovine serum albumin
  • PBMCs peripheral blood mononucial cells
  • PBMCs were purified from blood collected in the presence of EDTA anticoagulant (2.5 mM), using a density gradient on Histopaque 1077 (Sigma). 20 ml of diluted blood 1: 1 with PBS was deposited on a 4 ml mattress of Histopaque 1077 and, after centrifugation for 30 minutes at 500xg, the interface between the plasma and the Histopaque, containing the PBMCs, was collected and then lys erythrocytes.
  • ELISPOT immunosorbent assay for 30 minutes at 500xg
  • porcine IFN and secretory cells against VPPA was determined by an ELISPOT assay following the method described in Diaz and Mateu. 2005. Vet Immunol Immunopathol 106: 107-12. For this, 96-well plates (Costar 3590, Corning) were upholstered with 50 ⁇ / well of a porcine anti-IFN antibody (BD Pharmingen, clone P2G10) at a 1: 100 dilution in carbonate-bicarbonate buffer pH 9.6, for 14 hours at 4 S C.
  • porcine anti-IFN antibody BD Pharmingen, clone P2G10
  • COS cells grown in DMEM medium were infected with 2% fetal calf serum in the absence of the IPTG inducer at a concentration of 0.1 pfu / cell of the BA71 .v220i.TK-, BA71 .v62i.TK- and BA71 viruses.
  • pB438L.TK- The particles Empty virals were purified as described to purify the BA71 .v220i.TK-, BA71 .v62i.TK- and BA71 .vB438Li.TK- viruses.

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Abstract

La présente invention concerne une série de virus de la peste porcine africaine (VPPA) recombinants, caractérisés en ce qu'ils sont obtenus à partir de la souche virulente BA71 dont l'expression du gène pp220, pp62 o pB438L est réprimée. Dans le cas où le gène pp220 est réprimé, le gène TK est également supprimé, ou le gène CD2 éventuellement avec le pol X, alors que les souches dont le gène PP62 ou pB438L est réprimé ont également le gène TK supprimé. Par ailleurs, l'invention concerne les particules virales vides générées naturellement par l'infection par les virus recombinants pauvres en réplication et leur utilisation dans un vaccin contre la peste porcine africaine.
PCT/ES2012/070058 2011-02-08 2012-01-31 Vaccin contre le virus de la peste porcine africaine basé sur des virus recombinants pauvres en réplication Ceased WO2012107614A1 (fr)

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WO2016049101A1 (fr) * 2014-09-24 2016-03-31 The United States Of America, As Represented By The Secretary Of Agriculture La souche du virus atténué de la peste porcine africaine induit une protection contre une provocation par un produit isolé homologue virulent du virus parental de la géorgie de 2007
CN107937349A (zh) * 2017-11-27 2018-04-20 中国检验检疫科学研究院 稳定表达非洲猪瘟病毒p54蛋白的细胞系及其制备与应用
WO2020102370A1 (fr) * 2018-11-15 2020-05-22 Kansas State University Research Foundation Compositions immunogènes contre le virus de la peste porcine africaine
CN111569083A (zh) * 2020-05-25 2020-08-25 杭州勇诚睿生物科技有限公司 一种适用于抗非洲猪瘟病毒siRNA药物的靶向载体及其应用
WO2022090131A1 (fr) * 2020-10-26 2022-05-05 Consejo Superior De Investigaciones Científicas Virus de la peste porcine africaine recombinant utilisé en tant que vaccin vivant atténué contre la peste porcine africaine
CN114958782A (zh) * 2022-03-16 2022-08-30 中国农业科学院兰州兽医研究所 一种iptg诱导性缺失d1133l基因的非洲猪瘟病毒减毒株及其应用
CN116492455A (zh) * 2023-03-31 2023-07-28 中国人民解放军军事科学院军事医学研究院 非洲猪瘟病毒k421r基因及利用其制备的复制缺陷型非洲猪瘟疫苗
EP4137506A4 (fr) * 2020-04-14 2024-06-26 Plumbline Life Sciences, Inc. Composition de vaccin contre la peste porcine africaine
WO2024235368A1 (fr) * 2023-05-18 2024-11-21 Centro De Ingeniería Genética Y Biotecnología Composition vaccinale contre le virus de la peste porcine africaine
WO2024233996A3 (fr) * 2023-05-11 2025-04-03 The Wistar Institute Of Anatomy And Biology Vaccins contre le virus de la peste porcine africaine, et leurs procédés d'utilisation

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016049101A1 (fr) * 2014-09-24 2016-03-31 The United States Of America, As Represented By The Secretary Of Agriculture La souche du virus atténué de la peste porcine africaine induit une protection contre une provocation par un produit isolé homologue virulent du virus parental de la géorgie de 2007
CN107937349A (zh) * 2017-11-27 2018-04-20 中国检验检疫科学研究院 稳定表达非洲猪瘟病毒p54蛋白的细胞系及其制备与应用
CN107937349B (zh) * 2017-11-27 2021-01-15 中国检验检疫科学研究院 稳定表达非洲猪瘟病毒p54蛋白的细胞系及其制备与应用
WO2020102370A1 (fr) * 2018-11-15 2020-05-22 Kansas State University Research Foundation Compositions immunogènes contre le virus de la peste porcine africaine
EP4137506A4 (fr) * 2020-04-14 2024-06-26 Plumbline Life Sciences, Inc. Composition de vaccin contre la peste porcine africaine
CN111569083A (zh) * 2020-05-25 2020-08-25 杭州勇诚睿生物科技有限公司 一种适用于抗非洲猪瘟病毒siRNA药物的靶向载体及其应用
WO2022090131A1 (fr) * 2020-10-26 2022-05-05 Consejo Superior De Investigaciones Científicas Virus de la peste porcine africaine recombinant utilisé en tant que vaccin vivant atténué contre la peste porcine africaine
CN114958782A (zh) * 2022-03-16 2022-08-30 中国农业科学院兰州兽医研究所 一种iptg诱导性缺失d1133l基因的非洲猪瘟病毒减毒株及其应用
CN116492455A (zh) * 2023-03-31 2023-07-28 中国人民解放军军事科学院军事医学研究院 非洲猪瘟病毒k421r基因及利用其制备的复制缺陷型非洲猪瘟疫苗
CN116492455B (zh) * 2023-03-31 2024-04-23 中国人民解放军军事科学院军事医学研究院 非洲猪瘟病毒k421r基因及利用其制备的复制缺陷型非洲猪瘟疫苗
WO2024233996A3 (fr) * 2023-05-11 2025-04-03 The Wistar Institute Of Anatomy And Biology Vaccins contre le virus de la peste porcine africaine, et leurs procédés d'utilisation
WO2024235368A1 (fr) * 2023-05-18 2024-11-21 Centro De Ingeniería Genética Y Biotecnología Composition vaccinale contre le virus de la peste porcine africaine

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