Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/0003—Invertebrate antigens
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P33/00—Antiparasitic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
  • C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
  • C07K14/43527—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from ticks
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
  • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide

Definitions

• the present invention falls within the recombinant DNA technology and immunology, specifically, within the field of antigens capable of inducing immunity against infestations caused by hematophagous arthropods that act as vectors of pathogens, ticks, mosquitoes and sandflies, and, therefore, within the field of compositions, preferably vaccines, intended for the treatment or prevention of said infestations.
• Ticks are obligate hematophagous ectoparasites, which are distributed throughout the world, from the Arctic to tropical regions. Ticks transmit pathogens that affect animals and humans, whose transmitted diseases have a great impact on animal and human health.
• Tick-borne diseases include: Borreliosis, Anaplasmosis, Coxiqueliosis, Franciselliosis, Riquetsiosis, Theileriosis, Elriquesiosis and Babesiosis, as well as viral diseases such as tick-borne encephalitis. Tick infestations also have an economic impact on livestock, given that cause the loss of weight of the animals and the reduction of milk production. However, control of tick-borne infestations is difficult since these parasites have few natural enemies.
• tick infestations are carried out through a series of integrated measures in which various control methods adapted to a geographical area act.
• a main component of these control methods is the application of chemical acaricides.
• acaricides has shown limited efficacy in reducing infestations and these measures are often accompanied by serious adverse effects, such as the selection of acaricide-resistant ticks, environmental pollution and contamination of meat and meat. milk with residues of chemical agents (Graf, et al., 2004).
• Vaccines developed against arthropods are based on induction in the host of antigen-specific antibodies that are harmful to the arthropod vector during feeding (Almeida and Billingsley, 2002; de la Fuente, et al., 2007; de la Fuente, et al., 1998; Lal, et al., 2001; Milleron, et al., 2004; Suneja, et al., 2003; Titus, et al., 2006; Valenzuela, et al., 2001; Willadsen, 2004) .
• Bm86 tick antigen
• AKR is an evolutionarily conserved protein that regulates gene expression and, therefore, is involved in multiple cellular processes, such as digestion, immune response, reproduction and development (Almazán, et al., 2005a; Almazán, et al ., 2005b; de la Fuente, et al., 2006a; de la Fuente, et al., 2006b; Kocan, et al., 2009; Nijhof, et al., 2007). Therefore, vaccines have recently been developed that comprise the subolesina antigen and that induce an immunological protection against Tick infestations in vertebrate hosts (US2004 / 0022795 A1 and US2006 / 0040361 A1).
• SUB or AKR proteins are conserved in different arthropod species, thus providing a candidate for the production of a vaccine capable of protecting against infestation with different hematophagous vectors of pathogens that affect man and animals. These proteins have demonstrated their effectiveness as vaccines against infestation with different hematophagous vectors and the decrease in infection with pathogens transmitted by ticks.
• Vaccination experiments with orthologous ticks or mosquito recombinant antigens show a reduction in the fertility and / or survival of arthropods, such as ticks, mosquitoes and sand fleas. In ruminants, infestations of the Ixodes scapularis tick are reduced by vaccination with subolesina orthologs from ticks and mosquitoes with similar efficacy.
• chimeras based on protective epitopes identified in the tick SUB and the mosquito AKR have been constructed in the present invention.
• the construction of the chimeras is also based on the possible secondary structure of the resulting proteins.
• this apparently trivial approach is very complex because it is difficult to anticipate how the system Immune of the vaccinated host will recognize the epitopes included in the chimeric proteins, generating antibodies that are protective. Only the empirical test of different sequence combinations can lead to the discovery of protective antigens such as chimeras Q38 and Q41, object of the present invention.
• the present invention refers to an isolated peptide capable of inducing a protective and cross-reactive immune response against an infestation caused by at least one hematophagous arthropod, characterized in that it comprises at least one linear epitope and at least one conformational epitope, said epitopes. they are common to orthologs of the tickle and mosquito subolesina protein.
• said peptide is SEQ ID No. 1 and / or SEQ ID No. 2. More preferably, said peptide is useful in medicine. Said peptide is called "peptide of the invention”.
• the invention refers to the peptide described above, characterized in that it is useful for inducing a protective and cross-reactive immune response against an infestation caused by at least one hematophagous arthropod selected from: a tick, a mosquito, a sandfly , a red chicken mite, a flea, a louse and a sea louse. More preferably, said hematophagous arthropod is selected from: Ixodes ricinus, Aedes albopictus and Phiebotomus perniciosus.
• the present invention refers to an isolated polynucleotide that is characterized by coding for a peptide described above.
• said polynucleotide is SEQ ID No. 3 or SEQ ID No. 4.
• Said polynucleotide is called "polynucleotide of the invention”.
• the present invention refers to an expression vector characterized in that it comprises at least one polynucleotide described above. Said expression vector is called "expression vector of the invention”.
• the present invention refers to a host cell, characterized in that it comprises at least one expression vector described above.
• said cell is bacterial, and more preferably, said bacterial cell is Escherichia coli.
• Said host cell is called the "host cell of the invention.”
• the present invention refers to a non-human animal / organism, characterized in that it comprises at least one host cell described above.
• said animal is a mouse.
• Said non-human animal / organism is called the "non-human animal / organism of the invention”.
• the present invention refers to an isolated antibody characterized in that it reacts against a peptide described above. Said antibody is called "antibody of the invention”.
• the present invention refers to a composition characterized in that it comprises a peptide described above, or a polypeptide described above, or an expression vector described above, or a host cell described above.
• said composition may additionally comprise a pharmaceutically acceptable carrier or excipient. More preferably, said composition may additionally comprise an adjuvant. Even more preferably, said composition may comprise a second active ingredient. Said composition is called "composition of the invention”.
• the present invention refers to the use of a peptide described above, or a polypeptide described above, or an expression vector described above, or a host cell described above, or a non-human animal described above, or an antibody described above, for the Preparation of a medicine.
• said medicament is useful for the prevention and / or treatment of an infestation caused at least by a hematophagous arthropod.
• said hematophagous arthropod is selected from: a tick, a mosquito, a sandfly, a red hen mite, a flea, a louse and a sea louse.
• said hematophagous arthropod is a hematophagous arthropod selected from: Ixodes ricinus, Aedes albopictus and Phlebotomus perniciosus.
• the present invention refers to the use of a peptide described above, or a polypeptide described above, or an expression vector described above, or a host cell described above, or a non-human animal described above, or an antibody described above, for the control of the infestations transmitted by hematophagous arthropods described above.
• the present invention refers to a medicament capable of inducing a protective and cross-reactive immune response against an infestation caused by at least one hematophagous arthropod characterized in that it comprises a therapeutically effective amount of a peptide described above, or a polypeptide described above, or an expression vector described above, or a host cell described above.
• said medically is a vaccine.
• Said medicament is called "medicament of the invention”.
• the present invention refers to a method of prevention and / or treatment against an infestation caused by at least one hematophage arthropod characterized by the administration of a therapeutically acceptable amount of a peptide described above, or a polypeptide described above, or a vector of expression described above, or a host cell described above, or an antibody described above, or a medicament described above. Said method is called "method of the invention”.
• the present invention provides two chimera proteins: Q38 and Q41 (SEQ.ID N ° 1 and SEQ.ID. N ° 2), designed by combining a series of linear epitopes, conformational and structural or discontinuous mimetopes, from orthologs of the tickle and mosquito subolesina protein. These proteins are capable of inducing a protective and cross-reactive immune response against infestations caused by hematophagous arthropods (ticks, mosquitoes and sandflies).
• the invention also comprises the compositions of said chimera proteins and the use thereof and of these compositions for the preparation of medicaments, preferably vaccines, intended for the treatment or prevention of these infestations.
• the chimera proteins Q38 and Q41 share a linear epitope derived from SUB and another from AKR, two conformational epitopes derived from SUB and three derived from AKR.
• the predicted three-dimensional structure for both chimeras indicates that they are composed of alpha helices and beta plegamists that expose the protective epitopes on the surface.
• epitopope means the individual portion of an antigen capable of inducing an immune response; It is normally associated with the portion of the antigen that is recognized by an antibody.
• An "antigen” means a substance foreign to an organism that, once introduced into it, normally triggers an immune response.
• immune response or “immune response” means the activity performed by the immune system to fight foreign substances (antigens).
• immune system means the group of organs and cells that defend the body against infections, infestations and other diseases.
• chimera protein it is understood in the present invention a protein created from the union of two or more epitopes that originally code for separate proteins but that the translation of this fusion gene results in an individual polypeptide with functional properties derived from each of the original proteins.
• peptides or polypeptides whose amino acid sequence is 80% -95% identical or homologous to the sequences described in the present invention. Two sequences are identical if they are equal residue to residue. However, for sequences that are not identical, it is useful to have a measure of how far they are from being identical. For this, an alignment is made between the two sequences, the number of residues that are identical is counted and divided by the length of the alignment, giving the percentage of identity.
• Protein fusion is a technique that is frequently used in molecular biology. It is usually related to the production of proteins in living systems, such as, but not limited to, bacteria, yeast, mammalian cells, baculovirus or insect cells.
• the inclusion of the coding sequence of interest in phase, respecting the reading pattern, allows the production of a chimeric protein.
• Fusion or chimeric proteins such as those of the present invention, are easily purified, using the characteristics of the proteins to bind to a chromatographic matrix, or to precipitate under certain conditions.
• the purification of these fusion proteins is easy by, for example, but not limited to, sepharose-glutathione columns which are known to one skilled in the art.
• polynucleotide of the invention which codes for the peptide of the invention, or to the polynucleotide sequence complementary to said polynucleotide.
• SEQ sequences. ID. N ° 3 and SEQ.ID. No. 4 describe the nucleotide sequences from which the SEQs are derived. ID. N ° 1 and SEQ. ID. N ° 2, respectively.
• nucleotide sequence refers to a polymeric form of nucleotides of any length that may or may not be chemical or biochemically modified. They refer, therefore, to any polyiribonucleotide or polydeoxyribonucleotide, both single-stranded and double-stranded.
• the polynucleotide of the invention can be obtained artificially by conventional cloning and selection methods, or by sequencing.
• the polynucleotide in addition to the coding sequence, can carry other elements, such as, but not limited to, introns, non-coding sequences at the 5 'or 3' ends, ribosome binding sites, or stabilizing sequences. These polynucleotides can additionally also include coding sequences for additional amino acids that may be useful, for example, but not limited to increasing the stability of the peptide generated from it or allowing a better purification thereof.
• Cloning of the polynucleotide of the invention is preferably performed using an expression vector.
• These vectors include in the sequence of the polynucleotide of the invention or, where appropriate, between the polynucleotide sequence encoding a carrier protein and the polynucleotide sequence of the invention a series of amino acid sequences that are protease cleavage targets.
• the advantage of this structure is that once the fusion protein is produced, the expression system is lysed and purified by means of, for example, but not limited to, affinity chromatography, the carrier protein of the peptide of the invention can be cleaved by digestion with a protease and repurify the peptide of the invention by the same chromatographic system.
• the polynucleotide sequences of the invention can be inserted into a replicating vector for cloning (amplification of DNA) or for expression.
• expression vectors are, but are not limited to, plasmids, cosmids, viral particles, that is, viral DNA or RNA vectors, or phages.
• the polynucleotides of the invention can be inserted into these vectors by a series of procedures known to those skilled in the art. For example, the polynucleotide sequence is inserted at sites suitable for restriction endonuclease enzymes by standard techniques.
• Vector components generally include, but are not limited to, one or more signal sequences, an origin of replication, one or more genetic markers, an "enhancer” element, a promoter and / or a transcription termination sequence.
• the construction of appropriate vectors containing one or more of these components comprises standard ligation techniques, which are known to one skilled in the art.
• expression vector of the invention comprising the polynucleotides of the invention (SEQ. ID. No. 3 and SEQ. ID. N 4).
• the expression vector of the invention can be introduced, for example, although not limited, transfection or transformation, in host cells, such as, but not limited to, plant, mammalian, bacterial, yeast or insect cells, hereinafter "host cell of the invention".
• host cell of the invention The introduction of the expression vector of the invention into the cell can be carried out by any of the physical or biological methods to give rise to transformed or transfected cells.
• biological methods include, but are not limited to, the use of viral DNA and RNA vectors.
• the main advantage of physical methods is that they are not associated with oncogenic or pathological virus processes. However, physical methods are less accurate and often result in insertions of multiple copies, random integrations, disruption of own and foreign genetic sequences, as well as unpredictable expression.
• viral vectors for the introduction of genes into mammalian cells there are vectors of poxvirus, herpes simplex, adenovirus, vectors associated with adenovirus, etc.
• the peptides of the invention SEQ. ID. N ° 1 and SEQ.ID. N ° 2 have antigenic capacity and can be used to develop mono and polyclonal antibodies that specifically bind to it. This can be accomplished by various methods known to a person skilled in the art. Therefore, another aspect of the invention relates to an antibody, hereafter referred to as "antibody of the invention", specific to the peptide of the invention.
• antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, that is, molecules that contain an antigen binding site that specifically binds (immunoreacts) with the peptide of the invention.
• portions of immunologically active immunoglobulin molecules include F (ab) and F (ab ') 2 fragments, which can be generated by treating the antibody with an enzyme such as pepsin.
• the antibodies can be polyclonal (typically include different antibodies directed against different determinants or epitopes) or monoclonal (directed against a single determinant in the antigen).
• the monoclonal antibody can be altered biochemically or by genetic manipulation, or it can be synthetic, possibly lacking the antibody in whole or in parts, from portions that are not necessary for recognition of the peptide of the invention and being substituted by others that communicate additional advantageous properties to the antibody.
• the antibody can also be recombinant, chimeric, humanized, synthetic or a combination of any of the foregoing.
• a "recombinant antibody or polypeptide” is a antibody that has been produced in a host cell transformed or transfected with the nucleic acid encoding the antibody or peptide of the invention, or that produces the antibody or peptide of the invention as a result of homologous recombination.
• composition of the invention comprising an element selected from:
• the composition of the invention further comprises a pharmaceutically acceptable carrier or excipient.
• the composition of the invention further comprises an adjuvant.
• the composition of the invention further comprises another active ingredient.
• excipient refers to a substance that helps the absorption of the elements of the composition of the invention, stabilizes said elements, activates or aids the preparation of the composition in the sense of giving it consistency or providing flavors that make it nicer.
• the excipients could have the function of keeping the ingredients together, such as, for example, starches, sugars or cellulose, the sweetening function, the coloring function, the protective function of the composition, for example, to isolate it from air and / or moisture, the filling function of a tablet, capsule or any other form of presentation, such as, for example, is the case of dibasic calcium phosphate, the disintegrating function to facilitate the dissolution of the components and its absorption in the intestine, without excluding other types of excipients not mentioned in this paragraph.
• the "vehicle”, like the excipient, is a substance that is used in the composition to dilute any of the components of the present invention comprised therein to a certain volume or weight.
• the pharmacologically acceptable carrier is an inert substance or action analogous to any of the elements of the present invention.
• the function of the vehicle is to facilitate the incorporation of other elements, allow a better dosage and administration or give consistency and form to the composition.
• the pharmacologically acceptable carrier is the diluent.
• adjuvant refers to an agent that does not possess an antigenic effect by itself, but which can stimulate the immune system by increasing its response to the composition of the invention.
• adjuvants known in the state of the art, such as, but not limited to, aluminum phosphate, aluminum hydroxide, toll-like receptor agonists, cytokines, squalene, incomplete Freund's adjuvant or Freund's complete adjuvant.
• the proteins of the invention comprise one or several epitopes common to the two orthologs of tick and mosquito subolesina, so, as the examples of the present invention show, it is capable of inducing a protective immune response against infestations caused at least by both parasites in several hosts. Therefore, its administration to an organism is very useful to reduce the transmission of pathogens by these vectors.
• another aspect of the invention relates to the use of the polynucleotide, expression vector, cell, organism, antibody or composition of the invention for the preparation of a medicament, hereafter referred to as “medicament. of the invention "; or alternatively, to the peptide, polynucleotide, expression vector, cell, organism, antibody or composition of the invention for use as a medicament.
• medication refers to any substance used for prevention, relief, treatment or cure of infestations or diseases in man and animals.
• it refers to a composition comprising the peptide, the polynucleotide, the expression vector, the cell, the organism, the antibody or the composition of the invention.
• the medicament of the invention comprises the peptide, polynucleotide, expression vector, cell, organism, antibody or composition of the invention in a therapeutically effective amount, which is capable of inducing an immune response against infestations caused. by hematophagous arthropods, preferably, by ticks, mosquitoes and sandflies, in the organism to which it is administered.
• the term medication therefore includes what is known as a vaccine.
• the term "therapeutically effective amount” refers to the amount of peptide, polynucleotide, expression vector, cell, organism, antibody or composition of the invention, which produces the desired effect.
• Pharmaceutically acceptable adjuvants and vehicles that can be used in the medicament of the invention are those known to those skilled in the art.
• the medicaments of the invention can be used both alone and in combination with other medicaments or compositions for the treatment or prevention of infestations caused by hematophagous arthropods, preferably, by ticks, mosquitoes and sandflies.
• prevention as understood in the present invention, is to prevent the occurrence of damage whose cause is infestations caused by hematophagous arthropods, preferably, by ticks and mosquitoes.
• treatment means combating the effects caused as a result of infestations caused by hematophagous arthropods, preferably, by ticks, mosquitoes and sandflies, to stabilize the condition of individuals or prevent subsequent damage.
• subolesina is an evolutionarily conserved antigen between ticks and mosquitoes, it is to be expected that other genus of hematophagous arthropods possess orthologous proteins that present a global homology in their amino acid sequence with the sequence thereof, so that the peptides of the invention, when comprising at least one epitope of subolesina, also would be useful to induce immunity against the infestations caused by other hematophagous arthropod parasites that express orthologs proteins to the subolesina.
• another aspect of the invention relates to the use of the peptide, polynucleotide, expression vector, cell, organism, antibody or composition of the invention for the preparation of a medicament for treatment or prevention. of infestations caused by hematophagous arthropods, or alternatively, to the peptide, polynucleotide, expression vector, cell, organism, antibody or composition of the invention for use as a medicament for the treatment or prevention of infestations caused by hematophagous arthropods.
• infestation refers to the invasion of a living organism by external or internal parasitic agents.
• the objective of these parasites is their survival at the expense of the parasitizing host.
• Hematophagous arthropods means those invertebrate organisms, with segmented bodies, bilateral symmetry, chitinous exoskeleton, segmented locomotor appendages, presence of sense organs, dioecious, sexually reproduced and whose growth is metamorphosis.
• hematophagous arthropods are, but are not limited to, mites, flies, mosquitoes, simulids, sandflies, fleas, beetles, arbovirosis, trypanosomes, filarias or ticks.
• the hematophagous arthropods are the tick, the mosquito and the sandfly.
• a non-limiting example of some of the diseases transmitted by an infestation of hematophagous arthropods include: Borreliosis, Anaplasmosis, Coxiqueliosis, Franciselliosis, Riquetsiosis, Theileriosis, Elriquesiosis and Babesiosis, as well as viral diseases such as hematophage-borne arthropods. Infestations by hematophagous arthropods also have an economic impact on the livestock, since they cause the loss of weight of the animals and the reduction of milk production.
• the medicament of the invention is a vaccine.
• the term "vaccine” refers to an epitopic or antigen preparation used to elicit a response of the immune system to a parasitic infestation or to a disease. They are prepared of antigens or epitopes that, once inside the organism, provoke the response of the immune system through the production of antibodies, and generate immunological memory producing permanent or transient immunity.
• FIG. one Antibody response to SUB / AKR chimeras Q38 and Q41 (SEQ.ID.N 0 1 and SEQ.ID.N 0 2) in mice vaccinated with them.
• the antibody titer was determined by ELISA in vaccinated mice (A) in serum samples collected at different times and faced with each specific protein used in the vaccination and the control was evaluated against a mixture of all the proteins used in the experiment, and (B) at the end of the experiment (week 8) against each of the recombinant proteins used in the vaccination.
• the antibody titer was expressed as the mean ⁇ SD OD 45 onm (OD mouse serum - OD PB s control) and compared between vaccinated and control groups using an ANOVA test (A; * P ⁇ 0.05) ot Student's (B; * P ⁇ 0.05).
• the arrows in (A) indicate dates for the 1st, 2nd and 3rd immunization and vector infestation.
• FIG. 2 Effect of vaccination with SUB / AKR chimeras Q38 and Q41 (SEQ.ID.N 0 1 and SEQ.ID.N 0 2) in vector infestation.
• the reduction of the percentage in infestation, molting and oviposition and / or fertility in the individuals fed in vaccinated mice with respect to the control (mice fed in mice vaccinated with adjuvant saline) is shown for (A) ticks. ricinus, (B) A. albopictus mosquitoes and (C) P. perniciosus sandflies.
• SEQ ID No 1 Identify the sequence of amino acids corresponding to the synthetic protein "Chimera 38", of 238 residues, of artificial origin. Said sequence comprises a region rich in histidine, added to allow the purification of the recombinant proteins by affinity columns and which are known to those skilled in the art, located in the base range 6-1 1 of the sequence.
• Said sequence comprises a region rich in histidine, added to allow the purification of the recombinant proteins by affinity columns and which are known to those skilled in the art, located in the base range 6-1 1 of the sequence.
• SEQ ID No 3. DNA sequence of 657 nucleotides, of artificial or synthetic origin, used in the production of the synthetic protein "Chimera 38" and coding for SEQ. I D. No. 1.
• SEQ ID No 4. 645 nucleotide DNA sequence, of artificial or synthetic origin, used in the production of the synthetic protein "Chimera 41" and coding for the SEQ. I D. No. 2
• EXAMPLE 1 Control of infestations produced by multiple vectors by subolesina / akirina vaccines
• VTE vector-borne diseases
• Anaplasma phagocytophilum Rostiales: Anaplasmataceae
• Borrelia burgdor ⁇ eri Spirochaetales: Spirochaetaceae
• Flaviviridae Flavivirus
• Phlebotomes are also vectors of several pathogens, including Leishmania spp., Which causes various forms of human and canine leishmaniasis (Chumbles F et ai, 2007).
• VTE vector-borne diseases
• Vaccination is an alternative for the control of vectors that is not harmful to the environment that allows the control of several VTEs through the use of common targets among the vectors (de la Fuente, et ai, 1998; de la Fuente, et ai, 2007 ; Merino, O et ai, 201 1).
• Vaccination with antigens of arthropod vectors has been proposed as an alternative for controlling vector infestations and reducing their ability to transmit pathogens that impact on human and animal health (Kishore K et ai, 2006; de la Fuente , et al., 2007; Merino, O et al., 201 1; Valenzuela, et al., 2001; Lal, et al., 2001; Almeida and Billingsley, 2002; Suneja, et ai., 2003; Milleron, et ai., 2004; Titus, et ai., 2006; Kedzierski L et ai., 2006; Sa ⁇ l A 2007; Canales M. et ai., 2009).
• Subolesina and akirina (AKR) are orthologous proteins of ticks and insects (de la Fuente, et al., 2006a; Macqueen DJ and Johnston IA, 2009; Galindo RC et al., 2009; Mangold AJ et al., 2009 ) that affect the expression of signal transduction and innate response genes, as well as positive and negative transcriptional regulators (de la Fuente, et al., 2006; Macqueen DJ and Johnston IA, 2009; Galindo RC et al., 2009; Mangold AJ et al., 2009; Goto A et al., 2006).
• SUB intervenes in other molecular pathways including those necessary for the development and function of tissues, as well as for infection and multiplication of pathogens in ticks (de la Fuente, et al., 2006a; of the Source, et ai, 2006b; Kocan, et ai, 2007; Zivkovic Z et al., 2010).
• SUB was discovered as a protective antigen in Ixodes scapularis (Almazán C et al., 2003). Vaccination with recombinant SUB showed an efficient control of tick infestations by reducing their number, weight and oviposition, as well as in the infection of different tick-borne pathogens (Merino, O et al., 2011 a; Merino, O et al. , 201 1 b, from Source J et al., 201 1).
• the mosquitoes used in this study came from a colony of Baix Llobregat (Barcelona, Spain) maintained for 31 generations at the University of Zaragoza.
• the mosquitoes were raised in an environmental cabin at 25 ⁇ 1 ° C, 75 ⁇ 5% RH, and photoperiod of 12 h light: 12 h darkness.
• Phlebotomus perniciosus sandflies used in this study came from an indigenous colony (Madrid City, Spain) established in the Medical Entomology Unit of the Carlos III Health Institute (Spain).
• the P. perniciosus strain has been maintained in the laboratory for more than 200 generations.
• the specimens were raised in an environmental cabin at 28 ° C, 95-100% RH, and a photoperiod of 17 h light: 7 h darkness.
• the females were mixed with males after their emergence until the females were selected for the experiment. Under these breeding conditions mating occurs, as has been proven by dissection and examination of ovaries (Moreno-Cid JA et al., 2011).
• insects were supplemented with absorbent cotton saturated with 10-30% of a sucrose solution for four days. On day five, the insects were fasted for five hours before infestation in mice (Moreno-Cid JA et al., 201 1). Finally, the females were kept in new cages without males until the end of the experiment.
• the chimeras SUB / AKR Q38 and Q41 were designed by combining linear B-cell and discontinuous conformational protective epitopes identified in sheep and rabbit sera immunized with SUB from Ixodes scapularis and AKR from A. albopictus (Channels M, et ai , 2009; Prudencio, CR et ai, 2010).
• the Q38 chimera was designed by combining protective epitopes selected from SUB and AKR and introducing the GGGS spacer sequence to improve the exposure of these epitopes.
• the Q41 chimera was designed by adding cysteines between the protective epitopes of SUB and AKR for the formation of disulfide bridges and aiding in the exposure of epitopes. Amino acids R and K were also added to improve trypsin digestion and peptide analysis in future experiments.
• Recombinant chimeras of SUB / AKR Q38 and Q41 were expressed in Escherichia coli from synthetic genes optimized for codon use in E. coli and purified by affinity chromatography with Ni columns (Genscript Corporation, Piscatway, NJ, USA) . Recombinant antigens or saline solution were adjuvant in Montanide ISA 50 V2 (Seppic, Paris, France).
• E 100 (I - a k ), where a k represents the reduction in the development process observed (k) in the vectors fed in vaccinated mice when they were compared with those fed in control mice, vaccinated with saline and adjuvant.
• mice For each mouse infestation, larvae derived from batches of 100 tick eggs were used. The mice were kept at 22 ° C and with 80-90% RH. Fattened larvae were collected three times a day for 7 days, two days after the full larvae detached from the host. The larvae were collected, counted and incubated for molting at 24 ° C and 90% RH.
• Each mouse was deposited in a nylon cage, anesthetized with ketamine (100 mg / kg) and xylazine hydroxychloride (10 mg / kg) by intraperitoneal injection and was exposed to the infestation of 40 females of A. albopictus for 30 min (Moreno -Cid JA et ai, 2009). After exposure, 20 female mosquitoes were introduced into vials Individuals for the study of oviposition. The mortality of female mosquitoes was assessed at 10 days post-infestation (dpi). The parideras were removed at 15 dpi days and the eggs were counted to evaluate oviposition. These were incubated for 72 h at room temperature in water until emergency to assess fertility.
• mice were deposited in a nylon cage, anesthetized with ketamine (100 mg / kg) and xylazine hydroxychloride (10 mg / kg) by intraperitoneal injection and was exposed to the infestation of 35-40 females of P. perniciosus (Moreno- Cid JA et al., 2009). After exposure, 10 female sandflies were introduced into individual vials for the study of oviposition. The eggs of the females fed on the same mouse were transferred to a container for the study of adult phlebotome development and to evaluate fertility as previously described (Canales M et al., 2009; Moreno-Cid JA et al., 2009 ).
• mice immunized by ELISA Plasma samples were collected for each immunized and control animal at weeks 0, 3, 6 and 8 before each immunization and infestation. The serum was separated from the blood by centrifugation and stored individually at -20 ° C.
• the chimeras of SUB / AKR Q38 and Q41 have been designed by combining linear B epitopes and discontinuous conformational protective epitopes. Both chimeras comprise protective epitopes of both SUB and AKR but differ in their composition and position.
• the E of the vaccine is a parameter that takes into account all the effects of vector infestation that is observed for a given antigen. This parameter cannot be used to compare the effect of vaccination between different vector species because the development processes evaluated may be different for each vector. However, vaccine E is a good parameter to compare the effects of different antigens on infestation by a certain vector. Vaccine E analysis showed a reduction in vector infestations in vaccinated mice (Fig. 2D).
• the present invention shows the effect of vaccination with chimeras of SUB / AK Q38 and Q41 in infestations by ticks, mosquitoes and sand mosquitoes (sandflies), with effects on molting, oviposition and / or fertility.
• the hypothesis posed in these experiments is that the feeding of Hematophagous arthropods on immunized hosts entail the ingestion of specific antibodies to target antigens, which cause deleterious effects on their food and reproductive function.
• anti-SUB / AKR antibodies affect vector infestation and fertility is unknown, but it may include the reduction of the biological activity of SUB / AKR and / or the interaction of antibodies with conserved epitopes. in other proteins (de la Fuente et al. 2011; Moreno-Cid JA et al. 201 1).
• the control of pathogens transmitted by arthropod vectors that affect animal and human health is important for the eradication of vector-borne diseases.
• An important advantage of vaccines against arthropods is probably their ability to reduce or prevent the transmission of various pathogens through the immunization of reservoir hosts and susceptible animal populations (de la Fuente et al. 2003; de la Fuente et al. 1998; Merino, O.
• results of the present invention are the first that show the effect of SUB / AKR chimeras on different vector species, as well as the first experiments that show the effects of vaccination of tick / infestations.
• ricinus The comparison between the chimeras of SUB / AKR Q38 and Q41 in infestations of ticks, mosquitoes and sandflies showed that (a) vaccination with Q41 showed the highest E in mosquitoes by reducing the survival and fertility of females, and , (b) Vaccination with Q38 had the highest effect in reducing the oviposition of mosquitoes and sandflies.
• the SU B and AKR vaccines controlled vector infestations due to the high conservation of tick and mosquito proteins (Channels M et al.
• tick protective antigen, 4D8 is a conserved protein involved in modulation of tick blood ingestion and reproduction.
• Mangold AJ Galindo RC, from Source J. Response to the commentary of D. Macqueen on: Galindo RC, Doncel-Pérez E, Zivkovic Z, Naranjo V, Gortázar C, Mangold AJ, et al. Tick subolesin is an ortholog of the akirins described in insects and vertebrates [Dev. Comp. Immunol 33 (2009) 612-617]. Dev Comp Immunol 2009; 33: 878-879.
• Nijhof AM Taoufik A, from Source J, Kocan KM, from Vries E, Jongejan F. Gene silensing of the tick protective antigens, Bm86, Bm91, subolesin, in the one-host tick Boophilus microplus by RNA interference.
• Akirin links twist-regulated transcription with the Brahma chromatin remodeling complex during embryogenesis.

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