WO2009144353A1 - Attenuated vaccine for foot-and-mouth disease - Google Patents

Attenuated vaccine for foot-and-mouth disease Download PDF

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WO2009144353A1
WO2009144353A1 PCT/ES2009/070187 ES2009070187W WO2009144353A1 WO 2009144353 A1 WO2009144353 A1 WO 2009144353A1 ES 2009070187 W ES2009070187 W ES 2009070187W WO 2009144353 A1 WO2009144353 A1 WO 2009144353A1
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seq
amino acid
isolated
sequence
virus
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PCT/ES2009/070187
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Spanish (es)
French (fr)
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Esteban Domingo Solans
Cristina ESCARMÍS HOMS
Samuel Ojosnegros Martos
Juan Francisco GARCÍA ARRIAZA
Marta Sanz-Ramos Rojo
Noemí SEVILLA HIDALGO
Teresa RODRÍGUEZ CALVO
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Consejo Superior De Investigaciones Científicas
Instituto Nacional De Investigación Y Tecnología Agraria Y Alimentaria
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Publication of WO2009144353A1 publication Critical patent/WO2009144353A1/en

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    • C12N7/00Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C12N7/04Inactivation or attenuation; Producing viral sub-units
    • C12N7/045Pseudoviral particles; Non infectious pseudovirions, e.g. genetically engineered
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/12Viral antigens
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/12Viral antigens
    • A61K39/125Picornaviridae, e.g. calicivirus
    • A61K39/135Foot- and mouth-disease virus
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C12N7/04Inactivation or attenuation; Producing viral sub-units
    • C12N7/08Inactivation or attenuation by serial passage of virus
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/51Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525Virus
    • A61K2039/5254Virus avirulent or attenuated
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/55Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552Veterinary vaccine
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011Details
    • C12N2770/32011Picornaviridae
    • C12N2770/32111Aphthovirus, e.g. footandmouth disease virus
    • C12N2770/32134Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011Details
    • C12N2770/32011Picornaviridae
    • C12N2770/32111Aphthovirus, e.g. footandmouth disease virus
    • C12N2770/32161Methods of inactivation or attenuation
    • C12N2770/32164Methods of inactivation or attenuation by serial passage

Definitions

  • the present invention falls within the field of molecular biology, biotechnology, and veterinary medicine, and specifically refers to an attenuated vaccine, with foot-and-mouth disease virus presenting the deleted genome, which are capable of complementing Yes, and they act against foot and mouth disease.
  • VFA foot-and-mouth disease virus
  • AF glossophobic or foot-and-mouth disease
  • the VFA genome consists of a single polarity RNA molecule positive of about 8500 nucleotides that codes for a single polyprotein. This is cut by viral proteases to produce the 4 proteins of the capsid and the 9 non-structural proteins, involved in different steps of virus replication (Belsham, 1993, Prog. Biophys. Mol. Biol. 60: 241-260; Masón et al. 2003. Virus Res. 91: 9-32; Porter, 2003. J. Viral., 67: 6917-6921).
  • the viral capsid, of icosahedral symmetry is composed of 60 copies of each of the structural proteins VP1, VP2, VP3 and VP4 (review in Bachrach, 1977.
  • Vaccines against FA currently on the market are obtained from virus growth in BHK cell cell cultures (Radlett et al., 1985. Dev. Biol. Standard, 60: 163; Telling, 1975. Industrial production of FMD vaccine using BHK suspension cells. Some comparative results relating in vitro assays and cattle potency. In Report of the research Group of the Standing technical Committee of the European Commission for the control of Foot-and-Mouth Disease, Brescia, Italy. Organization, Rome, 95). This virus is chemically inactivated with a compound of aziridines, more specifically ethylene-binary amine (BEI) (review in Brown, F., 2001, Vaccine, 20: 322-327).
  • BEI ethylene-binary amine
  • Inactivated virus vaccines as in the case of AF, must carry adjuvants that confer sufficient immunity on animals. He The most commonly used adjuvant for cattle and sheep is aluminum hydroxide, but for pigs it is necessary to use mineral oil as Freund's incomplete adjuvant (Freund & Thompson, 1945, Science 101: 468). Generally, monovalent vaccines are prepared, but in countries where more than one type of virus circulates, the corresponding polyvalent vaccines can be used.
  • Vaccines currently in use have to meet the following requirements in their production: 1- the viral antigen must be produced in large quantities because each dose of vaccine needs to contain high levels of inactivated virus to be effective;
  • a live vaccine should preferably maintain the antigenic complement of the wild type strain.
  • the live vaccine must be sufficiently avirulent to avoid unacceptable pathological effects, but on the other hand it must cause a sufficient level of immunity in the host.
  • the live attenuated vaccine should preferably not have the probability of reverting to a virulent wild type strain.
  • the present invention provides a vaccine that has two safety barriers against the conventional vaccine: first, it is highly attenuated in vivo, which means that if there is an escape during its production, it does not entail any risk such as possible start of an outbreak; second, it is composed of defective viruses that need to complement to produce a productive infection, and this only happens at a very high multiplicity of infection (MOI), which in vivo is highly unlikely to happen. In any case, if this possibility occurred at the point of inoculation, even if the complete virus was obtained by recombination or by complementation, the resulting virus would be the C-S8p260 population, which is fully attenuated.
  • MOI multiplicity of infection
  • said vaccine being a complete virus, is capable of conferring an immunity that will be longer than that conferred by the current vaccine and more specific and robust than that conferred by recombinant vaccines. The reason for all this is that, being a complete virus, it will express a wide repertoire of T and B epitopes necessary for the complete elimination of the virus.
  • an isolated virus population is provided, henceforth a population of viruses of the invention, characterized in that it comprises a polynucleotide sequence that encodes an amino acid sequence homologous to SEQ ID NO: 1, and at least one of the sequences selected from: a) a polynucleotide sequence encoding an amino acid sequence homologous to SEQ ID NO: 2, b) a polynucleotide sequence encoding an amino acid sequence homologous to SEQ ID NO: 3 , for use as a medicine.
  • homologous refers to the similarity between two structures due to a common evolutionary ancestry, and more specifically, to the similarity between the amino acids of two or more proteins or amino acid sequences. Two proteins are considered homologous if they have the same evolutionary origin or if they have similar function and structure.
  • VFA Foot-and-mouth disease virus (FMDV) strain C1-Santa Pau or C-S8, belonging to serotype C or Aphthovirus C
  • FMDV Freot-and-mouth disease virus
  • identity refers to the proportion of identical amino acids between two amino acid sequences that are compared. Sequence comparison methods are known in the state of the art, and include, but are not limited to, the BLASTP or BLASTN program, and FASTA (Altschul et al., J. Mol. Biol. 215: 403-410 ( 1999) Since two proteins are considered homologous if they have the same evolutionary origin or if they have similar function and structure, in general, it is assumed that higher values of similarity or identity of 30% indicate homologous structures. Identity percentages of at least 80% will maintain the same properties of said peptide.
  • the putative VFA strains are identifiable by their homology at the genome level or of the polypeptides encoded by it. Generally, VFA strains they have an identity greater than 80%, preferably an identity greater than 90%, and even more preferably an identity greater than 95%, and even more preferably an identity greater than 99% at the level of the amino acid sequences.
  • the methods for determining the homology and percent identity of amino acid sequences are known in the art. For example, the amino acid sequence can be determined directly and can be compared with the sequences provided herein. For example also, the nucleotide sequence of the putative VFA genomic material can be determined (usually by means of an intermediate cDNA compound), and deduced the amino acid sequence encoded therein, to compare with the corresponding regions of the sequences provided in this memory.
  • the isolated virus population of the invention is characterized in that it comprises a polynucleotide sequence encoding an amino acid sequence, hereinafter the first amino acid sequence of the invention, which comprises a peptide with an identity of at least 80% with SEQ ID NO: 1, and at least one of the sequences selected from: a) a polynucleotide sequence encoding an amino acid sequence, hereinafter the second amino acid sequence of the invention, comprising a peptide with an identity of at least 80% with SEQ ID NO: 2, b) a polynucleotide sequence encoding an amino acid sequence, hereinafter third amino acid sequence of the invention, comprising a peptide with an identity of at least 80% with SEQ ID NO: 3, for use as a medicine.
  • the first amino acid sequence of the invention which comprises a peptide with an identity of at least 80% with SEQ ID NO: 1
  • the second amino acid sequence of the invention comprising a peptide with an identity of at least 80% with SEQ ID
  • Viruses with the amino acid sequence SEQ ID NO: 1 belong to the variant C-S8p260D417, have a deletion of 417 nucleotides in the region coding for protease L, with respect to the polynucleotide sequence of variant C-S8.
  • the ⁇ 417 deletion includes positions 1153 to 1517 (of the VFA genome, following the numbering of Escarm ⁇ s et al. 1996. Genetic lesions associated with Muller's ratchet in an RNA virus. J. Mol. Biol. 264: 255-267).
  • the viruses with the amino acid sequence SEQ ID NO: 2 belong to the variant C-S8p260D999, and have a deletion of 999 nucleotides in the region that codes for the structural proteins ( ⁇ 999), with respect to the polynucleotide sequence of the variant C- S8
  • the ⁇ 999 deletion includes positions 2793 to 3793 of the VFA genome, following the same numbering as in the previous case.
  • viruses with the amino acid sequence SEQ ID NO: 3 belong to the variant C-S8p260D1017, and have a deletion of 1017 nucleotides in the region that codes for the structural proteins ( ⁇ 1017), with respect to the polynucleotide sequence of the variant C- S8
  • the ⁇ 1017 deletion includes positions 1932 to 2950 of the VFA genome, following the same numbering as in the previous cases.
  • Viruses with the amino acid sequences SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 are incapable of generating, each individually, infectious virus with the ability to replicate. They can be caused by complementation (between SEQ ID NO: 1 and either SEQ ID NO: 2 or SEQ ID NO: 3) or by recombination. But although it is possible that the complementation occurs temporarily and that this favors the induction of antibodies and the cellular immune response, recombination is not a problem because the strain C-S8p260 is attenuated.
  • virus population or "viral population” to a plurality of viruses existing in any form, and comprising at least two viral variants of a virus of the same species.
  • a population of viruses may be a suspension of virus particles present in a cell culture medium or in another solution. It can also be a pellet or a lyophilized preparation containing the viruses.
  • isolated protein capsid hereinafter isolated protein capsid of the invention, comprising at least one of the amino acid sequences of the invention, for use as a medicine.
  • a genetic construction which would direct the in vitro or intracellular transcription of the polynucleotide sequences of the virus population of the invention, and comprises The polynucleotide sequence encoding the first amino acid sequence of the invention and another sequence selected from: a. polynucleotide sequence, which encodes the second amino acid sequence of the invention, b. polynucleotide sequence, which encodes the third amino acid sequence of the invention, c. nucleic acid molecules whose complementary hybrid chain with the polynucleotide sequence of a) and / or b), d.
  • This genetic construction includes the cloning and expression vectors that comprise the nucleic acid molecules of the expression system of the invention.
  • Such expression vectors include suitable control sequences, such as, for example, control elements for translation (such as start and stop codes) and for transcription (for example, promoter-operator regions, binding sites).
  • the vectors according to the invention can include plasmids and viruses (comprising bacteriophages and eukaryotic viruses), according to procedures well known and documented in the art, and can be expressed in a variety of different expression systems, also well known and documented in Ia technique. Many other viral and non-viral vectors are described and are known in the art.
  • polynucleotide and “nucleic acid” are used interchangeably herein, referring to polymeric forms of nucleotides of any length, both ribonucleotides and deoxyribonucleotides.
  • peptide refers to a polymeric form of amino acids of any length, which may be coding or non-coding, chemically or biochemically modified.
  • the population of viruses isolated from the invention, the sequences amino acids of the invention, the isolated protein capsid of the invention, the genetic construction of the invention, or any combination thereof can be formulated in compositions for use as an immunogen (hereinafter, immunogens of the invention). These immunogens can also be used as vaccines in animals, and more particularly in mammals, including humans, or produce a response in the production of antibodies in animals.
  • immunogens of the invention an immunologically effective amount of at least one of the viruses, amino acid sequences or capsids is mixed with a physiologically acceptable carrier suitable for administration to mammals including humans.
  • the immunogens may be covalently linked to each other, to other peptides, to a transporter protein or to other carriers, incorporated into liposomes or other similar vesicles, and / or mixed with an adjuvant or absorbent as is known in the field of vaccines.
  • they can be mixed with other immunostimulatory complexes.
  • the immunogens are not coupled and merely mixed with a physiologically acceptable transporter such as a normal buffer or saline compound suitable for administration to mammals including humans.
  • the immunogens of the invention have protective antigenic sequences.
  • These protective antigens are capable of generating a protective (immunogenic) host immune response, that is, a host response that leads to the generation of immune effector molecules, antibodies or cells that damage, inhibit or kill the invading biological entity, " thus protecting "the host from a clinical or sub-clinical disease and a loss of productivity.
  • a protective (immunogenic) host immune response that is, a host response that leads to the generation of immune effector molecules, antibodies or cells that damage, inhibit or kill the invading biological entity, " thus protecting "the host from a clinical or sub-clinical disease and a loss of productivity.
  • Such protective immune response can commonly be manifested by the generation of antibodies.
  • the antibodies produced after the immunization of the animal hereinafter antibodies of the invention, are used as medicine.
  • the antibodies of the present invention can be formulated for administration to an animal, and more preferably to a mammal, including man, in a variety of ways.
  • the antibodies may be in sterile aqueous solution or in biological fluids, such as serum.
  • Aqueous solutions may be buffered or unbuffered and have additional active or inactive components. Additional components include salts to modulate the ionic strength, preservatives including, but not limited to, antimicrobial agents, antioxidants, chelants, and the like, and nutrients including glucose, dextrose, vitamins and minerals.
  • the antibodies can be prepared for solid administration.
  • Antibodies can be combined with various inert carriers or excipients, including but not limited to; binders such as microcrystalline cellulose, gum tragacanth, or gelatin; excipients such as starch or lactose; dispersing agents such as alginic acid or corn starch; lubricants such as magnesium stearate, glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; or flavoring agents such as peppermint or methyl salicylate.
  • binders such as microcrystalline cellulose, gum tragacanth, or gelatin
  • excipients such as starch or lactose
  • dispersing agents such as alginic acid or corn starch
  • lubricants such as magnesium stearate, glidants such as colloidal silicon dioxide
  • sweetening agents such as sucrose or saccharin
  • flavoring agents such as peppermint or methyl salicylate.
  • Antibodies or their formulations can be administered to an animal, including a mammal and, therefore, to man, in a variety of ways.
  • Such means include, but are not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intracecal, intraventricular, oral, enteral, parenteral, intranasal or dermal.
  • composition of the invention which comprises the population of virus isolated from the invention, of the amino acid sequences of the invention, an isolated protein capsid of the invention, an antibody of The invention, the genetic construction of the invention, or any combination thereof, for use as a medicine.
  • the composition of the invention is used for the treatment or prevention of foot and mouth disease.
  • viruses, amino acid sequences, Ia or capsids, genetic constructs of the invention, or any combination thereof are found, or translated, in a therapeutically effective amount, capable of generating antibodies for use in Ia vaccine development
  • vaccine refers to an antigen preparation used to establish the immune system response to a disease. They are prepared of antigens that once inside the organism cause the response of the immune system, through the production of antibodies, and generate immunological memory producing permanent or transient immunity.
  • antigen refers to a cell surface molecule (generally a protein or a polysaccharide), which can induce the formation of antibodies.
  • a cell surface molecule generally a protein or a polysaccharide
  • antigens such as proteins or peptides, polysaccharides and, more rarely, other molecules such as nucleic acids.
  • medication refers to any substance used for prevention, diagnosis, relief, treatment or cure of diseases in man and animals.
  • it also refers to the viruses of the invention, the capsid of the invention, the genetic construction of the invention, the plasmid of the invention or the composition of the invention, which is capable of generating a immune response against a given organism, which is causing such disease in man or animals. It includes, therefore, what is known as a vaccine, as previously defined herein.
  • the composition of the invention is a vaccine, henceforth a vaccine of the invention.
  • the vaccine also comprises pharmacologically acceptable excipients.
  • the vaccine comprises an adjuvant.
  • the vaccine has a recombinant origin.
  • the vaccine is versatile.
  • adjuvant refers to an agent, as long as it does not have an antigenic effect in itself, which can stimulate the immune system by increasing its response to the vaccine.
  • aluminum salts “aluminum phosphate” and “aluminum hydroxide” are the two adjuvants most commonly used in vaccines. Other substances, such as squalene, can also be used as adjuvants.
  • the term "polyvalent” is used to refer to the vaccine comprising the combination of two or more antigens in total, including one or more of any of the serotypes, types, varieties or mutants that are they include within the classification of Foot and Mouth Disease Virus (VFA).
  • VFA Foot and Mouth Disease Virus
  • An alternative method of vaccine production is the use of molecular biology techniques to produce a fusion protein that contains one or more of the amino acid sequences of the present invention and a highly immunogenic peptide or protein, against a certain infection. . Therefore, in another aspect, the vaccine of the invention has a recombinant origin.
  • a "vector” is a replicon to which another polynucleotide segment has been attached, to perform the replication and / or expression of the bound segment.
  • a “replicon” is any genetic element that behaves as an autonomous unit of polynucleotide replication within a cell; that is, able to replicate under its own control.
  • Control sequence refers to polynucleotide sequences that are necessary to effect the expression of the coding sequences to which they are linked. The nature of such control sequences differs depending on the host organism; in prokaryotes, said control sequences generally include a promoter, a ribosomal binding site, and termination signals; in eukaryotes, generally, said control sequences include promoters, termination signals, enhancers and, sometimes, silencers. It is intended that the term “control sequences” includes, at a minimum, all components whose presence is necessary for expression, and may also include additional components whose presence is advantageous.
  • Operaationally linked refers to a juxtaposition in which the components thus described have a relationship that allows them to function in the intended way.
  • a control sequence "operatively linked" to a coding sequence is linked in such a way that the expression of the coding sequence is achieved under conditions compatible with control sequences.
  • a “free reading frame” is a region of a polynucleotide sequence that encodes a polypeptide; This region may represent a portion of a coding sequence or a complete coding sequence.
  • a "coding sequence” is a polynucleotide sequence that is transcribed into mRNA and / or translated into a polypeptide when under control of appropriate regulatory sequences. The limits of the coding sequence are determined by a translation start codon at the 5 'end and a translation end codon at the 3' end.
  • a coding sequence may include, but is not limited to mRNA, cDNA, and recombinant polynucleotide sequences.
  • transfection refers to the introduction or transfer of an exogenous nucleic acid molecule into a eukaryotic cell, including, but not limited to, a ribonucleic or deoxyribonucleic acid molecule (by example, naked RNA or DNA).
  • Plasmid refers to a circular fragment of double-stranded DNA, which is found inside almost all bacteria, and that act and replicate independently to bacterial chromosomal DNA and can be transferred from one bacterium to another. They are used as vectors in genetic manipulation.
  • the expression “therapeutically effective amount” refers to the amount of virus, amino acid sequences, capsids, antibodies or genetic constructs that allow its calculated expression to produce the desired effect and, in In general, it will be determined, among other causes, by the characteristics of said viruses, antibodies, sequences and constructions and the therapeutic effect to be achieved.
  • the pharmaceutically acceptable adjuvants and vehicles that can be used in said compositions are the vehicles known to those skilled in the art.
  • compositions provided by this invention can be provided by any route of administration, for which said composition will be formulated in the appropriate pharmaceutical form and with the pharmacologically acceptable excipients to the route of administration chosen.
  • VFA denotes a viral species of the PicoARNviridae family, which belong to group IV of the Baltimore classification, whose pathogenic forms cause foot-and-mouth disease, and whose attenuated, persistent or defective forms derived from They are related to this disease.
  • the VFA genome is made up of a simple, positive-sense RNA chain with a length between 7.2 and 9.0 kb. It is known that viruses containing RNA have relatively high mutation frequencies, of the order of 10 ⁇ 3 to 10 ⁇ 5 per incorporated nucleotide. Consequently, VFA populations are made up of a heterogeneous set of genetic variants undergoing rapid evolution, which have led to a great diversity of related genotypes and phenotypic variants, including seven different serotypes.
  • Figure 1 (Fig. 1) .- C-S8p260 vaccination scheme in C57BL / 6 mice.
  • Figure 2 (Fig. 2) .- Survival of mice vaccinated with C-S8p260.
  • Figure 3 (Fig. 3) .- ELISA titers in mice vaccinated with C-S8p260.
  • Figure 4 (Fig. 4) .- Pigs temperature after challenge with C-S8c1.
  • Figure 5 (Fig. 5) .- Scheme of the different regions of the VFA genome.
  • VP3 and VP4 which code for structural proteins, and regions
  • Figure 6 (Fig. 6) .- Alignment of the different VFA serotypes, including the deleted variants ⁇ 417, ⁇ 999 and ⁇ 1017, and indicating the correspondence of said regions in the different serotypes.
  • RNA molecules The analysis by RT-PCR of the RNA of the population C-S8p260 revealed the presence of three different RNA molecules: a molecule that contains a deletion of 417 nucleotides in the region that encodes protease L ( ⁇ 417); a second molecule with a deletion of 999 nucleotides in the region that codes for structural proteins ( ⁇ 999) and, - a third, with a deletion of 1017 nucleotides in the same region ( ⁇ 1017) (Garc ⁇ a-Arriaza et al., 2004, J. Virol. 78: 11678-11685). None of these deletions alter the open reading phase of the VFA genome. Different quantification analyzes made it possible to ensure that the C-S8p260 population consists mostly of deleted genomes, finding at least 10,000 times less standard virus RNA, or without delations.
  • VFA C-S8p260 is composed of defective viruses capable of complementing when the infection is performed at a very high MOI.
  • Example 2 Use of the viral population C-S8p260 in the elaboration of a live attenuated vaccine
  • the viral population C-S8p260 has been tested, initially, as a possible live attenuated vaccine in a mouse model developed in the inventors' laboratory (Salguero et al., 2005, Virology 332: 384-396). C57BL / 6 mice are highly susceptible to infection with VFA C-S8c1. Thus, the inoculation of VFA in the plantar pad, subcutaneous injection, (inoculation site equivalent to the coronary impeller of the pig, the natural host) produced the appearance of disease symptoms at 24 hours post-inoculation (pi) (bristling hair, apathy, hunched posture and depression), and death between 36-48 hours pi).
  • pi post-inoculation
  • a first objective was to assess whether the viral population C-S8p260 is attenuated in vivo. For this, 16 mice were inoculated in the plantar pad (AP) with 10 7 PFUs of C-S8p260 (the highest dose that can be inoculated with the volume allowed in the AP that are 50 ⁇ l). The animals were bled at different times pi (days 1, 2, 3 15 and 30 pi) to determine viremia and antibody titer against VFA ( Figure 1). Serum viral titer was determined by plating titration in susceptible BHK cells at short times pi, 1, 2 and 3 days, and no viral replication was detected. Likewise, the animals did not detect any symptoms of illness or death due to viral infection.
  • mice of the C-S8p260 population were inoculated intramuscularly with 10 7 PFUs of C-S8p260. At 60 days post-inoculation they were challenged with 10 4 PFUs of VFA C-S8c1 in the AP. The results indicated the complete absence of symptoms in the mice vaccinated intramuscularly.
  • mice were inoculated in the AP with 10 3 PFUs of C-S8p260. These mice bled on days 1, 2, 3, 15 and 30 pi ( Figure 1). The animals, as expected by the results shown above, did not show any type of disease or blood viremia at early post-inoculation times. These mice were challenged with 10 4 PFUs of C-S8c1 in the AP at day 90 pi. Four control animals without vaccinations were included. 100% of the animals previously inoculated with 10 3 PFUs of C-S8p260 survived the challenge with C-S8c1 while controls died at 48 h pi. ( Figure 2) Likewise, no viremia was detected in vaccinated animals after the challenge, which once again indicated sterile protection. These data indicate that lower doses of antigen protect against a lethal dose of VFA.
  • the vaccine proposed in this patent was also evaluated in pig, the natural host of VFA, in a preliminary trial.
  • the animals are They divided into five groups. In each of the groups the protocol followed is common to all of them.
  • the C-S8p260 virus was inoculated intramuscularly in 1 ml of PBS.
  • the challenge was performed at 500 ⁇ l in the coronary impeller. Blood samples were taken from OD, before the inoculation of C-S8p260, D2, D4, D7, D15 and D30 (before the challenge). After the challenge, the animals were bled every two days and subjected to observation every day to determine the appearance of AF symptoms until the 11th post-challenge day.
  • Group 2. 1 animal. In this group the same dose of C-S8p260 was used as in the previous group but adjuvant was added in the second dose.
  • the second dose of C-S8p260 was performed in a 1: 1 dilution with Freund's complete adjuvant. Group 3. 3 animals.
  • the dose of C-S8p260 was diluted 1: 100 in order to assess whether a high defective dose was needed.
  • Group 5 Animals. These animals are the positive control of the experiment in which the pigs were inoculated with PBS instead of the vaccine and challenged with the same dose of C-S8c1. These animals were not protected and showed the symptoms of AF.
  • Group 1 Temperature table expressed in 0 C. It has been included to compare animals # 21 and # 22 of group 5 which are the challenged but unvaccinated controls that developed all the expected symptoms of AF.
  • canker sores Appearance of canker sores. It is defined as the appearance of primary canker sores (at the site of inoculation) and canker sores on all legs (secondary). The day the animals were sacrificed is indicated.
  • This animal did not develop any type of symptom associated with AF or another disease. He remained completely healthy throughout the experiment. It is necessary to make the assessment that he was sharing a box with the control animals so that he was in contact with very sick pigs that exhaled viruses. This animal is considered for the purpose of fully protected vaccine evaluation.
  • animal # 13 has a basal temperature (measured for 25 consecutive days) of 40.2 0 C, very high for the average temperature of pigs. Appearance of canker sores.
  • the dose of defective VFA C-S8p260 of 5 x 10 6 PFUs with adjuvant completely protected a pig. That same dose without adjuvant has conferred partial protection.
  • the 10 5 PFU dose has only protective effect when inoculated with adjuvant.
  • the ability to protect C-S8p260 with the least dose but including adjuvant in the 2 inoculations is being evaluated in a new pig experiment.
  • the protective capacity of C-S8p260 inactivated with BEI is being evaluated.
  • the C-S8p260 population was inactivated following the inactivation protocol based on the use of 2-Bromoethylamine Hydrobromide (BEA), an azirin derivative.
  • BEA 2-Bromoethylamine Hydrobromide
  • 7 female C57BI / 6 mice of 8 weeks of age were inoculated with 50 microliters of intramuscularly inactivated C-S8p260, a dose equivalent to 10 7 PFUs of C-S8p260.

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Abstract

The invention relates to an attenuated vaccine against foot-and-mouth disease, with viral variants of the foot-and-mouth disease virus, in which one of the variants includes the deleted genome in the region coding for protease L and another of the variants in the region of the structural proteins. Said variants can complement one another.

Description

VACUNA ATENUADA PARA LA FIEBRE AFTOSA. VACCINE ATTENTIONED FOR FEVERY FEVER.
La presente invención se engloba dentro del campo de Ia biología molecular, de Ia biotecnología, y de Ia medicina veterinaria, y específicamente se refiere a una vacuna atenuada, con virus de Ia fiebre aftosa que presentan el genoma delecionado, que son capaces de complementar entre sí, y que actúan frente a Ia fiebre aftosa.The present invention falls within the field of molecular biology, biotechnology, and veterinary medicine, and specifically refers to an attenuated vaccine, with foot-and-mouth disease virus presenting the deleted genome, which are capable of complementing Yes, and they act against foot and mouth disease.
ESTADO DE LA TÉCNICA ANTERIORSTATE OF THE PREVIOUS TECHNIQUE
El virus de Ia fiebre aftosa (VFA) es un miembro de Ia familia Picornavirídae, género Aphthovirus, y es el agente causante de Ia glosopeda o fiebre aftosa (FA), una enfermedad muy contagiosa y económicamente devastadora que afecta a animales de pezuña hendida (vacas, cerdos, ovejas y cabras, entre otros animales), caracterizada por Ia aparición de vesículas en las patas y el hocico (Bachrach, H. L. 1978. Foot-and-Mouth disease: worldwide impact and control measures, p. 299- 310. In E. K. a. K. Maramorosch (ed.), Viruses and environment. Academic Press, Inc., New York, N.Y.; Rowlands, D. J. 2003. Virus Res 91 : 1-161 ; Sobrino, F., and E. Domingo. 2004. Foot-and-mouth disease. Horizon Press, London). El impacto social y económico de FA puede ser catastrófico cuando un brote aparece en países libres de VFA con animales inmunológicamente naive. Este fue el caso del brote de FA en Taiwan en 1997 y el del Reino Unido en 2001 , en el que millones de animales infectados y en contacto fueron sacrificados con un coste directo e indirecto de billones de Euros (Gibbens, J. C, y col. 2001. Vet Rec 149: 729-43; Knowles, N. J., y col. 2001. Vet Rec 148: 258-9; Yang, P. C, y col. 1999. Vet Rec 145: 731-4).The foot-and-mouth disease virus (VFA) is a member of the Picornavirídae family, genus Aphthovirus, and is the causative agent of glossophobic or foot-and-mouth disease (AF), a very contagious and economically devastating disease that affects animals of cleft hoof ( cows, pigs, sheep and goats, among other animals), characterized by the appearance of vesicles on the legs and snout (Bachrach, HL 1978. Foot-and-Mouth disease: worldwide impact and control measures, p. 299-310. In EK A. K. Maramorosch (ed.), Viruses and environment, Academic Press, Inc., New York, NY; Rowlands, DJ 2003. Virus Res 91: 1-161; Sobrino, F., and E. Domingo. 2004. Foot-and-mouth disease, Horizon Press, London). The social and economic impact of AF can be catastrophic when an outbreak appears in VFA-free countries with immunologically naive animals. This was the case of the AF outbreak in Taiwan in 1997 and that of the United Kingdom in 2001, in which millions of infected and contact animals were slaughtered at a direct and indirect cost of billions of Euros (Gibbens, J. C, and col. 2001. Vet Rec 149: 729-43; Knowles, NJ, et al. 2001. Vet Rec 148: 258-9; Yang, P. C, et al. 1999. Vet Rec 145: 731-4).
El genoma de VFA consiste en una sola molécula de ARN de polaridad positiva de unos 8500 nucleótidos que codifica para una sola poliproteína. Ésta es cortada por proteasas virales para producir las 4 proteínas de Ia cápsida y las 9 proteínas no-estructurales, implicadas en distintos pasos de Ia replicación del virus (Belsham, 1993, Prog. Biophys. Mol. Biol. 60: 241-260; Masón y col. 2003. Virus Res. 91 : 9-32; Porter, 2003. J. Viral., 67: 6917-6921 ). La cápsida viral, de simetría icosaédrica, está compuesta por 60 copias de cada una de las proteínas estructurales VP1 , VP2, VP3 y VP4 (revisión en Bachrach, 1977.Foot and Mouth Disease virus, properties, molecular biology and immunogenicity, JA (Ed.), Beltsville Symposia in Agricultural Research, I, Virology in Agriculture. Allanheld, Osmun, Montclair, NJ). Similar a otros virus ARN, las poblaciones virales de VFA presentan una gran heterogeneidad genética reflejada en Ia gran diversidad serológica que presenta, con siete serotipos antigénicamente distintos, O, A, C, South African Territories (SAT) 1 , SAT 2, SAT 3 y Asia 1 (Pereira 1981. Foot-and-mouth disease virus. Pg. 333-363. In G. RPG (Ed.). Virus diseases of food animáis, VoI. 2, Academic Press, NY). La inmunidad protectora frente a un serotipo no protege frente a otros serotipos, Io que complica el diseño de vacunas.The VFA genome consists of a single polarity RNA molecule positive of about 8500 nucleotides that codes for a single polyprotein. This is cut by viral proteases to produce the 4 proteins of the capsid and the 9 non-structural proteins, involved in different steps of virus replication (Belsham, 1993, Prog. Biophys. Mol. Biol. 60: 241-260; Masón et al. 2003. Virus Res. 91: 9-32; Porter, 2003. J. Viral., 67: 6917-6921). The viral capsid, of icosahedral symmetry, is composed of 60 copies of each of the structural proteins VP1, VP2, VP3 and VP4 (review in Bachrach, 1977. Photo and Mouth Disease virus, properties, molecular biology and immunogenicity, JA (Ed .), Beltsville Symposia in Agricultural Research, I, Virology in Agriculture, Allanheld, Osmun, Montclair, NJ). Similar to other RNA viruses, viral populations of VFA have a great genetic heterogeneity reflected in the great serological diversity that it presents, with seven antigenically different serotypes, O, A, C, South African Territories (SAT) 1, SAT 2, SAT 3 and Asia 1 (Pereira 1981. Foot-and-mouth disease virus. Pg. 333-363. In G. RPG (Ed.). Virus diseases of food animáis, VoI. 2, Academic Press, NY). Protective immunity against a serotype does not protect against other serotypes, which complicates vaccine design.
Las vacunas frente a Ia FA actualmente en el mercado se obtienen a partir del crecimiento de virus en cultivos celulares de células BHK (Radlett y col., 1985. Dev. Biol. Standard, 60: 163; Telling, 1975. Industrial production of FMD vaccine using BHK suspensión cells. Some comparative results relating in vitro assays and cattle potency. In Report of the research Group of the Standing technical Committee of the European Commission for the control of Foot-and-Mouth Disease, Brescia, Italy. Food and Agriculture Organization. Rome, 95). Este virus se inactiva químicamente con un compuesto de aziridinas, de manera más concreta etilen-amina binaria (BEI en sus siglas inglesas) (revisión en Brown, F., 2001 , Vaccine, 20: 322-327). Las vacunas de virus inactivados, como es el caso de FA, deben llevar adyuvantes que confiera Ia inmunidad suficiente en los animales. El adyuvante más utilizado para el ganado vacuno y ovino es hidróxido de aluminio, pero para el ganado porcino es preciso utilizar aceite mineral como adyuvante incompleto de Freund (Freund & Thompson, 1945, Science 101 : 468). Por Io general, se preparan vacunas monovalentes, pero en los países en los que circula más de un tipo de virus, se pueden utilizar las vacunas polivalentes correspondientes.Vaccines against FA currently on the market are obtained from virus growth in BHK cell cell cultures (Radlett et al., 1985. Dev. Biol. Standard, 60: 163; Telling, 1975. Industrial production of FMD vaccine using BHK suspension cells. Some comparative results relating in vitro assays and cattle potency. In Report of the research Group of the Standing technical Committee of the European Commission for the control of Foot-and-Mouth Disease, Brescia, Italy. Organization, Rome, 95). This virus is chemically inactivated with a compound of aziridines, more specifically ethylene-binary amine (BEI) (review in Brown, F., 2001, Vaccine, 20: 322-327). Inactivated virus vaccines, as in the case of AF, must carry adjuvants that confer sufficient immunity on animals. He The most commonly used adjuvant for cattle and sheep is aluminum hydroxide, but for pigs it is necessary to use mineral oil as Freund's incomplete adjuvant (Freund & Thompson, 1945, Science 101: 468). Generally, monovalent vaccines are prepared, but in countries where more than one type of virus circulates, the corresponding polyvalent vaccines can be used.
Las vacunas actualmente en uso tienen que cumplir los siguientes requisitos en su producción: 1- el antígeno viral se debe producir en grandes cantidades porque cada dosis de vacuna necesita contener altos niveles de virus inactivado para ser efectivas;Vaccines currently in use have to meet the following requirements in their production: 1- the viral antigen must be produced in large quantities because each dose of vaccine needs to contain high levels of inactivated virus to be effective;
2- Ia preparación del virus se debe inactivar de tal manera que no quede ninguna infectividad residual pero que al mismo tiempo mantenga Ia inmunogenicidad del virus;2- The preparation of the virus should be inactivated in such a way that there is no residual infectivity but at the same time it maintains the immunogenicity of the virus;
3- un adyuvante se debe añadir a Ia vacuna para potenciar Ia respuesta de anticuerpos frente a las proteínas virales.3- An adjuvant should be added to the vaccine to enhance the antibody response against viral proteins.
Una vacuna viva debe mantener preferiblemente el complemento antigénico de Ia cepa de tipo salvaje. Además, Ia vacuna viva debe ser suficientemente avirulenta para evitar efectos patológicos inaceptables, pero por otra parte debe provocar un nivel suficiente de inmunidad en el hospedador. Finalmente, Ia vacuna viva atenuada no debe tener, preferiblemente, probabilidad de revertir a una cepa de tipo salvaje virulenta.A live vaccine should preferably maintain the antigenic complement of the wild type strain. In addition, the live vaccine must be sufficiently avirulent to avoid unacceptable pathological effects, but on the other hand it must cause a sufficient level of immunity in the host. Finally, the live attenuated vaccine should preferably not have the probability of reverting to a virulent wild type strain.
Estas vacunas tienen inconvenientes grandes:These vaccines have big drawbacks:
1- el posible escape de virus durante Ia producción de grandes cantidades de virus virulento; 2- posibilidad de un brote debido a Ia incorrecta inactivación del virus virulento usado para Ia producción de Ia vacuna con Io que se puede producir una infección que, aunque asintomática, de lugar a un brote; 3- Ia inmunidad no es muy duradera por Io que los animales necesitan dosis de recuerdo cada 6 meses.1- the possible escape of virus during the production of large quantities of virulent virus; 2- possibility of an outbreak due to the incorrect inactivation of the virulent virus used for the production of the vaccine with what can be produce an infection that, although asymptomatic, results in an outbreak; 3 - The immunity is not very durable because animals need a dose of memory every 6 months.
Una manera de paliar estos inconvenientes sería el uso de virus atenuados que, aunque replicando, no provocan ningún tipo de enfermedad. Debido a Ia enorme variabilidad, ya comentada, de los virus ARN, no se ha elegido esta técnica como válida ya que puede resultar en Ia aparición de un revertiente de Ia atenuación y que se vuelva virulento, con el consiguiente peligro para Ia aparición de un nuevo brote de FA. Sin embargo, este tipo de vacunas daría una inmunidad larga y duradera, sin necesidad de dosis de recuerdo, ya que mimetizaría Io que ocurre en una infección natural.One way to alleviate these inconveniences would be the use of attenuated viruses that, although replicating, do not cause any type of disease. Due to the enormous variability, already commented, of the RNA viruses, this technique has not been chosen as valid since it can result in the appearance of a reversal of the attenuation and that becomes virulent, with the consequent danger for the appearance of a new outbreak of FA. However, this type of vaccine would give a long and lasting immunity, without the need for a souvenir dose, since it would mimic what happens in a natural infection.
Existe Ia necesidad de encontrar un sistema que proporcione soluciones completas al problema de encontrar nuevas vacunas frente a Ia FA y que carezca de los inconvenientes que presentan las vacunas actuales, principalmente el posible escape de virus virulento de las plantas de producción o Ia posibilidad de Ia presencia de virus virulento residual en Ia formulación de vacuna inactivada, Io que daría lugar a nuevos brotes de FA.There is a need to find a system that provides complete solutions to the problem of finding new vaccines against AF and that lacks the inconveniences of current vaccines, mainly the possible escape of virulent virus from production plants or the possibility of Ia presence of residual virulent virus in the inactivated vaccine formulation, which would lead to new outbreaks of AF.
BREVE DESCRIPCIÓN DE LA INVENCIÓNBRIEF DESCRIPTION OF THE INVENTION
La presente invención proporciona una vacuna que tiene dos barreras de seguridad frente a Ia vacuna convencional: primera, está altamente atenuada in vivo, Io que hace que en caso de que haya un escape durante Ia producción de Ia misma no suponga ningún tipo de riesgo como posible inicio de un brote; segunda, está compuesta por virus defectivos que necesitan complementar para producir una infección productiva, y esto sólo sucede a muy alta multiplicidad de infección (MOI), Io que in vivo es altamente improbable que suceda. En cualquier caso, si esta posibilidad se diera en el punto de inoculación, aunque se obtuviera el virus completo por recombinación o por complementación, el virus resultante sería Ia población C-S8p260, que está totalmente atenuada. Además, dicha vacuna, al ser un virus completo, es capaz de conferir una inmunidad que será más larga que Ia conferida por Ia actual vacuna y más específica y robusta que Ia conferida por las vacunas recombinantes. La razón para todo ello es que, al tratarse de un virus completo, expresará un amplio repertorio de epítopos T y B necesarios para Ia completa eliminación del virus.The present invention provides a vaccine that has two safety barriers against the conventional vaccine: first, it is highly attenuated in vivo, which means that if there is an escape during its production, it does not entail any risk such as possible start of an outbreak; second, it is composed of defective viruses that need to complement to produce a productive infection, and this only happens at a very high multiplicity of infection (MOI), which in vivo is highly unlikely to happen. In any case, if this possibility occurred at the point of inoculation, even if the complete virus was obtained by recombination or by complementation, the resulting virus would be the C-S8p260 population, which is fully attenuated. In addition, said vaccine, being a complete virus, is capable of conferring an immunity that will be longer than that conferred by the current vaccine and more specific and robust than that conferred by recombinant vaccines. The reason for all this is that, being a complete virus, it will express a wide repertoire of T and B epitopes necessary for the complete elimination of the virus.
DESCRIPCIÓN DE LA INVENCIÓNDESCRIPTION OF THE INVENTION
De acuerdo con un aspecto de Ia presente invención, se proporciona una población de virus aislada, de aquí en adelante población de virus de Ia invención, caracterizada porque comprende una secuencia de polinucleótidos que codifica una secuencia aminoacídica homologa a Ia SEQ ID NO: 1 , y al menos una de las secuencias seleccionada de entre: a) una secuencia de polinucleótidos que codifica una secuencia aminoacídica homologa a Ia SEQ ID NO: 2, b) una secuencia de polinucleótidos que codifica una secuencia aminoacídica homologa a Ia SEQ ID NO: 3, para su uso como medicamento.In accordance with one aspect of the present invention, an isolated virus population is provided, henceforth a population of viruses of the invention, characterized in that it comprises a polynucleotide sequence that encodes an amino acid sequence homologous to SEQ ID NO: 1, and at least one of the sequences selected from: a) a polynucleotide sequence encoding an amino acid sequence homologous to SEQ ID NO: 2, b) a polynucleotide sequence encoding an amino acid sequence homologous to SEQ ID NO: 3 , for use as a medicine.
El término "homología", tal y como se utiliza en esta memoria, hace referencia a Ia semejanza entre dos estructuras debida a una ascendencia evolutiva común, y más concretamente, a Ia semejanza entre los aminoácidos de dos o más proteínas o secuencias aminoacídicas. Dos proteínas se consideran homologas si tienen el mismo origen evolutivo o si tienen función y estructura similares. En el caso particular de las variantes víricas con deleciones en el genoma, aunque las variantes de Ia invención se han obtenido a partir de un solo serotipo de VFA (Foot-and-mouth disease virus (FMDV) strain C1 -Santa Pau ó C-S8, perteneciente al serotipo C ó Aphthovirus C ), a Ia que se hace referencia de aquí en adelante como VFA, es suficiente para permitir a un experto en Ia materia obtener variantes con genomas delecionados en las mismas regiones para otras cepas que estén comprendidas dentro de Ia especie. Así, entre los distintos tipos de VFA, y sin limitarse a estos, se incluye:The term "homology", as used herein, refers to the similarity between two structures due to a common evolutionary ancestry, and more specifically, to the similarity between the amino acids of two or more proteins or amino acid sequences. Two proteins are considered homologous if they have the same evolutionary origin or if they have similar function and structure. In the particular case of viral variants with deletions in the genome, although the variants of the invention they have been obtained from a single serotype of VFA (Foot-and-mouth disease virus (FMDV) strain C1-Santa Pau or C-S8, belonging to serotype C or Aphthovirus C), which is referred to here in Hereafter as VFA, it is sufficient to allow a person skilled in the art to obtain variants with genomes deleted in the same regions for other strains that are comprised within the species. Thus, among the different types of VFA, and not limited to these, it includes:
- Virus de Ia fiebre aftosa - tipo A (=Aphthovirus A). - Virus de Ia fiebre aftosa - tipo Asia 1 (=Aphthovirus Asia 1 ).- Foot and mouth disease virus - type A (= Aphthovirus A). - Foot and mouth disease virus - type Asia 1 (= Aphthovirus Asia 1).
- Virus de Ia fiebre aftosa - tipo C (=Aphthovirus C).- Foot and mouth disease virus - type C (= Aphthovirus C).
- Virus de Ia fiebre aftosa - tipo O (=Aphthovirus O).- Foot and mouth disease virus - type O (= Aphthovirus O).
- Virus de Ia fiebre aftosa - tipo SAT 1 (=Aphthovirus SAT1 ).- Foot and mouth disease virus - type SAT 1 (= Aphthovirus SAT1).
- Virus de Ia fiebre aftosa - tipo SAT 2 (=Aphthovirus SAT2). - Virus de Ia fiebre aftosa - tipo SAT 3 (=Aphthovirus SAT3).- Foot and mouth disease virus - type SAT 2 (= Aphthovirus SAT2). - Foot and mouth disease virus - type SAT 3 (= Aphthovirus SAT3).
El término "identidad", tal y como se utiliza en esta memoria, hace referencia a Ia proporción de aminoácidos idénticos entre dos secuencias aminoácidicas que se comparan. Los métodos de comparación de secuencias son conocidos en el estado de Ia técnica, e incluyen, aunque sin limitarse a ellos, el programa BLASTP o BLASTN, y FASTA (Altschul et al., J. Mol. Biol. 215: 403-410 (1999). Puesto que dos proteínas se consideran homologas si tienen el mismo origen evolutivo o si tienen función y estructura similares, en general, se asume que valores superiores de similitud o identidad del 30% indican estructuras homologas. Podemos considerar, por tanto, que porcentajes de identidad de, al menos, un 80%, mantendrán las mismas propiedades de dicho péptido.The term "identity", as used herein, refers to the proportion of identical amino acids between two amino acid sequences that are compared. Sequence comparison methods are known in the state of the art, and include, but are not limited to, the BLASTP or BLASTN program, and FASTA (Altschul et al., J. Mol. Biol. 215: 403-410 ( 1999) Since two proteins are considered homologous if they have the same evolutionary origin or if they have similar function and structure, in general, it is assumed that higher values of similarity or identity of 30% indicate homologous structures. Identity percentages of at least 80% will maintain the same properties of said peptide.
Debido a Ia interrelación evolutiva de las cepas de VFA, las cepas de VFA putativas son identificables por su homología al nivel de genoma o de los polipéptidos codificados por el mismo. Generalmente, las cepas de VFA tienen una identidad mayor de 80%, preferiblemente una identidad mayor de 90 %, y aún más preferiblemente una identidad mayor de 95%, y aún más preferiblemente una identidad mayor de 99% al nivel de las secuencias aminoacídicas. Los métodos para determinar Ia homología y porcentaje de identidad de las secuencias de aminoácidos son conocidos en Ia técnica. Por ejemplo, Ia secuencia de aminoácidos se puede determinar directamente y puede compararse con las secuencias que se proporcionan en esta memoria. Por ejemplo también, Ia secuencia de nucleótidos del material genómico del VFA putativo se puede determinar (usualmente por Ia vía de un compuesto intermedio de cDNA), y deducirse Ia secuencia de aminoácidos codificada en ella, para comparar con las regiones correspondientes de las secuencias proporcionadas en esta memoria.Due to the evolutionary interrelation of the VFA strains, the putative VFA strains are identifiable by their homology at the genome level or of the polypeptides encoded by it. Generally, VFA strains they have an identity greater than 80%, preferably an identity greater than 90%, and even more preferably an identity greater than 95%, and even more preferably an identity greater than 99% at the level of the amino acid sequences. The methods for determining the homology and percent identity of amino acid sequences are known in the art. For example, the amino acid sequence can be determined directly and can be compared with the sequences provided herein. For example also, the nucleotide sequence of the putative VFA genomic material can be determined (usually by means of an intermediate cDNA compound), and deduced the amino acid sequence encoded therein, to compare with the corresponding regions of the sequences provided in this memory.
De acuerdo con otro aspecto de Ia presente invención, Ia población de virus aislada de Ia invención está caracterizada porque comprende una secuencia de polinucleótidos que codifica una secuencia aminoacídica, de aquí en adelante primera secuencia aminoacídica de Ia invención, que comprende un péptido con una identidad de al menos un 80% con Ia SEQ ID NO: 1 , y al menos una de las secuencias seleccionada de entre: a) una secuencia de polinucleótidos que codifica una secuencia aminoacídica, de aquí en adelante segunda secuencia aminoacídica de Ia invención, que comprende un péptido con una identidad de al menos un 80% con Ia SEQ ID NO: 2, b) una secuencia de polinucleótidos que codifica una secuencia aminoacídica, de aquí en adelante tercera secuencia aminoacídica de Ia invención, que comprende un péptido con una identidad de al menos un 80% con Ia SEQ ID NO: 3, para su uso como medicamento. Los virus con Ia secuencia aminoacídica SEQ ID NO: 1 pertenecen a Ia variante C-S8p260D417, presentan una deleción de 417 nucleótidos en Ia región que codifica para Ia proteasa L, respecto a Ia secuencia de polinucleótidos de Ia variante C-S8. La deleción Δ417 incluye de las posiciones 1153 a 1517 (del genoma del VFA, siguiendo Ia numeración de Escarmís et al. 1996. Genetic lesions associated with Muller's ratchet in an ARN virus. J. Mol. Biol. 264:255-267).According to another aspect of the present invention, the isolated virus population of the invention is characterized in that it comprises a polynucleotide sequence encoding an amino acid sequence, hereinafter the first amino acid sequence of the invention, which comprises a peptide with an identity of at least 80% with SEQ ID NO: 1, and at least one of the sequences selected from: a) a polynucleotide sequence encoding an amino acid sequence, hereinafter the second amino acid sequence of the invention, comprising a peptide with an identity of at least 80% with SEQ ID NO: 2, b) a polynucleotide sequence encoding an amino acid sequence, hereinafter third amino acid sequence of the invention, comprising a peptide with an identity of at least 80% with SEQ ID NO: 3, for use as a medicine. Viruses with the amino acid sequence SEQ ID NO: 1 belong to the variant C-S8p260D417, have a deletion of 417 nucleotides in the region coding for protease L, with respect to the polynucleotide sequence of variant C-S8. The Δ417 deletion includes positions 1153 to 1517 (of the VFA genome, following the numbering of Escarmís et al. 1996. Genetic lesions associated with Muller's ratchet in an RNA virus. J. Mol. Biol. 264: 255-267).
Los virus con Ia secuencia aminoacídica SEQ ID NO: 2 pertenecen a Ia variante C-S8p260D999, y presentan una deleción de 999 nucleótidos en Ia región que codifica para las proteínas estructurales (Δ999), respecto a Ia secuencia de polinucleótidos de Ia variante C-S8. La deleción Δ999 incluye de las posiciones 2793 a 3793 del genoma del VFA, siguiendo Ia misma numeración que en el caso anterior. Los virus con Ia secuencia aminoacídica SEQ ID NO: 3 pertenecen a Ia variante C-S8p260D1017, y presentan una deleción de 1017 nucleótidos en Ia región que codifica para las proteínas estructurales (Δ1017), respecto a Ia secuencia de polinucleótidos de Ia variante C-S8. La deleción Δ1017 incluye de las posiciones 1932 a 2950 del genoma del VFA, siguiendo Ia misma numeración que en los casos anteriores.The viruses with the amino acid sequence SEQ ID NO: 2 belong to the variant C-S8p260D999, and have a deletion of 999 nucleotides in the region that codes for the structural proteins (Δ999), with respect to the polynucleotide sequence of the variant C- S8 The Δ999 deletion includes positions 2793 to 3793 of the VFA genome, following the same numbering as in the previous case. The viruses with the amino acid sequence SEQ ID NO: 3 belong to the variant C-S8p260D1017, and have a deletion of 1017 nucleotides in the region that codes for the structural proteins (Δ1017), with respect to the polynucleotide sequence of the variant C- S8 The Δ1017 deletion includes positions 1932 to 2950 of the VFA genome, following the same numbering as in the previous cases.
Los virus con las secuencias aminoacídicas SEQ ID NO: 1 , SEQ ID NO: 2 y SEQ ID NO: 3 son incapaces de generar, cada uno individualmente, virus infeccioso con capacidad para replicar. Lo pueden originar por complementación (entre SEQ ID NO: 1 y o bien SEQ ID NO: 2 o bien SEQ ID NO: 3) o por recombinación. Pero aunque es posible que Ia complementación se dé transitoriamente y que ello favorezca Ia inducción de anticuerpos y Ia respuesta inmune celular, Ia recombinación no es un problema porque Ia cepa C-S8p260 es atenuada.Viruses with the amino acid sequences SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 are incapable of generating, each individually, infectious virus with the ability to replicate. They can be caused by complementation (between SEQ ID NO: 1 and either SEQ ID NO: 2 or SEQ ID NO: 3) or by recombination. But although it is possible that the complementation occurs temporarily and that this favors the induction of antibodies and the cellular immune response, recombination is not a problem because the strain C-S8p260 is attenuated.
En esta memoria se entiende por "población de virus" o "población viral" a una pluralidad de virus existente en cualquier forma, y que comprenda al menos dos variantes virales de un virus de Ia misma especie. Por ejemplo, una población de virus puede ser una suspensión de partículas de virus presentes en un medio de cultivo celular o en otra solución. Puede ser también un pellet o una preparación liofilizada conteniendo los virus.In this report it is understood by "virus population" or "viral population" to a plurality of viruses existing in any form, and comprising at least two viral variants of a virus of the same species. For example, a population of viruses may be a suspension of virus particles present in a cell culture medium or in another solution. It can also be a pellet or a lyophilized preparation containing the viruses.
Otro aspecto de Ia invención se refiere a una cápside proteica aislada, de aquí en adelante cápside proteica aislada de Ia invención, que comprende al menos una de las secuencias aminoacídicas de Ia invención, para su uso como medicamento.Another aspect of the invention relates to an isolated protein capsid, hereinafter isolated protein capsid of the invention, comprising at least one of the amino acid sequences of the invention, for use as a medicine.
De acuerdo con otro aspecto de Ia presente invención, se proporciona una construcción genética, de aquí en adelante construcción genética de Ia invención, que dirigiría Ia trascripción in vitro o intracelular de las secuencias de polinucleótidos de Ia población de virus de Ia invención, y comprende Ia secuencia de polinucleótidos que codifica Ia primera secuencia aminoacídica de Ia invención y otra secuencia seleccionada de entre: a. secuencia de polinucleótidos, que codifica Ia segunda secuencia aminoacídica de Ia invención, b. secuencia de polinucleótidos, que codifica Ia tercera secuencia aminoacídica de Ia invención, c. moléculas de ácido nucleico cuya cadena complementaria híbrida con Ia secuencia polinucleotídica de a) y/o b), d. secuencia de nucleótidos de a), b), ó c), preferiblemente de doble cadena, correspondiente a un sistema o vector de expresión génica, operativamente enlazada con, al menos, un promotor que dirija Ia transcripción de dicha secuencia de nucleótidos de interés, y con otras secuencias necesarias o apropiadas para Ia transcripción y su regulación adecuada en tiempo y lugar, por ejemplo, señales de inicio y terminación, sitios de corte, señal de poliadenilación, origen de replicación, activadores transcripcionales (enhancers), silenciadores transcripcionales (silencers), etc..In accordance with another aspect of the present invention, a genetic construction is provided, henceforth genetic construction of the invention, which would direct the in vitro or intracellular transcription of the polynucleotide sequences of the virus population of the invention, and comprises The polynucleotide sequence encoding the first amino acid sequence of the invention and another sequence selected from: a. polynucleotide sequence, which encodes the second amino acid sequence of the invention, b. polynucleotide sequence, which encodes the third amino acid sequence of the invention, c. nucleic acid molecules whose complementary hybrid chain with the polynucleotide sequence of a) and / or b), d. nucleotide sequence of a), b), or c), preferably double stranded, corresponding to a gene expression system or vector, operably linked to at least one promoter that directs the transcription of said nucleotide sequence of interest, and with other sequences necessary or appropriate for the transcription and its adequate regulation in time and place, for example, start and end signals, cut-off sites, signal of polyadenylation, origin of replication, transcriptional activators (enhancers), transcriptional silencers (silencers), etc.
Esta construcción genética incluye los vectores de clonación y expresión que comprenden las moléculas de ácidos nucleicos del sistema de expresión de Ia invención. Tales vectores de expresión incluyen secuencias de control adecuadas, tales como, por ejemplo, elementos de control de Ia traducción (como códigos de iniciación y de parada) y de Ia transcripción (por ejemplo, regiones de promotor-operador, sitios de unión). Los vectores conforme a Ia invención pueden incluir plásmidos y virus (comprendiendo bacteriófagos y virus eucarióticos), de acuerdo con procedimientos bien conocidos y documentados en Ia técnica, y pueden expresarse en una variedad de sistemas de expresión diferentes, asimismo bien conocidos y documentados en Ia técnica. Muchos otros vectores virales y no virales están descritos y son conocidos en Ia técnica.This genetic construction includes the cloning and expression vectors that comprise the nucleic acid molecules of the expression system of the invention. Such expression vectors include suitable control sequences, such as, for example, control elements for translation (such as start and stop codes) and for transcription (for example, promoter-operator regions, binding sites). The vectors according to the invention can include plasmids and viruses (comprising bacteriophages and eukaryotic viruses), according to procedures well known and documented in the art, and can be expressed in a variety of different expression systems, also well known and documented in Ia technique. Many other viral and non-viral vectors are described and are known in the art.
Se conoce, así mismo, una variedad de técnicas que pueden utilizarse para introducir tales vectores en células procarióticas o eucarióticas para su expresión. Técnicas adecuadas de transformación o transfección están bien descritas en Ia bibliografía.A variety of techniques that can be used to introduce such vectors into prokaryotic or eukaryotic cells for expression are also known. Appropriate transformation or transfection techniques are well described in the literature.
Los términos "polinucleótido" y "ácido nucleico" se usan aquí de manera intercambiable, refiriéndose a formas poliméricas de nucleótidos de cualquier longitud, tanto ribonucleótidos como desoxiribonucleótidos.The terms "polynucleotide" and "nucleic acid" are used interchangeably herein, referring to polymeric forms of nucleotides of any length, both ribonucleotides and deoxyribonucleotides.
Los términos "péptido", "oligopéptido", "polipéptido" y "proteína" se usan aquí de manera intercambiable, y se refieren a una forma polimérica de aminoácidos de cualquier longitud, que pueden ser codificantes o no codificantes, química o bioquímicamente modificados.The terms "peptide", "oligopeptide", "polypeptide" and "protein" are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which may be coding or non-coding, chemically or biochemically modified.
La población de virus aislados de Ia invención, las secuencias aminoacídicas de Ia invención, Ia cápsida proteica aislada de Ia invención, Ia construcción genética de Ia invención, o cualquiera de sus combinaciones se pueden formular en composiciones para usar como inmunógeno (de aquí en adelante, inmunógenos de Ia invención). Estos inmunógenos pueden también ser usados como vacunas en animales, y más particularmente en mamíferos, incluyendo humanos, o producir una respuesta en Ia producción de anticuerpos en animales. Para Ia formulación de tales composiciones, una cantidad efectiva inmunológicamente de al menos uno de los virus, de las secuencias aminoacídicas o de las cápsides es mezclado con un transportador adecuado aceptable fisiológicamente para Ia administración a mamíferos incluyendo humanos. Los inmunógenos pueden estar covalentemente ligados entre ellos, a otros péptidos, a una proteína transportadora o con otros transportadores, incorporados en liposomas u otras vesículas similares, y/o mezclados con un adyuvante o absorbente como es conocido en el campo de las vacunas. Por ejemplo, pueden ser mezclados con otros complejos inmunoestimuladores. Alternativamente, los inmunógenos no están acoplados y meramente mezclados con un transportador aceptable fisiológicamente tal como un compuesto tampón o salino normal adecuado para Ia administración a mamíferos incluyendo humanos.The population of viruses isolated from the invention, the sequences amino acids of the invention, the isolated protein capsid of the invention, the genetic construction of the invention, or any combination thereof can be formulated in compositions for use as an immunogen (hereinafter, immunogens of the invention). These immunogens can also be used as vaccines in animals, and more particularly in mammals, including humans, or produce a response in the production of antibodies in animals. For the formulation of such compositions, an immunologically effective amount of at least one of the viruses, amino acid sequences or capsids is mixed with a physiologically acceptable carrier suitable for administration to mammals including humans. The immunogens may be covalently linked to each other, to other peptides, to a transporter protein or to other carriers, incorporated into liposomes or other similar vesicles, and / or mixed with an adjuvant or absorbent as is known in the field of vaccines. For example, they can be mixed with other immunostimulatory complexes. Alternatively, the immunogens are not coupled and merely mixed with a physiologically acceptable transporter such as a normal buffer or saline compound suitable for administration to mammals including humans.
Por tanto, y como se ha descrito anteriormente, los inmunógenos de Ia invención presentan secuencias antigénicas protectoras. Estos antígenos protectores son capaces de generar una respuesta inmune (inmunogénica) protectora del hospedador, es decir, una respuesta del hospedador que conduce a Ia generación de moléculas efectoras inmunes, anticuerpos o células que dañan, inhiben o matan a Ia entidad biológica invasora, "protegiendo" así al hospedador de una enfermedad clínica o sub-clínica y de una pérdida de productividad. Tal respuesta inmune protectora puede manifestarse comúnmente por Ia generación de anticuerpos. En otro aspecto de Ia invención, los anticuerpos producidos tras Ia inmunización del animal, de aquí en adelante anticuerpos de Ia invención, son usados como medicamento.Therefore, and as described above, the immunogens of the invention have protective antigenic sequences. These protective antigens are capable of generating a protective (immunogenic) host immune response, that is, a host response that leads to the generation of immune effector molecules, antibodies or cells that damage, inhibit or kill the invading biological entity, " thus protecting "the host from a clinical or sub-clinical disease and a loss of productivity. Such protective immune response can commonly be manifested by the generation of antibodies. In another aspect of the invention, the antibodies produced after the immunization of the animal, hereinafter antibodies of the invention, are used as medicine.
Los anticuerpos de Ia presente invención pueden formularse para su administración a un animal, y más preferiblemente a un mamífero, incluyendo al hombre, en una variedad de formas. Así, los anticuerpos pueden estar en disolución acuosa estéril o en fluidos biológicos, tal como suero. Las disoluciones acuosas pueden estar tamponadas o no tamponadas y tienen componentes activos o inactivos adicionales. Los componentes adicionales incluyen sales para modular Ia fuerza iónica, conservantes incluyendo, pero sin limitarse a, agentes antimicrobianos, antioxidantes, quelantes, y similares, y nutrientes incluyendo glucosa, dextrosa, vitaminas y minerales. Alternativamente, los anticuerpos pueden prepararse para su administración en forma sólida. Los anticuerpos pueden combinarse con varios vehículos o excipientes inertes, incluyendo pero sin limitarse a; aglutinantes tales como celulosa microcristalina, goma tragacanto, o gelatina; excipientes tales como almidón o lactosa; agentes dispersantes tales como ácido algínico o almidón de maíz; lubricantes tales como estearato de magnesio, deslizantes tales como dióxido de silicio coloidal; agentes edulcorantes tales como sacarosa o sacarina; o agentes aromatizantes tales como menta o salicilato de metilo.The antibodies of the present invention can be formulated for administration to an animal, and more preferably to a mammal, including man, in a variety of ways. Thus, the antibodies may be in sterile aqueous solution or in biological fluids, such as serum. Aqueous solutions may be buffered or unbuffered and have additional active or inactive components. Additional components include salts to modulate the ionic strength, preservatives including, but not limited to, antimicrobial agents, antioxidants, chelants, and the like, and nutrients including glucose, dextrose, vitamins and minerals. Alternatively, the antibodies can be prepared for solid administration. Antibodies can be combined with various inert carriers or excipients, including but not limited to; binders such as microcrystalline cellulose, gum tragacanth, or gelatin; excipients such as starch or lactose; dispersing agents such as alginic acid or corn starch; lubricants such as magnesium stearate, glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; or flavoring agents such as peppermint or methyl salicylate.
Los anticuerpos o sus formulaciones pueden administrarse a un animal, incluyendo un mamífero y, por tanto, al hombre, en una variedad de formas. Tales medios incluyen, pero sin limitarse a, intraperitoneal, intravenoso, intramuscular, subcutáneo, intracecal, intraventricular, oral, enteral, parenteral, intranasal o dérmico.Antibodies or their formulations can be administered to an animal, including a mammal and, therefore, to man, in a variety of ways. Such means include, but are not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intracecal, intraventricular, oral, enteral, parenteral, intranasal or dermal.
La dosificación de anticuerpos para obtener una cantidad farmacéuticamente eficaz depende de una variedad de factores, como por ejemplo, Ia edad, peso, sexo, tolerancia,... del animal. Otro aspecto de Ia invención se refiere a una composición, de aquí en adelante composición de Ia invención, que comprende Ia población de virus aislada de Ia invención, de las secuencias aminoacídicas de Ia invención, una cápside proteica aislada de Ia invención, un anticuerpo de Ia invención, Ia construcción genética de Ia invención, o cualquiera de sus combinaciones, para su uso como medicamento. En una realización preferida de este aspecto de Ia invención, Ia composición de Ia invención se usa para el tratamiento o prevención de Ia fiebre aftosa.The dosage of antibodies to obtain a pharmaceutically effective amount depends on a variety of factors, such as, for example, the age, weight, sex, tolerance, ... of the animal. Another aspect of the invention relates to a composition, hereinafter composition of the invention, which comprises the population of virus isolated from the invention, of the amino acid sequences of the invention, an isolated protein capsid of the invention, an antibody of The invention, the genetic construction of the invention, or any combination thereof, for use as a medicine. In a preferred embodiment of this aspect of the invention, the composition of the invention is used for the treatment or prevention of foot and mouth disease.
Más preferiblemente, los virus, las secuencias aminoacídicas, Ia o las cápsides, las construcciones genéticas de Ia invención, o cualquiera de sus combinaciones, se encuentran, o se traducen, en una cantidad terapéuticamente efectiva, capaz de generar anticuerpos para su uso en Ia elaboración de vacunas.More preferably, viruses, amino acid sequences, Ia or capsids, genetic constructs of the invention, or any combination thereof, are found, or translated, in a therapeutically effective amount, capable of generating antibodies for use in Ia vaccine development
En el contexto de Ia presente invención el término "vacuna" se refiere a una preparación antigénica empleada para establecer Ia respuesta del sistema inmune a una enfermedad. Son preparados de antígenos que una vez dentro del organismo provocan Ia respuesta del sistema inmunitario, mediante Ia producción de anticuerpos, y generan memoria inmunológica produciendo inmunidad permanente o transitoria.In the context of the present invention the term "vaccine" refers to an antigen preparation used to establish the immune system response to a disease. They are prepared of antigens that once inside the organism cause the response of the immune system, through the production of antibodies, and generate immunological memory producing permanent or transient immunity.
El término "antígeno" en esta memoria se refiere a una molécula (generalmente una proteína o un polisacárido) de superficie celular, que puede inducir Ia formación de anticuerpos. Hay muchos tipos de moléculas diferentes que pueden actuar de antígenos, como las proteínas o péptidos, los polisacáridos y, más raramente, otras moléculas como los ácidos nucleicos.The term "antigen" herein refers to a cell surface molecule (generally a protein or a polysaccharide), which can induce the formation of antibodies. There are many different types of molecules that can act as antigens, such as proteins or peptides, polysaccharides and, more rarely, other molecules such as nucleic acids.
El término "medicamento", tal y como se usa en esta memoria, hace referencia a cualquier sustancia usada para prevención, diagnóstico, alivio, tratamiento o curación de enfermedades en el hombre y los animales. En el contexto de Ia presente invención se refiere, también, a los virus de Ia invención, Ia cápside de Ia invención, Ia construcción genética de Ia invención, el plásmido de Ia invención o Ia composición de Ia invención, que es capaz de generar una respuesta inmune frente a un organismo dado, que está causando dicha enfermedad en el hombre o los animales. Incluye, por tanto, Io que se conoce como vacuna, tal y como se ha definido previamente en esta memoria.The term "medication", as used herein, refers to any substance used for prevention, diagnosis, relief, treatment or cure of diseases in man and animals. In the context of the present invention, it also refers to the viruses of the invention, the capsid of the invention, the genetic construction of the invention, the plasmid of the invention or the composition of the invention, which is capable of generating a immune response against a given organism, which is causing such disease in man or animals. It includes, therefore, what is known as a vaccine, as previously defined herein.
Por tanto, en otra realización preferida, Ia composición de Ia invención es una vacuna, de aquí en adelante vacuna de Ia invención. En una realización más preferida, Ia vacuna además comprende excipientes farmacológicamente aceptables. En otra realización aún más preferida, Ia vacuna comprende un adyuvante. En otra realización Ia vacuna presenta un origen recombinante. En otra realización preferida Ia vacuna es polivalente.Therefore, in another preferred embodiment, the composition of the invention is a vaccine, henceforth a vaccine of the invention. In a more preferred embodiment, the vaccine also comprises pharmacologically acceptable excipients. In another even more preferred embodiment, the vaccine comprises an adjuvant. In another embodiment, the vaccine has a recombinant origin. In another preferred embodiment, the vaccine is versatile.
En esta memoria, el término "adyuvante" se refiere a un agente, mientras no posea un efecto antigénico por si mismo, que puede estimular el sistema inmune incrementando su respuesta a Ia vacuna. Aunque sin limitarse a ellas, las sales de aluminio "fosfato de aluminio" e "hidróxido de aluminio" son los dos adyuvantes más comúnmente empleados en las vacunas. Otras sustancias, como por ejemplo el escualeno, también se pueden emplear como adyuvantes.In this report, the term "adjuvant" refers to an agent, as long as it does not have an antigenic effect in itself, which can stimulate the immune system by increasing its response to the vaccine. Although not limited to them, aluminum salts "aluminum phosphate" and "aluminum hydroxide" are the two adjuvants most commonly used in vaccines. Other substances, such as squalene, can also be used as adjuvants.
Tal y como se define en esta memoria, el término "polivalente" se usa para referirse a Ia vacuna que comprende Ia combinación de dos o más antígenos en total, incluyendo uno o más de cualquiera de los serotipos, tipos, variedades o mutantes que se incluyen dentro de Ia clasificación de Virus de Ia Fiebre Aftosa (VFA). Un método alternativo de Ia producción de vacunas es el uso de técnicas de biología molecular para producir una proteína de fusión que contiene una o varias de las secuencias aminoacídicas de Ia presente invención y un péptido o proteína altamente inmunogénico/a, frente a una determinada infección. Por tanto, en otro aspecto, Ia vacuna de Ia invención presenta un origen recombinante.As defined herein, the term "polyvalent" is used to refer to the vaccine comprising the combination of two or more antigens in total, including one or more of any of the serotypes, types, varieties or mutants that are they include within the classification of Foot and Mouth Disease Virus (VFA). An alternative method of vaccine production is the use of molecular biology techniques to produce a fusion protein that contains one or more of the amino acid sequences of the present invention and a highly immunogenic peptide or protein, against a certain infection. . Therefore, in another aspect, the vaccine of the invention has a recombinant origin.
Un "vector" es un replicón al que se ha unido otro segmento polinucleótido, para realizar Ia replicación y/o expresión del segmento unido.A "vector" is a replicon to which another polynucleotide segment has been attached, to perform the replication and / or expression of the bound segment.
Un "replicón" es cualquier elemento genético que se comporta como una unidad autónoma de replicación polinucleótida dentro de una célula; esto es, capaz de replicarse bajo su propio control.A "replicon" is any genetic element that behaves as an autonomous unit of polynucleotide replication within a cell; that is, able to replicate under its own control.
"Secuencia de control" se refiere a secuencias de polinucleótidos que son necesarias para efectuar Ia expresión de las secuencias codificadoras a las que están ligadas. La naturaleza de dichas secuencias de control difiere dependiendo del organismo huésped; en procariotas, dichas secuencias de control generalmente incluyen un promotor, un sitio de unión ribosomal, y señales de terminación; en eucariotas, generalmente, dichas secuencias de control incluyen promotores, señales de terminación, intensificadores y, en ocasiones, silenciadores. Se pretende que el término "secuencias de control" incluya, como mínimo, todos los componentes cuya presencia es necesaria para Ia expresión, y también puede incluir componentes adicionales cuya presencia sea ventajosa."Control sequence" refers to polynucleotide sequences that are necessary to effect the expression of the coding sequences to which they are linked. The nature of such control sequences differs depending on the host organism; in prokaryotes, said control sequences generally include a promoter, a ribosomal binding site, and termination signals; in eukaryotes, generally, said control sequences include promoters, termination signals, enhancers and, sometimes, silencers. It is intended that the term "control sequences" includes, at a minimum, all components whose presence is necessary for expression, and may also include additional components whose presence is advantageous.
"Unidos de forma operativa" se refiere a una yuxtaposición en Ia que los componentes así descritos tienen una relación que les permite funcionar en Ia manera intencionada. Una secuencia de control "unida de forma operativa" a una secuencia codificadora está ligada de tal manera que Ia expresión de Ia secuencia codificadora se consigue en condiciones compatibles con las secuencias de control."Operationally linked" refers to a juxtaposition in which the components thus described have a relationship that allows them to function in the intended way. A control sequence "operatively linked" to a coding sequence is linked in such a way that the expression of the coding sequence is achieved under conditions compatible with control sequences.
Un "marco de lectura libre" (ORF) es una región de una secuencia de polinucleótidos que codifica un polipéptido; esta región puede representar una porción de una secuencia codificadora o una secuencia codificadora completa.A "free reading frame" (ORF) is a region of a polynucleotide sequence that encodes a polypeptide; This region may represent a portion of a coding sequence or a complete coding sequence.
Una "secuencia codificadora" es una secuencia de polinucleótidos que se transcribe a ARNm y/o se traduce a un polipéptido cuando está bajo control de secuencias reguladoras apropiadas. Los límites de Ia secuencia codificadora se determinan mediante un codón de inicio de traducción en el extremo 5' y un codón de finalización de Ia traducción en el extremo 3'. Una secuencia codificadora puede incluir, pero no se limita a ARNm, ADNc, y secuencias de polinucleótidos recombinantes.A "coding sequence" is a polynucleotide sequence that is transcribed into mRNA and / or translated into a polypeptide when under control of appropriate regulatory sequences. The limits of the coding sequence are determined by a translation start codon at the 5 'end and a translation end codon at the 3' end. A coding sequence may include, but is not limited to mRNA, cDNA, and recombinant polynucleotide sequences.
Tal y como se usa en esta memoria, el término "transfección" se refiere a Ia introducción o transferencia de una molécula de ácido nucleico exógena en una célula eucariota, incluyendo, pero no limitándose a ella, una molécula de ácido ribonucleico o desoxiribonucleico (por ejemplo, ARN ó ADN desnudo).As used herein, the term "transfection" refers to the introduction or transfer of an exogenous nucleic acid molecule into a eukaryotic cell, including, but not limited to, a ribonucleic or deoxyribonucleic acid molecule (by example, naked RNA or DNA).
El término "plásmido" se refiere a fragmento circular de ADN bicatenario, que se encuentra en el interior de casi todas las bacterias, y que actúan y se replican de forma independiente al ADN cromosómico bacteriano y pueden transferirse de unas bacterias a otras. Se utilizan como vectores en manipulación genética.The term "plasmid" refers to a circular fragment of double-stranded DNA, which is found inside almost all bacteria, and that act and replicate independently to bacterial chromosomal DNA and can be transferred from one bacterium to another. They are used as vectors in genetic manipulation.
En el sentido utilizado en esta descripción, Ia expresión "cantidad terapéuticamente efectiva" se refiere a Ia cantidad de virus, secuencias aminoacídicas, cápsides, anticuerpos o construcciones genéticas que permitan su expresión calculada para producir el efecto deseado y, en general, vendrá determinada, entre otras causas, por las características propias de dichos virus, anticuerpos, secuencias y construcciones y el efecto terapéutico a conseguir. Los adyuvantes y vehículos farmacéuticamente aceptables que pueden ser utilizados en dichas composiciones son los vehículos conocidos por los técnicos en Ia materia.In the sense used in this description, the expression "therapeutically effective amount" refers to the amount of virus, amino acid sequences, capsids, antibodies or genetic constructs that allow its calculated expression to produce the desired effect and, in In general, it will be determined, among other causes, by the characteristics of said viruses, antibodies, sequences and constructions and the therapeutic effect to be achieved. The pharmaceutically acceptable adjuvants and vehicles that can be used in said compositions are the vehicles known to those skilled in the art.
Las composiciones proporcionadas por esta invención pueden ser facilitadas por cualquier vía de administración, para Io cual dicha composición se formulará en Ia forma farmacéutica adecuada y con los excipientes farmacológicamente aceptables a Ia vía de administración elegida.The compositions provided by this invention can be provided by any route of administration, for which said composition will be formulated in the appropriate pharmaceutical form and with the pharmacologically acceptable excipients to the route of administration chosen.
El término "VFA", como aquí se usa, denota una especie vírica de Ia familia PicoARNviridae, que pertenecen al grupo IV de Ia clasificación de Baltimore, cuyas formas patógenas causan Ia fiebre aftosa, y cuyas fomas atenuadas, persistentes o defectivas derivadas de las mismas están relacionadas con esta enfermedad. El genoma del VFA está constituido por una cadena simple de ARN, de sentido positivo, de una longitud entre 7.2 y 9.0 kb. Se sabe que los virus que contienen ARN presentan frecuencias de mutación relativamente elevadas, del orden de 10~3 a 10~5 por nucleótido incorporado. En consecuencia, las poblaciones de VFA están constituidas por un conjunto heterogéneo de variantes genéticos sometidos a rápida evolución, que han originado una gran diversidad de genotipos relacionados y de variantes fenotípicos, incluyendo siete serotipos diferentes.The term "VFA", as used herein, denotes a viral species of the PicoARNviridae family, which belong to group IV of the Baltimore classification, whose pathogenic forms cause foot-and-mouth disease, and whose attenuated, persistent or defective forms derived from They are related to this disease. The VFA genome is made up of a simple, positive-sense RNA chain with a length between 7.2 and 9.0 kb. It is known that viruses containing RNA have relatively high mutation frequencies, of the order of 10 ~ 3 to 10 ~ 5 per incorporated nucleotide. Consequently, VFA populations are made up of a heterogeneous set of genetic variants undergoing rapid evolution, which have led to a great diversity of related genotypes and phenotypic variants, including seven different serotypes.
A Io largo de Ia descripción y las reivindicaciones Ia palabra "comprende" y sus variantes no pretenden excluir otras características técnicas, aditivos, componentes o pasos. Para los expertos en Ia materia, otros objetos, ventajas y características de Ia invención se desprenderán en parte de Ia descripción y en parte de Ia práctica de Ia invención. Los siguientes ejemplos y dibujos se proporcionan a modo de ilustración, y no se pretende que sean limitativos de Ia presente invención.Throughout the description and the claims, the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and characteristics of the invention will emerge partly from the description and partly from the practice of the invention. The following Examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention.
BREVE DESCRIPCIÓN DE LAS FIGURASBRIEF DESCRIPTION OF THE FIGURES
Figura 1 (Fig. 1 ).- Esquema vacunación C-S8p260 en ratones C57BL/6.Figure 1 (Fig. 1) .- C-S8p260 vaccination scheme in C57BL / 6 mice.
Figura 2 (Fig. 2).- Supervivencia de ratones vacunados con C-S8p260.Figure 2 (Fig. 2) .- Survival of mice vaccinated with C-S8p260.
Figura 3 (Fig. 3).- Títulos de ELISA en ratones vacunados con C-S8p260.Figure 3 (Fig. 3) .- ELISA titers in mice vaccinated with C-S8p260.
Figura 4 (Fig. 4).- Temperatura de los cerdos después del desafío con C- S8c1.Figure 4 (Fig. 4) .- Pigs temperature after challenge with C-S8c1.
Figura 5 (Fig. 5).- Esquema de las distintas regiones del genoma del VFA.Figure 5 (Fig. 5) .- Scheme of the different regions of the VFA genome.
La región L que codifica para Ia proteasa Leader, las regiones VP1 , VP2,The L region coding for the Leader protease, the VP1, VP2 regions,
VP3 y VP4, que codifican para las proteínas estructurales, y las regionesVP3 and VP4, which code for structural proteins, and regions
2A, 2B, 2C, 3A, 3B, 3C y 3D, que codifican para las proteínas no estructurales. Se indican las regiones en las que se producen las deleciones de las variantes víricas Δ417, Δ999 y Δ1017.2A, 2B, 2C, 3A, 3B, 3C and 3D, which code for non-structural proteins. The regions where deletions of the viral variants Δ417, Δ999 and Δ1017 are indicated are indicated.
Figura 6 (Fig. 6).- Alineamiento de los distintos serotipos de VFA, incluyendo las variantes delecionadas Δ417, Δ999 y Δ1017, e indicando Ia correspondencia de dichas regiones en los distintos serotipos.Figure 6 (Fig. 6) .- Alignment of the different VFA serotypes, including the deleted variants Δ417, Δ999 and Δ1017, and indicating the correspondence of said regions in the different serotypes.
EXPOSICIÓN DETALLADA DE MODOS DE REALIZACIÓNDETAILED EXHIBITION OF REALIZATION MODES
A continuación se ilustrará Ia invención mediante unos ensayos realizados por los inventores, que pone de manifiesto Ia especificidad y efectividad de Ia vacuna.Next, the invention will be illustrated by tests carried out by the inventors, which shows the specificity and effectiveness of the vaccine.
Ejemplo 1.- Aislamiento y posterior creación de Ia población de VFA C-S8p260 de Ia invenciónExample 1.- Isolation and subsequent creation of the VFA population C-S8p260 of the invention
Los experimentos siguientes se llevaron a cabo con una población de VFA clonada, C-S8c1 , de serotipo C (Sobrino, F.; Dávila, M.; Ortín, J. & Domingo, E. (1983) Virology, 128: 310-318.). Este virus fue pasado 260 veces a alta multiplicidad de infección (MOI) en células BHK, denominando a Ia población viral resultante C-S8p260. El análisis mediante RT-PCR del ARN de Ia población C-S8p260 reveló Ia presencia de tres moléculas distintas de ARN: una molécula que contiene una deleción de 417 nucleótidos en Ia región que codifica para Ia proteasa L (Δ417); una segunda molécula con una deleción de 999 nucleótidos en Ia región que codifica para las proteínas estructurales (Δ999) y, - una tercera, con una deleción de 1017 nucleótidos en Ia misma región (Δ1017) (García-Arriaza y col., 2004, J. Virol. 78: 11678-11685). Ninguna de estas deleciones altera Ia fase de lectura abierta del genoma de VFA. Distintos análisis de cuantificación permitieron asegurar que Ia población C-S8p260 está formada mayoritariamente por los genomas delecionados, encontrándose al menos 10,000 veces menos ARN de virus estándar, o sin delaciones.The following experiments were carried out with a population of cloned VFA, C-S8c1, of serotype C (Sobrino, F .; Dávila, M .; Ortín, J. & Domingo, E. (1983) Virology, 128: 310-318.). This virus was passed 260 times at high multiplicity of infection (MOI) in BHK cells, naming the resulting viral population C-S8p260. The analysis by RT-PCR of the RNA of the population C-S8p260 revealed the presence of three different RNA molecules: a molecule that contains a deletion of 417 nucleotides in the region that encodes protease L (Δ417); a second molecule with a deletion of 999 nucleotides in the region that codes for structural proteins (Δ999) and, - a third, with a deletion of 1017 nucleotides in the same region (Δ1017) (García-Arriaza et al., 2004, J. Virol. 78: 11678-11685). None of these deletions alter the open reading phase of the VFA genome. Different quantification analyzes made it possible to ensure that the C-S8p260 population consists mostly of deleted genomes, finding at least 10,000 times less standard virus RNA, or without delations.
Mediante técnicas de biología molecular las deleciones Δ417 y Δ999 fueron generadas en un clon infeccioso del VFA C-S8c1 (pMT28), de tal manera que se generaron los transcritos VFA pMTΔ417 y VFA pMTΔ999. Estos transcritos se introdujeron simultáneamente dentro de células BHK mediante co-electroporación y se obtuvo un efecto citopático (muerte celular) a las 72 horas post-electroporación, Io que indicó que ambos ARNs pueden infectar por complementación en ausencia de genoma estándar. Asimismo, mediante análisis de microscopía electrónica se determinó que cada genoma defectivo era capaz de encapsidarse en una sola partícula viral (García-Arriaza y col., 2004, J. Virol. 78: 11678-11685). Estos virus defectivos son estables en cultivos celulares ya que se mantuvieron por complementación en ausencia de virus estándar durante 200 pases adicionales en cultivos de células BHK (García-Arriaza y col., 2006, J. Mol. Biol. 360:558-572). Es decir, todos estos datos permiten concluir que Ia población de VFA C- S8p260 está compuesta por virus defectivos capaces de complementar cuando Ia infección se realiza a una MOI muy alta.Using molecular biology techniques the deletions Δ417 and Δ999 were generated in an infectious clone of the VFA C-S8c1 (pMT28), such that the transcripts VFA pMTΔ417 and VFA pMTΔ999 were generated. These transcripts were simultaneously introduced into BHK cells by co-electroporation and a cytopathic effect (cell death) was obtained at 72 hours post-electroporation, which indicated that both RNAs can be infected by complementation in the absence of a standard genome. Likewise, by electron microscopy analysis it was determined that each defective genome was capable of encapsidating into a single viral particle (García-Arriaza et al., 2004, J. Virol. 78: 11678-11685). These defective viruses are stable in cell cultures as they were maintained by complementation in the absence of standard virus for 200 additional passes in BHK cell cultures (García-Arriaza et al., 2006, J. Mol. Biol. 360: 558-572) . That is, all these data allow us to conclude that the population of VFA C-S8p260 is composed of defective viruses capable of complementing when the infection is performed at a very high MOI.
Ejemplo 2.- Utilización de Ia población viral C-S8p260 en Ia elaboración de una vacuna viva atenuadaExample 2.- Use of the viral population C-S8p260 in the elaboration of a live attenuated vaccine
La población viral C-S8p260 se ha probado, inicialmente, como posible vacuna viva atenuada en un modelo de ratón desarrollado en el laboratorio de los inventores (Salguero y col., 2005, Virology 332: 384-396). Ratones C57BL/6 son altamente susceptibles a Ia infección con VFA C-S8c1. Así, Ia inoculación de VFA en Ia almohadilla plantar, inyección subcutánea, (sitio de inoculación equivalente al rodete coronario del cerdo, el hospedador natural) produjo Ia aparición de síntomas de enfermedad a las 24 horas post-inoculación (pi) (pelo erizado, apatía, postura encorvada y depresión), y muerte entre 36-48 horas pi).The viral population C-S8p260 has been tested, initially, as a possible live attenuated vaccine in a mouse model developed in the inventors' laboratory (Salguero et al., 2005, Virology 332: 384-396). C57BL / 6 mice are highly susceptible to infection with VFA C-S8c1. Thus, the inoculation of VFA in the plantar pad, subcutaneous injection, (inoculation site equivalent to the coronary impeller of the pig, the natural host) produced the appearance of disease symptoms at 24 hours post-inoculation (pi) (bristling hair, apathy, hunched posture and depression), and death between 36-48 hours pi).
2.1. - Vacunación vía en Ia almohadilla plantar2.1. - Vaccination via the plantar pad
Un primer objetivo fue evaluar si Ia población viral C-S8p260 está atenuada in vivo. Para ello, 16 ratones fueron inoculados en Ia almohadilla plantar (AP) con 107 PFUs de C-S8p260 (Ia dosis más alta que se puede inocular con el volumen permitido en Ia AP que son 50μl). Los animales fueron sangrados a distintos tiempos pi (días 1 , 2, 3 15 y 30 pi) para determinar viremia y título de anticuerpos frente a VFA (Figura 1 ). Se determinó título viral en suero mediante titulación por plaqueo en células susceptibles BHK a tiempos cortos pi, 1 , 2 y 3 días, y no se detectó replicación viral. Asimismo, los animales no detectaron ningún síntoma de enfermedad o muerte debido a Ia infección viral. Estos datos demuestran que Ia población C-S8p260 está atenuada en ratones. Este mismo esquema de experimento se utilizó para determinar Ia protección de los ratones inoculados con C-S8p260 frente a un desafío con una dosis letal de nuestro virus de referencia C-S8c1. Así, los 16 animales fueron desafiados al día 90 pi con 104 PFUs de C-S8c1 en Ia AP. Se incluyeron 4 animales control sin vacunar. Los resultados (Figura 2) fueron que el 100% de los animales inoculados con C-S8p260 sobrevivieron al desafío con C-S8c1 (tiempo de observación de 30 días post-desafío) mientras que los animales control murieron a las 48 horas pi. Se determinó Ia viremia de los animales inoculados con C-S8p260 después del desafío con C-S8c1 con el objetivo de ver si, aunque los animales estaban protegidos porque no desarrollaron ningún síntoma de enfermedad, el virus había replicado. Los resultados mostraron ausencia completa de replicación de C-S8c1 en ratones previamente inoculados con C-S8p260. Estos datos indican que Ia población de VFA C-S8p260 está atenuada e induce protección estéril en ratones, Io que sugiere Ia posible utilización de C-S8p260 como vacuna.A first objective was to assess whether the viral population C-S8p260 is attenuated in vivo. For this, 16 mice were inoculated in the plantar pad (AP) with 10 7 PFUs of C-S8p260 (the highest dose that can be inoculated with the volume allowed in the AP that are 50μl). The animals were bled at different times pi (days 1, 2, 3 15 and 30 pi) to determine viremia and antibody titer against VFA (Figure 1). Serum viral titer was determined by plating titration in susceptible BHK cells at short times pi, 1, 2 and 3 days, and no viral replication was detected. Likewise, the animals did not detect any symptoms of illness or death due to viral infection. These data demonstrate that the C-S8p260 population is attenuated in mice. This same experiment scheme was used to determine the protection of mice inoculated with C-S8p260 against a challenge with a lethal dose of our reference virus C-S8c1. Thus, the 16 animals were challenged daily 90 pi with 10 4 PFUs of C-S8c1 in the AP. Four control animals without vaccinations were included. The results (Figure 2) were that 100% of the animals inoculated with C-S8p260 survived the challenge with C-S8c1 (observation time of 30 days post-challenge) while the control animals died at 48 pi hours. The viremia of the animals inoculated with C-S8p260 after the challenge with C-S8c1 was determined in order to see if, although the animals were protected because they did not develop any disease symptoms, the virus had replicated. The results showed complete absence of C-S8c1 replication in mice previously inoculated with C-S8p260. These data indicate that the population of VFA C-S8p260 is attenuated and induces sterile protection in mice, which suggests the possible use of C-S8p260 as a vaccine.
2.2.- Vacuna intramuscular2.2.- Intramuscular vaccine
Dado que Ia población C-S8p260 fue inoculada inicialmente en Ia AP y las vacunas en hospedadores naturales se inoculan generalmente por vía intramuscular, se evaluó si Ia inoculación intramuscular en ratones de Ia población C-S8p260 también protegía a los ratones frente a un desafío con una dosis letal de C-S8c1. Para ello se inocularon 12 ratones C57BL/6 intramuscularmente con 107 PFUs de C-S8p260. A los 60 días postinoculación se les desafío con 104 PFUs de VFA C-S8c1 en Ia AP. Los resultados indicaron Ia completa ausencia de síntomas en los ratones vacunados intramuscularmente.Since the C-S8p260 population was initially inoculated into the AP and vaccines in natural hosts are generally inoculated intramuscularly, it was evaluated whether the intramuscular inoculation in mice of the C-S8p260 population also protected the mice against a challenge with a lethal dose of C-S8c1. For this, 12 C57BL / 6 mice were inoculated intramuscularly with 10 7 PFUs of C-S8p260. At 60 days post-inoculation they were challenged with 10 4 PFUs of VFA C-S8c1 in the AP. The results indicated the complete absence of symptoms in the mice vaccinated intramuscularly.
2.3.- Vacunas con menor dosis de antígeno Con el objetivo de determinar si una menor dosis de antígeno viral también inducía protección, 15 ratones se inocularon en Ia AP con 103 PFUs de C- S8p260. Estos ratones se sangraron a días 1 , 2, 3, 15 y 30 pi (Figura 1 ). Los animales, como se esperaba por los resultados mostrados anteriormente, no mostraron ningún tipo de enfermedad ni viremia en sangre a tiempos tempranos post-inoculación. Estos ratones fueron desafiados con 104 PFUs de C-S8c1 en Ia AP a día 90 pi. Se incluyeron 4 animales control sin vacunar. El 100% de los animales inoculados previamente con 103 PFUs de C-S8p260 sobrevivieron al desafío con C- S8c1 mientras que los controles murieron a las 48 h pi. (Figura 2) Asimismo, no se detectó viremia en los animales vacunados después del desafío Io que una vez más indicaba protección estéril. Estos datos indican que menores dosis de antígeno protegen frente a una dosis letal de VFA.2.3.- Vaccines with a lower dose of antigen In order to determine if a lower dose of viral antigen also induced protection, 15 mice were inoculated in the AP with 10 3 PFUs of C-S8p260. These mice bled on days 1, 2, 3, 15 and 30 pi (Figure 1). The animals, as expected by the results shown above, did not show any type of disease or blood viremia at early post-inoculation times. These mice were challenged with 10 4 PFUs of C-S8c1 in the AP at day 90 pi. Four control animals without vaccinations were included. 100% of the animals previously inoculated with 10 3 PFUs of C-S8p260 survived the challenge with C-S8c1 while controls died at 48 h pi. (Figure 2) Likewise, no viremia was detected in vaccinated animals after the challenge, which once again indicated sterile protection. These data indicate that lower doses of antigen protect against a lethal dose of VFA.
Como medida de Ia inducción de una respuesta inmune frente a VFA por Ia población C-S8p260 se determinó Ia cantidad de anticuerpos anti-VFA totales mediante ensayo de ELISA (Figura 3) y por ensayo de neutralización (Tabla 1 ). Los resultados de ELISA muestran una alta cantidad de anticuerpos específicos frente a VFA que se incrementó después del desafío con C-S8c1 , tanto en animales inoculados con 107 PFUs como con 103 PFUs de C-S8p260. Los títulos de neutralización en el momento del desafío y 72 h después del desafío son del orden de 2 (expresado como Ia inversa del logaritmo de Ia concentración de suero que produce Ia reducción de un 70% de Ia infectividad, PRN70). Estos datos indican que C-S8p260 es capaz de inducir una respuesta humoral frente a VFA capaz de inducir protección.As a measure of the induction of an immune response against VFA by the population C-S8p260, the amount of total anti-VFA antibodies was determined by ELISA assay (Figure 3) and by neutralization assay (Table 1). The ELISA results show a high amount of specific antibodies against VFA that was increased after challenge with C-S8c1, both in animals inoculated with 10 7 PFUs and with 10 3 PFUs of C-S8p260. The neutralization titers at the time of the challenge and 72 h after the challenge are of the order of 2 (expressed as the inverse of the logarithm of the serum concentration that produces the 70% reduction of the infectivity, PRN70). These data indicate that C-S8p260 is capable of inducing a humoral response against VFA capable of inducing protection.
2.4.- Vacunación de cerdos2.4.- Vaccination of pigs
La vacuna propuesta en esta patente se evaluó también en cerdo, el hospedador natural de VFA, en un ensayo preliminar. Los animales se dividieron en cinco grupos. En cada uno de los grupos el protocolo seguido es común para todos ellos. El virus C-S8p260 se inoculó por vía intramuscular en 1 mi de PBS. El desafío se realizó en 500 μl en el rodete coronario. Se tomaron muestras de sangre a DO, antes de Ia inoculación de C-S8p260, D2, D4, D7, D15 y D30 (antes del desafío). Después del desafío los animales fueron sangrados cada dos días y sometidos a observación cada día para determinar aparición de síntomas de FA hasta el día 11 post-desafío. Se determinó aparición de aftas primarias (en el sitio de inoculación) y aftas secundarias, así como estado general de los cerdos (síntomas de FA: apatía, depresión, imposibilidad de andar...). Asimismo, se tomó temperatura diaria de cada uno de los cerdos.The vaccine proposed in this patent was also evaluated in pig, the natural host of VFA, in a preliminary trial. The animals are They divided into five groups. In each of the groups the protocol followed is common to all of them. The C-S8p260 virus was inoculated intramuscularly in 1 ml of PBS. The challenge was performed at 500 μl in the coronary impeller. Blood samples were taken from OD, before the inoculation of C-S8p260, D2, D4, D7, D15 and D30 (before the challenge). After the challenge, the animals were bled every two days and subjected to observation every day to determine the appearance of AF symptoms until the 11th post-challenge day. Appearance of primary canker sores (at the site of inoculation) and secondary canker sores, as well as general condition of pigs (symptoms of AF: apathy, depression, inability to walk ...). Also, daily temperature of each of the pigs was taken.
Grupo 1. 4 animales. Se siguió el siguiente protocolo:Group 1. 4 animals. The following protocol was followed:
Figure imgf000024_0001
Figure imgf000024_0001
Grupo 2. 1 animal. En este grupo se utilizó Ia misma dosis de C-S8p260 que en el grupo anterior pero se añadió adyuvante en Ia segunda dosis.Group 2. 1 animal. In this group the same dose of C-S8p260 was used as in the previous group but adjuvant was added in the second dose.
Figure imgf000024_0002
Figure imgf000024_0002
La segunda dosis de C-S8p260 se realizó en una dilución 1 :1 con adyuvante completo de Freund. Grupo 3. 3 animales. La dosis de C-S8p260 se diluyó 1 :100 con el objetivo de evaluar si se necesitaba una dosis de defectivos alta.The second dose of C-S8p260 was performed in a 1: 1 dilution with Freund's complete adjuvant. Group 3. 3 animals. The dose of C-S8p260 was diluted 1: 100 in order to assess whether a high defective dose was needed.
Figure imgf000025_0001
Figure imgf000025_0001
Grupo 4. 2 animales. Se utilizó una dosis de defectivos 100 veces menor que inicialmente (igual que el grupo 3) pero se añadió adyuvante en Ia segunda dosis.Group 4. 2 animals. A 100 times lower dose of defects was used than initially (same as group 3) but adjuvant was added in the second dose.
Figure imgf000025_0002
Figure imgf000025_0002
Se diluyó 1 :1 con adyuvante completo de Freund en Ia segunda dosis de C-S8p260.It was diluted 1: 1 with Freund's complete adjuvant in the second dose of C-S8p260.
Grupo 5. 2 animales. Estos animales son el control positivo del experimento en el que los cerdos fueron inoculados con PBS en vez de Ia vacuna y desafiados con Ia misma dosis de C-S8c1. Estos animales no estaban protegidos y mostraron los síntomas de FA.Group 5. 2 animals. These animals are the positive control of the experiment in which the pigs were inoculated with PBS instead of the vaccine and challenged with the same dose of C-S8c1. These animals were not protected and showed the symptoms of AF.
Figure imgf000025_0003
Estos animales se pusieron muy enfermos y al día post-desafío fueron sacrificados por razones humanitarias.
Figure imgf000025_0003
These animals became very ill and the post-challenge day were sacrificed for humanitarian reasons.
Los resultados se pueden describir desde dos puntos de vista:The results can be described from two points of view:
1. Temperaturas1. Temperatures
2. Síntomas medido como Ia aparición de aftas.2. Symptoms measured as the appearance of thrush.
Grupo 1. Tabla de temperaturas expresada en 0C. Se ha incluido para comparar los animales #21 y #22 del grupo 5 que son los controles desafiados pero sin vacunar que desarrollaron todos los síntomas esperados de FA.Group 1. Temperature table expressed in 0 C. It has been included to compare animals # 21 and # 22 of group 5 which are the challenged but unvaccinated controls that developed all the expected symptoms of AF.
Figure imgf000026_0001
Figure imgf000026_0001
Estos datos de temperatura están también recogidos en forma de gráfica en Ia figura 4. Se puede apreciar como todos los cerdos desarrollaron una temperatura alta. Sólo el animal #5 mostró un retraso en el pico de viremia.These temperature data are also collected in graph form in Figure 4. It can be seen how all pigs developed a high temperature. Only animal # 5 showed a delay in peak viremia.
Aparición de aftas. Se define como Ia aparición de aftas primarias (en el sitio de inoculación) y de aftas en todas las patas (secundarias). Se indica el día que se sacrificaron los animales.Appearance of canker sores. It is defined as the appearance of primary canker sores (at the site of inoculation) and canker sores on all legs (secondary). The day the animals were sacrificed is indicated.
Figure imgf000026_0002
Figure imgf000027_0001
Figure imgf000026_0002
Figure imgf000027_0001
Se puede observar que en general todos los animales mostraron un retraso en Ia aparición de síntomas en relación a los controles (animales #21 y #22). En especial los animales #3 y #5 mostraron protección parcial ya que sólo aparecieron aftas primarias, no observándose una progresión de Ia infección.It can be observed that in general all the animals showed a delay in the appearance of symptoms in relation to the controls (animals # 21 and # 22). Especially animals # 3 and # 5 showed partial protection since only primary sores appeared, not observing a progression of the infection.
Grupo 2. Se compara el animal #11 con los controles positivos.Group 2. Animal # 11 is compared with positive controls.
Figure imgf000027_0002
Se puede observar como el animal #11 no desarrolla fiebre.
Figure imgf000027_0002
It can be seen how animal # 11 does not develop fever.
Aparición de aftas.Appearance of canker sores.
Este animal no desarrolló ningún tipo de síntoma asociado con FA u otra enfermedad. Se mantuvo completamente sano durante todo el experimento. Hay que hacer Ia apreciación de que se encontraba compartiendo box con los animales control por Io que estuvo en contacto con cerdos muy enfermos que exhalaban virus. Este animal se considera a efectos de evaluación de vacuna totalmente protegido.This animal did not develop any type of symptom associated with AF or another disease. He remained completely healthy throughout the experiment. It is necessary to make the assessment that he was sharing a box with the control animals so that he was in contact with very sick pigs that exhaled viruses. This animal is considered for the purpose of fully protected vaccine evaluation.
Grupo 3. Se comparan los animales #7, #8 y #10 con los controles.Group 3. Animals # 7, # 8 and # 10 are compared with controls.
Figure imgf000028_0002
Figure imgf000029_0001
Figure imgf000028_0002
Figure imgf000029_0001
Todos los animales mostraron temperaturas similares a los controles.All animals showed temperatures similar to controls.
Aparición de aftas.Appearance of canker sores.
Figure imgf000029_0002
Figure imgf000029_0002
Estos animales no mostraron ningún tipo de protección.These animals did not show any protection.
Grupo 4. Se comparan los animales #12 y #13 con los controles.
Figure imgf000030_0001
Group 4. Animals # 12 and # 13 are compared with controls.
Figure imgf000030_0001
Estos animales no desarrollaron fiebre. Hay que tener en cuenta que el animal #13 tiene una temperatura basal (medida durante 25 días consecutivos) de 40.20C, muy alta para Ia media de temperatura de cerdos. Aparición de aftas.These animals did not develop fever. Keep in mind that animal # 13 has a basal temperature (measured for 25 consecutive days) of 40.2 0 C, very high for the average temperature of pigs. Appearance of canker sores.
Figure imgf000030_0002
Estos animales mostraron protección parcial uno (#12) y total el otro (#13) ya que no tuvieron aftas o sólo en el sitio de inoculación.
Figure imgf000030_0002
These animals showed partial protection one (# 12) and total protection the other (# 13) since they had no thrush or only at the site of inoculation.
Conclusiones del experimento de vacunación en cerdos.Conclusions of the vaccination experiment in pigs.
La dosis de VFA defectivo C-S8p260 de 5 x 106 PFUs con adyuvante protegió completamente a un cerdo. Esa misma dosis sin adyuvante ha conferido protección parcial. La dosis 105 PFUs sólo tiene efecto protector cuando se inocula con adyuvante. Ahora mismo se está evaluando en un nuevo experimento de cerdos Ia capacidad de proteger de C-S8p260 con Ia menos dosis pero incluyendo adyuvante en las 2 inoculaciones. Asimismo, se está evaluando Ia capacidad protectora de C-S8p260 inactivado con BEI.The dose of defective VFA C-S8p260 of 5 x 10 6 PFUs with adjuvant completely protected a pig. That same dose without adjuvant has conferred partial protection. The 10 5 PFU dose has only protective effect when inoculated with adjuvant. Right now, the ability to protect C-S8p260 with the least dose but including adjuvant in the 2 inoculations is being evaluated in a new pig experiment. Likewise, the protective capacity of C-S8p260 inactivated with BEI is being evaluated.
Ejemplo 3.- Vacuna inactivadaExample 3.- Inactivated vaccine
La población C-S8p260 se inactivo siguiendo el protocolo de inactivación basado en Ia utilización de 2-Bromoetilamina Hidrobromuro (BEA), un derivado de azirinas. Se inocularon 7 ratones hembras C57BI/6 de 8 semanas de edad con 50 microlitros de C-S8p260 inactivado por vía intramuscular, una dosis equivalente a 107 PFUs de C-S8p260. A los 14 días se les inoculó con un segundo booster. En ninguno de los boosters se usó adyuvante.The C-S8p260 population was inactivated following the inactivation protocol based on the use of 2-Bromoethylamine Hydrobromide (BEA), an azirin derivative. 7 female C57BI / 6 mice of 8 weeks of age were inoculated with 50 microliters of intramuscularly inactivated C-S8p260, a dose equivalent to 10 7 PFUs of C-S8p260. At 14 days they were inoculated with a second booster. In none of the boosters was adjuvant used.
A los 30 días se realizó el desafío con 50 microlitros de 104 PFUs de C- S8c1 inoculado en Ia almohadilla plantar. Se utilizaron 2 controles positivos inoculados también con Ia misma cantidad de virus C-S8c1 y dos controles negativos inoculados con 50 microlitros de PBS en el mismo lugar. Los ratones inoculados con defectivos estuvieron protegidos después del desafío, hasta un periodo de observación de 60 días. Los controles positivos murieron al cabo de 36h.After 30 days, the challenge was performed with 50 microliters of 10 4 PFUs of C-S8c1 inoculated in the plantar pad. Two positive controls inoculated with the same amount of C-S8c1 virus and two negative controls inoculated with 50 microliters of PBS were used in the same place. Mice inoculated with defects were protected after challenge, up to an observation period of 60 days. Positive controls died after 36h.
Títulos de neutralizaciones en ratón C57BL/6 inmunizados con C-S8p260.C57BL / 6 mouse neutralization titres immunized with C-S8p260.
10' PFUs pre- desafío 1:100 pre-desafío 72h post-desafío 72h post-desafío10 'PFUs pre-challenge 1: 100 pre-challenge 72h post-challenge 72h post-challenge
Ratón PRN 7# Ratón PRN .7# Ratón PRN 7# Ratón PRN 7#PRN 7 mouse # PRN mouse .7 # PRN 7 mouse # PRN 7 mouse #
5 2,0 5 2,0 5 1,8 5 1,95 2.0 5 2.0 5 1.8 5 1.9
6 2,0 6 2,1 6 2,0 6 1,86 2.0 6 2.1 6 2.0 6 1.8
8 2,4 8 0 8 2,2 7 1,88 2.4 8 0 8 2.2 7 1.8
9 2,1 9 1,7 9 1,9 8 1,89 2.1 9 1.7 9 1.9 8 1.8
10 1,3 10 1,4 10 1,5 9 2,410 1.3 10 1.4 10 1.5 9 2.4
11 1,7 11 1,6 11 1,8 10 1,811 1.7 11 1.6 11 1.8 10 1.8
12 0,4 12 1,4 12 1,9 11 1,812 0.4 12 1.4 12 1.9 11 1.8
13 1,7 13 0,6 13 1,8 12 1,113 1.7 13 0.6 13 1.8 12 1.1
14 2,7 14 1,4 14 2,4 14 1,514 2.7 14 1.4 14 2.4 14 1.5
15 1,9 15 1,7 15 1,9 15 1,815 1.9 15 1.7 15 1.9 15 1.8
1,8 1,4 1,9 1,81.8 1.4 1.9 1.8
+/-0,6 +/- 0,6 +/- 0,2 +/- 0,3 +/- 0.6 +/- 0.6 +/- 0.2 +/- 0.3

Claims

REIVINDICACIONES
1. Población de virus aislada, caracterizada porque comprende una secuencia de polinucleótidos capaz de traducirse a una secuencia aminoacídica homologa a Ia SEQ ID NO: 1 , y al menos una secuencia seleccionada de entre: a) una secuencia de polinucleótidos capaz de traducirse a una secuencia aminoacídica homologa a Ia SEQ ID NO: 2, ó b) una secuencia de polinucleótidos capaz de traducirse a una secuencia aminoacídica homologa a Ia SEQ ID NO: 3, para su uso como medicamento.1. Isolated virus population, characterized in that it comprises a polynucleotide sequence capable of translating into an amino acid sequence homologous to SEQ ID NO: 1, and at least one sequence selected from: a) a polynucleotide sequence capable of translating into a amino acid sequence homologous to SEQ ID NO: 2, or b) a polynucleotide sequence capable of being translated into an amino acid sequence homologous to SEQ ID NO: 3, for use as a medicine.
2. Población de virus aislada caracterizada porque comprende una secuencia de polinucleótidos capaz de traducirse a una secuencia aminoacídica que comprende un péptido con una identidad de al menos un 80% con Ia SEQ ID NO: 1 , y al menos una secuencia seleccionada de entre: a) una secuencia de polinucleótidos capaz de traducirse a una secuencia aminoacídica que comprende un péptido con una identidad de al menos un 80% con Ia SEQ ID NO: 2, o b) una secuencia de polinucleótidos capaz de traducirse a una secuencia aminoacídica que comprende un péptido con una identidad de al menos un 80% con Ia SEQ ID NO: 3, para su uso como medicamento.2. Isolated virus population characterized in that it comprises a polynucleotide sequence capable of being translated into an amino acid sequence comprising a peptide with an identity of at least 80% with SEQ ID NO: 1, and at least one sequence selected from: a) a polynucleotide sequence capable of being translated into an amino acid sequence comprising a peptide with an identity of at least 80% with SEQ ID NO: 2, or b) a polynucleotide sequence capable of being translated into an amino acid sequence comprising a peptide with an identity of at least 80% with SEQ ID NO: 3, for use as a medicine.
3. Población de virus aislada según cualquiera de las reivindicaciones 1-2, donde las secuencias aminoacídicas presentan una identidad de al menos un 90% con Ia SEQ ID NO: 1 , SEQ ID NO: 2 o SEQ ID NO: 3, respectivamente.3. Virus population isolated according to any of claims 1-2, wherein the amino acid sequences they have an identity of at least 90% with SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, respectively.
4. Población de virus aislada según cualquiera de las reivindicaciones 1-3, donde las secuencias aminoacídicas presentan una identidad de al menos un 95% con Ia SEQ ID NO: 1 , SEQ ID NO: 2 o SEQ ID NO: 3, respectivamente.4. Virus population isolated according to any of claims 1-3, wherein the amino acid sequences have an identity of at least 95% with SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, respectively.
5. Población de virus aislada según cualquiera de las reivindicaciones 1-4, donde las secuencias aminoacídicas presentan una identidad de al menos un 99% con Ia SEQ ID NO: 1 , SEQ ID NO: 2 o SEQ ID NO: 3, respectivamente.5. Virus population isolated according to any of claims 1-4, wherein the amino acid sequences have an identity of at least 99% with SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, respectively.
6. Población de virus aislada según cualquiera de las reivindicaciones 1-5, donde las secuencias aminoacídicas son6. Virus population isolated according to any of claims 1-5, wherein the amino acid sequences are
Ia SEQ ID NO: 1 , SEQ ID NO: 2 o SEQ ID NO: 3, respectivamente.SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, respectively.
7. Uso de Ia población de virus aislada según cualquiera de las reivindicaciones 1-6, para Ia elaboración de un medicamento.7. Use of the isolated virus population according to any of claims 1-6, for the preparation of a medicament.
8. Cápside proteica aislada que comprende una secuencia aminoacídica según cualquiera de las reivindicaciones 1-6, para su uso como medicamento.8. Isolated protein capsid comprising an amino acid sequence according to any one of claims 1-6, for use as a medicament.
9. Construcción genética que comprende una secuencia de polinucleótidos que codifica una secuencia aminoacídica homologa a Ia SEQ ID NO: 1 y al menos otra secuencia seleccionada de entre: a) secuencia de polinucleótidos, que codifica una secuencia homologa a Ia SEQ ID NO: 2, b) secuencia de polinucleótidos, que codifica una secuencia homologa a Ia SEQ ID NO: 3, c) moléculas de ácido nucleico cuya cadena complementaria híbrida con Ia secuencia polinucleotídica de a) y/o b), d) secuencia de nucleótidos de a), b), ó c), preferiblemente de doble cadena, correspondiente a un sistema o vector de expresión génica, operativamente enlazada con, al menos, un promotor que dirija Ia transcripción de dicha secuencia de nucleótidos de interés, y con otras secuencias necesarias o apropiadas para Ia transcripción.9. Genetic construction comprising a polynucleotide sequence encoding an amino acid sequence homologous to SEQ ID NO: 1 and at least one other sequence selected from: a) polynucleotide sequence, which encodes a sequence homologous to SEQ ID NO: 2 , b) polynucleotide sequence, which encodes a sequence homologous to SEQ ID NO: 3, c) nucleic acid molecules whose complementary hybrid chain with the polynucleotide sequence of a) and / or b), d) nucleotide sequence of a), b), or c), preferably double-stranded, corresponding to a gene expression system or vector, operably linked with at least one promoter that directs the transcription of said nucleotide sequence of interest, and with other necessary or appropriate sequences for the transcription.
10. Anticuerpos producidos tras Ia inmunización de un animal, con Ia población de virus según cualquiera de las reivindicaciones 1-6, una cápside proteica aislada según Ia reivindicación 8, una construcción genética según Ia reivindicación 9, o cualquiera de sus combinaciones.10. Antibodies produced after the immunization of an animal, with the virus population according to any of claims 1-6, an isolated protein capsid according to claim 8, a genetic construct according to claim 9, or any combination thereof.
11. Anticuerpos según Ia reivindicación anterior, para su uso como medicamento.11. Antibodies according to the preceding claim, for use as a medicine.
12. Composición que comprende: a) una población de virus aislada según cualquiera de las reivindicaciones 1-6, b) una cápsida proteica aislada según Ia reivindicación 8, c) una construcción genética según Ia reivindicación 9, d) un anticuerpo según cualquiera de las reivindicaciones 10-11 , o cualquiera de sus combinaciones, para su uso como medicamento. 12. Composition comprising: a) an isolated virus population according to any of claims 1-6, b) an isolated protein capsid according to claim 8, c) a genetic construct according to claim 9, d) an antibody according to any of claims 10-11, or any combination thereof, for use as a medicament.
13. Composición según Ia reivindicación anterior donde el medicamento es una vacuna.13. Composition according to the preceding claim wherein the medicament is a vaccine.
14. Composición según cualquiera de las reivindicaciones 12-13, para su uso en el tratamiento y/o Ia prevención de Ia fiebre aftosa.14. Composition according to any of claims 12-13, for use in the treatment and / or prevention of foot and mouth disease.
15. Composición según cualquiera de las reivindicaciones 12-14, que además comprende excipientes farmacológicamente aceptables.15. Composition according to any of claims 12-14, further comprising pharmacologically acceptable excipients.
16. Composición según cualquiera de las reivindicaciones 12-15, que además comprende un adyuvante.16. Composition according to any of claims 12-15, further comprising an adjuvant.
17. Composición según cualquiera de las reivindicaciones 12-16, donde las secuencias aminoacídicas de Ia población de virus aislado según cualquiera de las reivindicaciones 1-6, Ia cápsida proteica aislada según Ia reivindicación 8, o el anticuerpo según cualquiera de las reivindicaciones 9-10, presenta un origen recombinante.17. Composition according to any of claims 12-16, wherein the amino acid sequences of the isolated virus population according to any of claims 1-6, the isolated protein capsid according to claim 8, or the antibody according to any of claims 9- 10, has a recombinant origin.
18. Composición según cualquiera de las reivindicaciones 12-17, que comprende dos o más tipos, serotipos, variedades o mutantes de virus de Ia aftosa.18. Composition according to any of claims 12-17, comprising two or more types, serotypes, varieties or mutants of FMD virus.
19. Uso de Ia composición según cualquiera de las reivindicaciones 12-18, para Ia elaboración de un medicamento destinado al tratamiento o prevención de Ia fiebre aftosa. 19. Use of the composition according to any of claims 12-18, for the preparation of a medicament for the treatment or prevention of foot and mouth disease.
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