WO2013097022A2 - Recombinant proteins, polynucleotides and vaccines to combat bovine herpesvirus - Google Patents

Recombinant proteins, polynucleotides and vaccines to combat bovine herpesvirus Download PDF

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WO2013097022A2
WO2013097022A2 PCT/BR2013/000006 BR2013000006W WO2013097022A2 WO 2013097022 A2 WO2013097022 A2 WO 2013097022A2 BR 2013000006 W BR2013000006 W BR 2013000006W WO 2013097022 A2 WO2013097022 A2 WO 2013097022A2
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bohv
bovine
proteins
epitopes
recombinant
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WO2013097022A3 (en
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Edel FIGUEIREDO BARBOSA STANCIOLI
Flavio GUIMARÃES DA FONSECA
Mateus LAGUARDIA NASCIMENTO
Amanda BORGES FERRARI
Aline Aparecida SILVA BARBOSA
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Universidade Federal De Minas Gerais - Ufmg
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    • 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
    • A61K39/12—Viral antigens
    • A61K39/245—Herpetoviridae, e.g. herpes simplex virus
    • A61K39/265—Infectious rhinotracheitis virus
    • 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
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • 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
    • A61K2039/70—Multivalent vaccine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
    • C12N2710/16722—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
    • C12N2710/16734—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/023—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a poxvirus

Definitions

  • the present technology describes the production of a vaccine containing recombinant proteins comprising 04 epitopes of the main antigenic proteins of each of two bovine herpesviruses (Bovine herpesvirus 1 - BoHV-1 and Bovine Herpesvirus 5 - BoHV-5), produced in prokaryote system.
  • each recombinant protein contains 04 linearly produced epitopes, one with BoHV-1 epitopes and one with BoHV-5 epitopes, resulting in two multiepitope proteins that have the potential to immunize and control viral spread in herds.
  • ruminants for two bovine diseases of major importance to the world livestock sector Infectious Bovine Rhinotracheitis and Bovine Encephalitis.
  • BoHV-1 and BoHV-5 Due to the great economic importance of cattle herds, fighting diseases that reduce productivity and can increase mortality rates is a priority in the affected countries.
  • the complex of diseases caused by BoHV-1 and BoHV-5 is accompanied by significant economic losses, such as reduced milk production, infertility, miscarriages and animal deaths, and for this reason, several attempts to eradicate these viruses - mainly BoHV. -1, due to their geographical distribution - have already been made in several countries. Some countries in Europe (Switzerland, Sweden, Austria, Denmark, Finland and Norway) have achieved IBR eradication without the use of vaccines, sacrificing seropositive animals and controlling the entry of potential contaminants into the country.
  • BoHV-1 / BoHV-5 are quite low, otherwise much of the country's cattle herd would have to be sacrificed, which would represent an even greater economic loss than those caused by viruses.
  • JONES, C; CHOWDHURY, S. A review of the biology of bovine herpesvirus type 1 -BHV-1, its role as a cofactor in the bovine respiratory disease complex and development of improved vaccines.
  • Subunit vaccines can be quite satisfactory in these respects. Using only immunogenic components of the virus, it is possible to induce the development of a protective response against infection. In this approach, risks of vaccine handling, latency and reactivation, recombination, and dissemination are eliminated, and one can easily discriminate between vaccinated and infected individuals. It is also possible, using in silico analyzes, to find immunogenic regions in the viral components and
  • the vaccine can be manipulated to stimulate cellular and humoral responses, providing real protection to vaccinated animals.
  • the use of artificially constructed multiepitope protein vaccines has been increasing in recent years, but their use against herpesviruses has not been reported.
  • BoHV-1 and BoHV-5 VP8 protein also induces, according to literature reports,
  • each protein contains 04 linearly produced epitopes, one with BoHV-1 epitopes and one with BoHV-5 epitopes, resulting in two multiepitope proteins that have the potential to immunize ruminants for two major bovine diseases.
  • Bovine Infectious Rhinotracheitis and Bovine Encephalitis are the major antigenic proteins of each of the two bovine herpesviruses, BoHV-1 and BoHV-5, produced in prokaryotic and / or eukaryotic system using recombinant vaccinia virus generated on the MVA platform (Modified Ankara Virus) .
  • each protein contains 04 linearly produced epitopes, one with BoHV-1 epitopes and one with BoHV-5 epitopes, resulting in two multiepitope proteins that have the potential to immunize ruminants for two major bovine diseases. for the world livestock sector: Bovine Infectious Rhinotracheitis and Bovine Encephalitis.
  • BoHV-1 proteins and peptides derived from it as a bovine herpesvirus vaccine, however all differ from the present technology and are cited below:
  • CA1338069 describes a vaccine to protect the bovine host from BHV-1 infection.
  • a vaccine comprises at least one antigenic subunit consisting of an epitope of a BHV-1 glycoprotein selected from the group consisting of gl, glll and glV.
  • EP0888777 describes the production of recombinant antigens derived from bovine herpesvirus type 1, especially a BHV-1 glV glycoprotein derived antigen, for use in the protection of
  • PI248650 describes a DNA vaccine comprising a plasmid containing a nucleic acid encoding a type 1 bovine herpes virus immunogen and the elements necessary for its expression in vivo.
  • US54622734 describes a kit and protocol for inducing immunity against herpesvirus infection.
  • the kit comprises a formulation containing an isolated viral glycoprotein that can be injected intramuscularly to induce systemic immunity.
  • a second formulation comprises a recombinant viral glycoprotein to be administered intranasally with an adjuvant.
  • US5858989 describes recombinant subunit vaccines against bovine herpesvirus type I, as well as vaccination methods and methods of producing the antigens or nucleotides employed in the vaccines.
  • Fusion proteins comprise a bovine herpesvirus protein linked to a biotherapeutic protein or reporter protein.
  • WO0134184 describes method and compositions for eliciting an immune response against bovine herpesvirus type 1 epitopes.
  • the composition comprises a type 1 bovine herpesvirus epitope complexed to a thermal shock protein and pharmaceutically acceptable carriers.
  • Figure 1- 1A Analysis of BoHV-1 protein eluates 1-9. M - Molecular Weight Standard. El 1 to 9 - Eluates 1 to 9. Seta points to proteins of interest; 1 B - Analysis of BoHV-5 protein eluates 1-9. M - Molecular Weight Standard. El 1 to 9 - Eluates 1 to 9. Arrow points to proteins of interest.
  • FIG. 2- Cell viability after 24 (A), 48 (B) and 72 (C) hours.
  • PBMC cells were inoculated with recombinant BoHV-1, BoHV-5 and BoHV-1 + BoHV-5 proteins and incubated for 24, 48 and 72 hours at 37 ° C in a CO 2 oven.
  • cells were evaluated for cell viability by the MTT colorimetric method. The results confirmed the absence of cytotoxicity of multiepitope recombinant proteins produced for bovine cells under "ex vivo" analysis.
  • FIG. 3- Dosage of Nitric Oxide (NO) using the Griess method after 24 (A), 48 (B) and 72 (C) hours of treatment.
  • PBMC cells were inoculated with recombinant BoHV-1, BoHV-5 and BoHV-1 + proteins.
  • BoHV-5 At the end of the incubation period, the supernatant of each group was collected and evaluated for nitric oxide production. There was no change in NO production for any of the protein treatments produced at any of the times tested, once again proving the absence of cytotoxicity of multiepitope recombinant proteins produced for bovine cells under "ex vivo" analysis.
  • FIG 4 Scheme of rabbit immunization with the recombinant proteins produced, where the dose booster system was performed.
  • the purified proteins were prepared with aluminum hydroxide adjuvant (70% protein, 5% aluminum hydroxide and 25% emulsigen) and inoculated into New Zealand rabbits.
  • FIG. 5 Western assay of RecPBoHV-1 recombinant protein (10 ⁇ g) and purified BoHV-1 ⁇ g virus). Electrophoretic fractionation was performed on 12% SDS-PAGE gel and transferred to PVDF membrane. A - RecBoHV-1 membrane; primary antibodies used: sera from animals strongly reactive to BoHV-1, BoHV-5 or both viruses (1 - Mab 1240, 3 - rabbits negative for BoHV-1 and 5, 5 - Bay 3, 6 - Mix 1, 7 - Mix 5, 8 - aBoHV-1, 9 - P1, 10 - P5, 11 - PN, 12 - Pab 1, 13 - P1 and 14 - P5).
  • B - BoHV-1 membranes B - BoHV-1 membranes; primary antibody used: sera from animals strongly reactive to BoHV-1, BoHV-5 or both viruses (1 - Mab 1240, 3 - rabbits negative for BoHV-1 and 5, 5 - Bay 3, 6 - Mix 1, 7 - Mix 5, 8 - aBoHV-1, 9 - P1, 10 - P5, 11 - PN, 12 - Pab 1, 13 - P1 and 14 - Bay 3, 15 - P1, 16 - Pab1, 17 - PN, 18 - PN, 19 - P5 and 20 - P5).
  • Kaleidoscope molecular weight marker BioRad).
  • FIG. 6- Western assay of RecPBoHV-5 recombinant protein (10 ⁇ g) and purified BoHV-5 virus ( ⁇ ). Electrophoretic fractionation was performed on 12% SDS-PAGE gel and transferred to PVDF membrane.
  • B - BoHV-5 membrane B - BoHV-5 membrane; primary antibody used: animal sera strongly reactive to BoHV-1, BoHV-5 or both viruses (1 - Mab 1240, 3 - BoHV-1 and 5, 5 - negative rabbit serum - Bay 3, 6 - Mix 1, 7 - Mix 5 , 8 - aBoHV-1, 9 - P1, 10 - P5, 11 - PN, 12 - Pab 1, 13 - P1 and 14 - P5, 15 - PN, 16 - Pab1). Kaleidoscope molecular weight marker (BioRad).
  • Electrophoretic fractionation was performed on 12% SDS-PAGE gel and transferred to PVDF membrane.
  • FIG. 8 Western assay of RecPBoHV-5 recombinant protein (10 ⁇ g) and purified BoHV-5 ⁇ g virus). Electrophoretic fractionation was performed on 12% SDS-PAGE gel and transferred to PVDF membrane. (1) Pool rabbits 1 and 2 cont. - zero; (2) Pool rabbits 1 and 2 cont. - Prime; (3) Pool rabbits
  • FIG. 9 - ELISA (rabbit and bovine total anti-IgG) assay using purified BoHV-1 virus (AgV-BoHV-1) and recombinant protein (Ag-RecBoHV-1) on
  • samples from a previously tested bovine library were used.
  • Figure 10 - ELISA (rabbit and bovine total anti-IgG) assay using purified BoHV-5 virus 3 (AgV-BoHV-5) and recombinant protein (Ag-RecBoHV-5) at a concentration of 250ng / well.
  • A, C, E AgV-BoHV-5 X rabbit sera pool 5 (starting dose, booster 1 and booster 2 respectively);
  • G AgV-BoHV-5 X bovine serum pool.
  • samples from a previously tested bovine library were used.
  • Figure 11 VERO cells infected with recombinant Modified Ankara Virus expressing BoHV-1 multiepitope recombinant protein. 12A - visualization of clones under optical microscope and 12B - visualization of clones under fluorescence microscope.
  • the present technology describes the production of a recombinant protein-containing vaccine comprising 04 epitopes of the main antigenic proteins of each of the two bovine herpesviruses, BoHV-1 and BoHV-5, produced in prokaryotic and / or eukaryotic system using recombinant virus generated in MVA (Modified Ankara Virus) platform.
  • each recombinant protein contains 04 linearly produced epitopes, one with BoHV-1 epitopes and the other with BoHV-5 epitopes, and epitopes linked using flexible glycine and serine loops to join the regions.
  • Example 1 In silico analysis of targeting protein coding genes, synthetic gene construction, and expression vector cloning In silico analyzes were the initial focus of the work, where the genes encoding the target proteins and their possible regions recognized by T and B cells were evaluated.
  • BoHV-5 gB important epitopes were determined by comparison with the BoHV-1 sequence:
  • BoHV-1 and BoHV-5 in their gB and VP8 proteins allowed the use of the same fragments for both viruses. There is only difference between proteins in gD protein fragments. This fact increases the possibility of protein cross-protection. Inoculation of both proteins in the same animal may enhance immunity against each virus.
  • Synthetic genes containing the BoHV-1 and BoHV-5 protein coding fragments were produced, and using a developed PCR system a restriction site for the BamHI and HindIII endonucleases (at the 5 'end of each primer) was added to each primer. Promega, USA) - For sense and antisense primers, respectively - as well as two extreme bases to facilitate the binding of enzymes to the DNA strand. Then, they were cloned into a commercial vector pQE 30, which has as its main feature the fusion of the sequence of interest to another histidine tail coder to facilitate post expression purification.
  • the clones selected from the above experiment were subjected to sequencing by the didesoxi method (Sanger et al., 1977) using automatic sequencer (Megabace) with primers flanking. Insert or the vector.
  • M15 strain Escherichia coli bacteria were transformed with pQE plasmids cloned with the inserts of interest - the gene cassettes. The chosen clones were grown and stored or screened.
  • Example 3- Denaturing Purification of Recombinant Proteins by Affinity Chromatography After determining the type of purification to which the proteins would be subjected, the purification itself was performed. The principle of this process is based on the affinity of the histidine tail, inserted into proteins by cloning the synthetic gene to vector pQE 30, to the resin containing nitrile tri-acetic acid (NTA) and charged with Ni 2+ ions (Ni- NTA). This high affinity causes the proteins to be immobilized on the support and separated from the total protein extract, in the principle of immobilized metal ion affinity chromatography (IMAC) already demonstrated in the literature. Washing the column with basic pH buffers removes the other proteins, but not those with histidine tail.
  • NTA nitrile tri-acetic acid
  • IMAC immobilized metal ion affinity chromatography
  • the "extra” proteins followed the expression profile of the "target” proteins and are present, albeit in smaller amounts, in almost all eluates where the major bands are found. This corroborates the hypothesis that they are the same protein, since hardly a contaminating protein, even if it contains a sequence of histidines, would obtain such a similar expression profile, and, as the gene cassette design included flexible loops (GSGSG), the proteins can express differences in folding during expression, producing more than one "population".
  • GGSGSG flexible loops
  • RPMI 1640 culture medium with L-glutamine (Sigma) was used.
  • the contents of each vial were reconstituted in 1 L of ultra pure water and sodium bicarbonate (Merck), 10mM HEPES (N-2 hydroxyethylpiperazine, N-2 ethanesulfonic acid) (Sigma) added.
  • the pH of the solution was adjusted to 7.1 and was called non-supplemented RPMI.
  • the medium used in the cultures was supplemented with 10% fetal bovine serum (Cultilab), 100 U / mL penicillin and 100 Mg / mL streptomycin (Sigma), and was called supplemented RPMI.
  • the solution was filtered with 0.22 ⁇ pore membrane and stored at 4 ° C.
  • bovine blood was performed at the School of Veterinary Medicine of the Federal University of Minas Gerais (UFMG) in Belo Horizonte. The animals were contained, then the blood was collected totaling a final volume of approximately 28 ml of blood from each animal in heparin-containing collection tubes.
  • PBMC was purified in Fico solution ! (GE Healthcare) at a ratio of 2: 1 (blood: Ficoll).
  • PBMC PBMC
  • recombinant proteins RecPBoHV-1, RecPBoHV-5 and RecPBoHV-1 + RecPBoHV-5
  • the PMBCs were resuspended in culture medium after counting and distributed into 96-well cell culture plates at a concentration of 1.5x10 5 cells / well.
  • PBMC were stimulated or not with the quadruplicate recombinant proteins (100ng / well) for 24, 48 and 72 hours at 37 ° C in a 5% CO 2 atmosphere. At the end of each culture time, samples were evaluated for cell viability and nitric oxide production.
  • the MTT colorimetric method 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium (Sigma) was used. Final concentration equal to 5 mg / mL.
  • solution A 3 ml phosphoric acid - 5% (H 3 PO 4 ), 47 ml distilled water and 1 g sulfanilamide.
  • Solution B 0.05g of alpha-naphytil-ethylenodiane (NEED) and 25ml of distilled water.
  • RecPBoHV-1, RecPBoHV-5 and RecPBoHV-1 + RecPBoHV-5 multiepitope proteins from the cells 24, 48 and 72 hours at 37 ° C in a CO2 oven, 20 ⁇ _ of MTT solution ( Light and incubated for 4 hours at 37 ° C in a C0 2 oven .
  • the inoculations and experimental procedures involving the rabbits were carried out in the animal room of the Veterinary School (Federal University of Minas Gerais), which has controlled ventilation and temperature. The rabbits were kept in individual cages and fed ration and filtered water at will.
  • Rabbit blood was collected in the marginal vein of the ear, with the aid of xylol-soaked cotton to promote local vasodilation, at the beginning of the experiment, after each immunization and at the end cardiac puncture (under pharmacological control - Ketamine (20 to 40mg / kg). kg) and Xylazine (3 to 4 mg / kg) intravenously).
  • the purified proteins were prepared with aluminum hydroxide adjuvant (70% protein, 5% aluminum hydroxide and 25% emulsigen) and inoculated into New Zealand rabbits (dose-boosting system consisting of three 20-day interval doses). ), as shown in Figure 4.
  • BoHV-1 ELISA An indirect ELISA was developed and standardized to analyze the reactivity of rabbit sera (New Zealand) against recombinant proteins. As control of the assays a bovine soroteca was used previously tested by viral seroneutralization methods and a commercial BoHV-1 ELISA containing sera from animals strongly reactive to BoHV-1, 5, both virus-negative or both.
  • plaques were sensitized with 250 ng / well of the recombinant antigen produced or with total purified virus proteins. Serum from New Zealand rabbits inoculated with recombinant proteins (RecBoHV-1, RecBoHV-5 or both) was used as the primary antibody using a dose-boosting system consisting of three 20-day interval doses.
  • a bovine library previously tested by the viral seroneutralization methods and by a commercial BoHV-1 ELISA containing sera from animals highly reactive to BoHV-1, 5, both virus or negative for both, was used.
  • the anti-bovine IgG and anti-rabbit IgG were used as secondary antibodies ( Figures 9 and Figure 10).
  • Modified Ankara Virus is a strain of Vaccinia virus that has been attenuated by serial passage for more than 500 times in embryonic chicken fibroblasts. Its name is because the parental virus was isolated from a horse injury in Ankara, Turkey (BLANCHARD, TJ, ALCAMI, A., PA AYIOTA, A., SMITH, GL. Modified vaccinia virus Ankara undergoes limited replication in human cells and lacks several immunomodulatory proteins: implications for use as a human vaccine. Journal of General Virology.v.79, p.1 159-1167, 1998).
  • MVA During its attenuation, MVA suffered genomic losses totaling approximately 30kb, resulting in a total loss of 26 ORFs along with the fragmentation of 21 more ORFs, as well as numerous small-scale mutations. MVA was used in the final stages of the smallpox eradication campaign and was administered intracutaneously, subcutaneously and intramuscularly to over 120,000 people in southern Germany and Turkey, and no significant side effects were reported. MVA is a promising candidate for vaccine use due to its restricted host spectrum, immunogenicity and avirulence in animal models and its excellent safety prognosis as a smallpox vaccine.
  • Plasmid pLW44 has 5028 bp, the p11 and mH5 promoters flanked by MVA virus gene regions, a coding region for GFP protein (Green fluorescent protein) and the enzymatic restriction sites of Sma I, Sal I and Pst I. gene cassettes have been inserted into the region controlled by the mH5 promoter between the restriction sites of the Sal I and Pst I enzymes.
  • the p11 promoter (late promoter) controls GFP production.
  • Both p11 and mH5 promoters are under the control of early and / or late MVA virus transcriptional activators.
  • transcriptional activators present together with the virion and / or produced after viral infection will associate with promoters leading to the production and expression of GFP and BoHV-1 and BoHV-multiepitope recombinant proteins. 5 Confirmation of cloning of recombinant colonies containing the worked gene fragments was done by PCR at from bacterial cultures using the primers PST-R and SAL - F. Subsequent sequencing confirmed the correct cloning and positioning of the gene cassettes.
  • the method used for the generation of recombinant MVA was performed by homologous recombination.
  • Susceptible cells were infected with "wild" MVA and transfected with transfer plasmids containing the constructed gene cassettes (pLW44) under the control of a VACV (Vaccinia virus) promoter, the selectable marker gene, and flanks corresponding to nonessential regions of the MVA genome.
  • VACV Vaccinia virus
  • Transfer plasmid transfection in cells previously infected with the MVA virus allows homologous recombination between the plasmid and virus genome to occur naturally at relatively high rates (-0.1%).
  • Recombinant viruses expressing the proteins of interest were selected in successive passages in a cell culture system and were cloned biologically using the fluorescence labeling of the selected green fluorescent protein (GFP) as a marker, depending on the selection marker used ( Figure 11 ).
  • GFP green fluorescent protein

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Abstract

The present technology describes the production of a vaccine containing recombinant proteins comprising 4 epitopes of the main antigenic proteins of each of two bovine herpesviruses, namely BoHV-1 (Bovine herpesvirus 1) and BoHV-5 (Bovine herpesvirus 5), produced in a prokaryotic system and/or a eukaryotic system using recombinant virus generated on the MVA (Modified Vaccinia Ankara) platform. Specifically, each recombinant protein contains 4 epitopes produced linearly, one with the BoHV-1 epitopes and the other with the BoHV-5 epitopes, and the epitopes linked with the use of flexible glycine and serine rings to connect the desired regions and to allow suitable tertiary conformation of each fragment, resulting in two multi-epitope proteins with potential to immunize ruminants (especially bovines) against the BoHV-1 and BoHV-5 viruses, for controlling infection and viral dissemination and reducing the level of financial loss caused by two bovine diseases of great significance for the worldwide livestock sector, namely bovine infectious rhinotracheitis and bovine encephalitis.

Description

PROTEÍNAS RECOMBINANTES, POLINUCLEOTÍDEOS E VACINAS  RECOMBINANT PROTEINS, POLINUCLEOTIDS AND VACCINES
CONTRA HERPESVÍRUS BOVINOS  AGAINST BOVINE HERPESVIRUS
A presente tecnologia descreve a produção de uma vacina contendo proteínas recombinantes compreendendo 04 epitopos das principais proteínas antigênicas de cada um de dois herpesvírus bovinos (Bovine herpesvirus 1 - BoHV-1 e Bovine Herpesvirus 5 - BoHV-5), produzidas em sistema procarioto. Especificamente, cada proteína recombinante contém 04 epitopos produzidos de forma linear, uma com os epitopos de BoHV-1 e a outra com os epitopos de BoHV-5, resultando em duas proteínas multiepitopo que têm o potencial de imunizar e controlar a disseminação virai em rebanhos de ruminantes para duas doenças bovinas de grande importância para o setor pecuário mundial: Rinotraqueíte Infecciosa bovina e Encefalite bovina. The present technology describes the production of a vaccine containing recombinant proteins comprising 04 epitopes of the main antigenic proteins of each of two bovine herpesviruses (Bovine herpesvirus 1 - BoHV-1 and Bovine Herpesvirus 5 - BoHV-5), produced in prokaryote system. Specifically, each recombinant protein contains 04 linearly produced epitopes, one with BoHV-1 epitopes and one with BoHV-5 epitopes, resulting in two multiepitope proteins that have the potential to immunize and control viral spread in herds. of ruminants for two bovine diseases of major importance to the world livestock sector: Infectious Bovine Rhinotracheitis and Bovine Encephalitis.
Devido à grande importância económica dos rebanhos bovinos, o combate às doenças que reduzem a produtividade e podem aumentar os índices de mortalidade é prioridade nos países afetados. O complexo de doenças causadas por BoHV-1 e BoHV-5 é acompanhado por perdas económicas significativas, como redução na produção de leite, infertilidade, abortos e morte dos animais e, por esta razão, várias tentativas de erradicação desses vírus - principalmente do BoHV-1 , por sua distribuição geográfica - já foram feitas em diversos países. Alguns países na Europa (Suíça, Suécia, Áustria, Dinamarca, Finlândia e Noruega) alcançaram a erradicação da IBR (Rinotraqueíte Infecciosa Bovina) sem o uso de vacinas, sacrificando os animais soropositivos e controlando a entrada de possíveis contaminantes no país. Obviamente, tal medida só é possível quando a prevalência de BoHV- 1/BoHV-5 é bastante baixa, do contrário, grande parte do efetivo bovino do país teria que ser sacrificado, o que representaria uma perda económica ainda maior que as causadas pelos vírus (JONES, C; CHOWDHURY, S. A review of the biology of bovine herpesvirus type 1 -BHV-1 , its role as a cofactor in the bovine respiratory disease complex and development of improved vaccines. Anim Health Res Rev 8(2) (2008)187-205; DEL MÉDICO ZAJAC, M.P.; LADELFA, M.F.; KOTSIAS, F.; MUYLKENS, B.; THIRY, J.; THIRY, E.; ROMERA, S.A. Biology of bovine herpesvirus 5. Vet J 184 (20 0) p. 138-145). Para os países com altas taxas de prevalência de IBR, a vacinação é a melhor escolha no combate ao vírus. Uma vacina protetora eficiente, que conferisse imunidade duradoura, poderia diminuir a circulação virai, reduzindo sua prevalência o suficiente para que estratégias de eliminação do gado s infectado pudessem ser usadas. Outro ponto a favor da vacinação é que outros ruminantes como búfalos, ovelhas e cabras podem ser infectados pelos herpesvírus bovinos. Nestes animais o vírus também consegue estabelecer latência e se reativar com sucesso, indicando possíveis reservatórios virais em locais onde há contato entre os diferentes rebanhos. Curiosamente, segundo o i levantamento de Ackermann e Engels em 2006 (ACKERMANN, M.; ENGELS, M. Pro and contra IBR-eradication. Vet Microbiol, vol.113, p.293-302, 2006), até aquele momento nenhum dos países que utilizaram a estratégia da vacinação alcançaram a erradicação do vírus. A justificativa para este fato é baseada na ineficiência das vacinas, comerciais ou experimentais, até hoje produzidas na indução de respostas humoral e celular protetoras e duradouras. Due to the great economic importance of cattle herds, fighting diseases that reduce productivity and can increase mortality rates is a priority in the affected countries. The complex of diseases caused by BoHV-1 and BoHV-5 is accompanied by significant economic losses, such as reduced milk production, infertility, miscarriages and animal deaths, and for this reason, several attempts to eradicate these viruses - mainly BoHV. -1, due to their geographical distribution - have already been made in several countries. Some countries in Europe (Switzerland, Sweden, Austria, Denmark, Finland and Norway) have achieved IBR eradication without the use of vaccines, sacrificing seropositive animals and controlling the entry of potential contaminants into the country. Obviously, such a measure is only possible when the prevalence of BoHV-1 / BoHV-5 is quite low, otherwise much of the country's cattle herd would have to be sacrificed, which would represent an even greater economic loss than those caused by viruses. (JONES, C; CHOWDHURY, S. A review of the biology of bovine herpesvirus type 1 -BHV-1, its role as a cofactor in the bovine respiratory disease complex and development of improved vaccines. Anim Health Res Rev 8 (2) ( 2008) 187-205; DEL MEDICAL ZAJAC, MP; LADELFA, MF; KOTSIAS, F .; MUYLKENS, B.; THIRY, J .; THIRY, E.; ROMERA, SA Biology of bovine herpesvirus 5. Vet J 184 (20) 0) pp 138-145). For countries with high rates of IBR prevalence, vaccination is the best choice for fighting the virus. An efficient protective vaccine, which would confer lasting immunity, could decrease viral circulation, reducing its prevalence enough that strategies for eliminating infected cattle could be used. Another point in favor of vaccination is that other ruminants such as buffalo, sheep and goats can be infected with bovine herpesviruses. In these animals the virus can also establish latency and successfully reactivate, indicating possible viral reservoirs in places where there is contact between different herds. Interestingly, according to the 2006 survey by Ackermann and Engels (ACKERMANN, M.; ENGELS, M. Pro and v IBR-eradication. Vet Microbiol, vol.113, p.293-302, 2006), so far none of the countries who used the vaccination strategy achieved virus eradication. The justification for this fact is based on the inefficiency of vaccines, commercial or experimental, to date produced in the induction of protective and lasting humoral and cellular responses.
Várias vacinas experimentais com vírus vivos modificados, inativados ou recombinantes, bem como vacinas de subunidade e vacinas com marcadores foram recentemente testadas (SPILKI, F.R.; SILVA, A.D.; BATISTA, H.B.C.R.; OLIVEIRA, A.P.; WINKELMANN, E.; FRANCO, A.C.; PORCIÚNCULA, J.A.; i ROEHE, P.M. Field evaluation of safety during gestation and horizontal spread of a recombinant differential bovine herpesvírus 1 (BoHV-1) vaccine. Pesq Vet Bras 25(1) (2005) p. 54-58). Muitas delas apresentaram resultados favoráveis, mas apenas com redução na disseminação do vírus e na apresentação clínica da doença. Nenhuma delas foi capaz de prevenir a infecção por BoHV-1 , ou impedir eficientemente sua disseminação. Outras vacinas também foram desenvolvidas, mas não testadas no campo. Algumas delas não apresentaram, ou não tiveram demonstradas respostas celular e humoral satisfatórias nos modelos animais experimentais. Para BoHV-5, as poucas vacinas experimentais testadas apresentaram os mesmos problemas que para BoHV-1.  Several experimental modified, inactivated or recombinant live virus vaccines as well as subunit vaccines and marker vaccines have recently been tested (SPILKI, FR; SILVA, AD; BATISTA, HBCR; OLIVEIRA, AP; WINKELMANN, E.; FRANCO, AC; PORCIÚNCULA, JA; i ROEHE, PM Field evaluation of safety during pregnancy and horizontal spread of a recombinant differential bovine herpesvirus 1 (BoHV-1) vaccine Pesq Vet Bras 25 (1) (2005) pp. 54-58). Many of them showed favorable results, but only with a reduction in virus spread and clinical presentation of the disease. None of them was able to prevent BoHV-1 infection or to effectively prevent its spread. Other vaccines have also been developed but not field tested. Some of them did not show or did not have satisfactory cellular and humoral responses in experimental animal models. For BoHV-5, the few experimental vaccines tested had the same problems as for BoHV-1.
A grande maioria das vacinas comerciais licenciadas pelo Ministério da Agricultura, Pecuária e Abastecimento (MAPA) do governo brasileiro é composta por formulações inativadas, multivalentes, envolvendo patógenos virais e bacterianos na mesma suspensão, distribuídas por empresas estrangeiras e que precisam de dose de reforço anual, aumentando o custo de manutenção do rebanho. A via de inoculação varia entre intramuscular e subcutânea, embora a via natural seja a intranasal, e alguns trabalhos tenham ressaltado a eficiência desta via na indução de resposta de anticorpos de mucosa, eficientes no combate à infecção e à excreção do vírus. Apenas uma é exclusiva para o combate da IBR, e apenas três combatem tanto o BoHV-5 quanto o BoHV-1. Algumas destas vacinas foram testadas experimentalmente, e evidenciou-se a necessidade de melhoria das vacinas atualmente disponíveis em nosso mercado, e também dos critérios de avaliação destes produtos. The vast majority of commercial vaccines licensed by the Brazilian government's Ministry of Agriculture, Livestock and Supply (MAPA) are inactivated, multivalent formulations involving pathogens. Viral and bacterial bacteria in the same suspension, distributed by foreign companies and requiring an annual booster dose, increasing the herd maintenance cost. The inoculation pathway varies between intramuscular and subcutaneous, although the natural pathway is intranasal, and some studies have emphasized the efficiency of this pathway in inducing mucosal antibody response, effective in combating virus infection and excretion. Only one is exclusive to IBR combat, and only three combat either BoHV-5 or BoHV-1. Some of these vaccines were experimentally tested, and there was a need to improve the vaccines currently available in our market, as well as the criteria for evaluating these products.
Muitos questionamentos envolvem as estratégias de vacinação, visto que vacinas vivas contendo o vírus "original" podem permitir latência e a disseminação do vírus entre o rebanho, mutações poderiam torná-lo novamente uma ameaça, além de ser um risco para o gado jovem, com sistema imune ainda em amadurecimento. Vacinas com marcadores ainda mantêm características imunossupressoras do vírus original, e podem se recombinar com o fenótipo selvagem, se presente no campo. Vacinas inativadas são mais seguras, porém geralmente não induzem uma imunidade celular adequada e vacinas de DNA normalmente geram apenas respostas celulares satisfatórias. No contexto da infecção pelo BoHV-1 e BoHV-5, patógenos intracelulares de grande importância económica e que estabelecem latência, uma vacina protetora deverá induzir respostas celular e humoral eficientes - a fim de combater o vírus e as células infectadas e prevenir a reativação e disseminação do vírus. Além disto, deverá induzir memória imunológica, bem como impedir a manifestação clínica da doença, que seria associada às perdas económicas. Uma exigência de mercado é a discriminação sorológica entre os animais infectados e os animais vacinados, desde que os países europeus citados alcançaram a erradicação do BoHV-1. Essa discriminação é importante também para avaliações epidemiológicas.  Many questions involve vaccination strategies, as live vaccines containing the "original" virus may allow latency and spread of the virus among the herd, mutations could again make it a threat, and a risk to young cattle with immune system still maturing. Labeled vaccines still retain immunosuppressive characteristics of the original virus, and may recombine with the wild phenotype if present in the field. Inactivated vaccines are safer, but generally do not induce adequate cellular immunity, and DNA vaccines usually generate only satisfactory cellular responses. In the context of BoHV-1 and BoHV-5 infection, very economically important and latent intracellular pathogens, a protective vaccine should induce efficient cellular and humoral responses - in order to fight the virus and infected cells and prevent reactivation and spread of the virus. In addition, it should induce immunological memory as well as prevent the clinical manifestation of the disease, which would be associated with economic losses. A market requirement is serological discrimination between infected and vaccinated animals since the European countries cited achieved the eradication of BoHV-1. This discrimination is also important for epidemiological evaluations.
Vacinas de subunidade podem ser bastante satisfatórias nestes aspectos. Utilizando apenas componentes imunogênicos do vírus, é possível induzir o desenvolvimento de uma resposta protetora contra a infecção. Nesta abordagem, riscos de manuseio, latência e reativação, recombinação e disseminação da vacina são eliminados, e pode-se facilmente discriminar entre os indivíduos vacinados e infectados. É possível também, utilizando-se das análises in silico, encontrar regiões imunogênicas nos componentes virais eSubunit vaccines can be quite satisfactory in these respects. Using only immunogenic components of the virus, it is possible to induce the development of a protective response against infection. In this approach, risks of vaccine handling, latency and reactivation, recombination, and dissemination are eliminated, and one can easily discriminate between vaccinated and infected individuals. It is also possible, using in silico analyzes, to find immunogenic regions in the viral components and
5 utilizá-las como indutoras da imunidade. Assim, a vacina pode ser manipulada para estimular respostas celular e humoral, conferindo proteção real aos animais vacinados. Em função disso, o uso de vacinas contendo proteínas multiepitopo artificialmente construídas vem crescendo nos últimos anos, mas sua utilização contra os herpesvirus ainda não foi relatada. 5 use them as immunity inducers. Thus, the vaccine can be manipulated to stimulate cellular and humoral responses, providing real protection to vaccinated animals. As a result, the use of artificially constructed multiepitope protein vaccines has been increasing in recent years, but their use against herpesviruses has not been reported.
) Imunoepitopos de células B e T das glicoproteínas B e D do herpesvirus bovino 1 já foram relatadas por autores, tanto utilizando análises de sequência in silico quanto em ensaios experimentais, fornecendo informações de grande importância na construção de novas vacinas para estes agentes. A proteína VP8 dos BoHV-1 e BoHV-5, induz também, segundo relatos da literatura, ) B and T cell immunoepitopes of bovine herpesvirus B and D glycoproteins 1 have been reported by authors using both in silico sequence analysis and experimental assays, providing important information in the construction of new vaccines for these agents. BoHV-1 and BoHV-5 VP8 protein also induces, according to literature reports,
> resposta celular protetora. O uso de uma proteína contendo epitopos múltiplos das proteínas aqui relatadas possui grande chance de induzir uma imunidade tanto celular quanto humoral protetora. > protective cellular response. The use of a protein containing multiple epitopes of the proteins reported herein has a high chance of inducing both cellular and humoral protective immunity.
O desenvolvimento da tecnologia dos poxvirus recombinantes como vetores representam um enorme avanço biotecnológico. Entre as vantagens da i utilização desse sistema destacam-se: (i) os métodos de construção e manipulação genéticos são relativamente simples, baseados principalmente no fenómeno de recombinação homóloga, processo que ocorre naturalmente durante a replicação dos poxvirus ; (ii) o vírus é capaz de infectar uma variada gama de tipos celulares eficientemente; (iii) seu genoma suporta a inserção de enormes quantidades de DNA exógeno, podendo tolerar até 25.000 pb de DNA adicional; (iv) a expressão de proteínas recombinantes ocorre naturalmente no citoplasma, o que elimina requerimentos especiais para processamento nuclear e transporte de RNA; (v) proteínas produzidas são processadas e transportadas de maneira idêntica ao que ocorre nas células de origem do gene estudado, de forma que as proteínas resultantes são biologicamente ativas; (vi) a síntese protéica ocorre em níveis relativamente altos; (vii) a manipulação do vetor é segura, sendo que o vírus vaccínia é facilmente contido dentro de padrões de bio-segurança de nível 2 (Moss.B. Genetically engineered poxviruses for recombinant gene expression.vaccination, and safety. Proc Natl Acad Sei U S A, v. 93, n. 21 , p.11341 -348). The development of recombinant poxvirus technology as vectors represents a huge biotechnological advance. Advantages of using such a system include: (i) genetic construction and manipulation methods are relatively simple, based mainly on the homologous recombination phenomenon, a process that occurs naturally during poxvirus replication; (ii) the virus is capable of infecting a wide range of cell types efficiently; (iii) its genome supports the insertion of huge amounts of exogenous DNA and can tolerate up to 25,000 bp of additional DNA; (iv) recombinant protein expression occurs naturally in the cytoplasm, which eliminates special requirements for nuclear processing and RNA transport; (v) produced proteins are processed and transported in the same way as in the cells of origin of the studied gene, so that the resulting proteins are biologically active; (vi) protein synthesis occurs at relatively high levels; (vii) vector manipulation is safe, with vaccinia virus easily contained within level 2 biosafety standards (Moss.B. Genetically engineered poxviruses for recombinant expression.vaccination gene, and safety. Proc Natl Acad Sci USA, v. 93, no. 21, p.11341 -348).
Assim, é objetivo da presente tecnologia a produção de uma vacina Thus, it is the objective of the present technology to produce a vaccine
5 compreendendo proteínas recombinantes contendo 04 epitopos das principais proteínas antigênicas de cada um dos dois herpesvírus bovinos, BoHV-1 e BoHV-5, produzidas em sistema procarioto e/ou em sistema eucarioto utilizando vírus vaccínia recombinante gerado na plataforma MVA (Vírus Ankara Modificado). Especificamente, cada proteína contém 04 epitopos o produzidos de forma linear, uma com os epitopos de BoHV-1 e a outra com epitopos de BoHV-5, resultando em duas proteínas multiepitopo que têm o potencial de imunizar ruminantes para duas doenças bovinas de grande importância para o setor pecuário mundial: Rinotraqueíte Infecciosa bovina e Encefalite bovina. 5 comprising recombinant proteins containing 04 epitopes of the major antigenic proteins of each of the two bovine herpesviruses, BoHV-1 and BoHV-5, produced in prokaryotic and / or eukaryotic system using recombinant vaccinia virus generated on the MVA platform (Modified Ankara Virus) . Specifically, each protein contains 04 linearly produced epitopes, one with BoHV-1 epitopes and one with BoHV-5 epitopes, resulting in two multiepitope proteins that have the potential to immunize ruminants for two major bovine diseases. for the world livestock sector: Bovine Infectious Rhinotracheitis and Bovine Encephalitis.
5 Alguns documentos descrevem o uso de proteínas de BoHV-1 e peptídeos derivados da mesma como vacina contra herpesvírus bovino, no entanto todos se diferem da presente tecnologia e são citados a seguir:  5 Some documents describe the use of BoHV-1 proteins and peptides derived from it as a bovine herpesvirus vaccine, however all differ from the present technology and are cited below:
O documento CA1338069 descreve uma vacina para proteger o hospedeiro bovino da infecção por BHV-1. Essa vacina compreende pelo o menos uma subunidade antigênica consistindo de um epitopo de uma glicoproteína de BHV-1 , selecionada do grupo consistindo de gl, glll e glV.  CA1338069 describes a vaccine to protect the bovine host from BHV-1 infection. Such a vaccine comprises at least one antigenic subunit consisting of an epitope of a BHV-1 glycoprotein selected from the group consisting of gl, glll and glV.
O documento EP0888777 descreve a produção de antígenos recombinantes derivados de herpesvírus bovino do tipo 1 , especialmente um antígeno derivado da glicoproteína glV de BHV-1 , para uso na proteção do EP0888777 describes the production of recombinant antigens derived from bovine herpesvirus type 1, especially a BHV-1 glV glycoprotein derived antigen, for use in the protection of
5 gado contra a infecção por esse vírus. 5 cattle against infection by this virus.
O documento PI248650 descreve uma vacina de DNA compreendendo um plasmídeo contendo um ácido nucléico que codifica um imunógeno do vírus de herpes bovino do tipo 1 e os elementos necessários à sua expressão in vivo.  PI248650 describes a DNA vaccine comprising a plasmid containing a nucleic acid encoding a type 1 bovine herpes virus immunogen and the elements necessary for its expression in vivo.
) O documento US54622734 descreve um kit e protocolo para induzir imunidade contra infecção por herpesvírus. O kit compreende uma formulação contendo uma glicoproteína virai isolada que pode ser injetada intramuscularmente para induzir imunidade sistémica. Uma segunda formulação compreende uma glicoproteína virai recombinante para ser administrada via intranasal com um adjuvante. ) US54622734 describes a kit and protocol for inducing immunity against herpesvirus infection. The kit comprises a formulation containing an isolated viral glycoprotein that can be injected intramuscularly to induce systemic immunity. A second formulation comprises a recombinant viral glycoprotein to be administered intranasally with an adjuvant.
O documento US5858989 descreve vacinas de subunidades recombinantes contra herpesvirus bovino tipo I, bem como métodos de vacinação e métodos de produzir os antígenos ou nucleotídeos empregados nas vacinas.  US5858989 describes recombinant subunit vaccines against bovine herpesvirus type I, as well as vaccination methods and methods of producing the antigens or nucleotides employed in the vaccines.
O documento US6316252 descreve proteínas de fusão, sequências de nucleotídeos para expressá-las e vetores contendo as sequências de i nucleotídeos. As proteínas de fusão compreendem uma proteína de herpesvirus bovino ligada a uma proteína bioterapêutica ou proteína repórter.  US6316252 describes fusion proteins, nucleotide sequences for expressing them and vectors containing the nucleotide sequences. Fusion proteins comprise a bovine herpesvirus protein linked to a biotherapeutic protein or reporter protein.
O documento WO0134184 descreve método e composições para desencadear uma resposta imune contra epitopos de herpesvirus bovino tipo 1. A composição compreende, um epítopo de herpesvirus bovino do tipo 1 complexado a uma proteína de shock térmico e carreadores farmacêuticamente aceitáveis.  WO0134184 describes method and compositions for eliciting an immune response against bovine herpesvirus type 1 epitopes. The composition comprises a type 1 bovine herpesvirus epitope complexed to a thermal shock protein and pharmaceutically acceptable carriers.
Breve descrição das Figuras Brief Description of the Figures
Figura 1- 1A -Análise dos eluatos de 1 a 9 da proteína de BoHV-1. M - Padrão de peso molecular. El 1 a 9 - Eluatos de 1 a 9. Seta aponta as proteínas de > interesse; 1 B - Análise dos eluatos de 1 a 9 da proteína de BoHV-5. M - Padrão de peso molecular. El 1 a 9 - Eluatos de 1 a 9. Seta aponta as proteínas de interesse.  Figure 1- 1A - Analysis of BoHV-1 protein eluates 1-9. M - Molecular Weight Standard. El 1 to 9 - Eluates 1 to 9. Seta points to proteins of interest; 1 B - Analysis of BoHV-5 protein eluates 1-9. M - Molecular Weight Standard. El 1 to 9 - Eluates 1 to 9. Arrow points to proteins of interest.
Figura 2- Viabilidade celular após 24 (A), 48 (B) e 72 (C) horas. As células PBMC foram inoculadas com proteínas recombinantes BoHV-1 , BoHV-5 e BoHV-1+ BoHV-5 e incubadas por 24, 48 e 72 horas a 37°C em estufa de C02. Ao término do período de incubação, as células foram avaliadas quanto à viabilidade celular pelo método colorimétrico de MTT. Os resultados comprovaram a ausência de citotoxicidade das proteínas recombinantes multiepitopos produzidas para células de bovinos em análise "ex-vivo". Figure 2- Cell viability after 24 (A), 48 (B) and 72 (C) hours. PBMC cells were inoculated with recombinant BoHV-1, BoHV-5 and BoHV-1 + BoHV-5 proteins and incubated for 24, 48 and 72 hours at 37 ° C in a CO 2 oven. At the end of the incubation period, cells were evaluated for cell viability by the MTT colorimetric method. The results confirmed the absence of cytotoxicity of multiepitope recombinant proteins produced for bovine cells under "ex vivo" analysis.
Figura 3- Dosagem do Óxido Nítrico (NO) utilizando o método de Griess após 24 (A) , 48 (B) e 72 (C) horas de tratamento. As células PBMC foram inoculadas com as proteínas recombinantes BoHV-1 , BoHV-5 e BoHV-1 + BoHV-5. Ao término do período de incubação, o sobrenadande de cada grupo foi coletado e avaliado quanto à produção de óxido nítrico. Não houve alteração da produção de NO para nenhum dos tratamentos com as proteínas produzidas, em nenhum dos tempos testados, comprovando mais uma vez a ausência de citotoxicidade das proteínas recombinantes multiepitopos produzidas para células de bovinos em análise "ex-vivo". Figure 3- Dosage of Nitric Oxide (NO) using the Griess method after 24 (A), 48 (B) and 72 (C) hours of treatment. PBMC cells were inoculated with recombinant BoHV-1, BoHV-5 and BoHV-1 + proteins. BoHV-5. At the end of the incubation period, the supernatant of each group was collected and evaluated for nitric oxide production. There was no change in NO production for any of the protein treatments produced at any of the times tested, once again proving the absence of cytotoxicity of multiepitope recombinant proteins produced for bovine cells under "ex vivo" analysis.
Figura 4 - Esquema da imunização dos coelhos com as proteínas recombinantes produzidas, onde foi realizado o sistema dose-reforço. As proteínas purificadas foram preparadas com o adjuvante hidróxido de alumínio (70% de proteína, 5% de hidróxido de alumínio e 25% de emulsigen) e inoculadas em coelhos da raça Nova Zelândia. Figure 4 - Scheme of rabbit immunization with the recombinant proteins produced, where the dose booster system was performed. The purified proteins were prepared with aluminum hydroxide adjuvant (70% protein, 5% aluminum hydroxide and 25% emulsigen) and inoculated into New Zealand rabbits.
Figura 5 - Ensaio de Western da proteína recombinante RecPBoHV-1 (10>g) e do vírus purificado BoHV-1 ^g). O fracionamento eletroforético foi realizado em gel de SDS-PAGE 12% e transferido para membrana de PVDF. A - Membrana RecBoHV-1 ; anticorpos primários utilizados: soros de animais fortemente reativos para BoHV-1 , para BoHV-5 ou para ambos os vírus (1 - Mab 1240, 3 -.soros de coelhos negativos para BoHV-1 e 5, 5 - Baia 3, 6 - Mix 1 , 7 - Mix 5, 8 - aBoHV-1 , 9 - P1 , 10 - P5, 11 - PN, 12 - Pab 1 , 13 - P1 e 14 - P5). B - Membranas BoHV-1 ; anticorpo primário utilizado: soros de animais fortemente reativos para BoHV-1 , para BoHV-5 ou para ambos os vírus (1 - Mab 1240, 3 -.soros de coelhos negativos para BoHV-1 e 5, 5 - Baia 3, 6 - Mix 1 , 7 - Mix 5, 8 - aBoHV-1 , 9 - P1 , 10 - P5, 11 - PN, 12 - Pab 1 , 13 - P1 e 14 - Baia 3, 15 - P1 , 16 - Pab1, 17 - PN, 18 - PN, 19 - P5 e 20 - P5). Marcador de peso molecular Kaleidoscope (BioRad).  Figure 5 - Western assay of RecPBoHV-1 recombinant protein (10 µg) and purified BoHV-1 µg virus). Electrophoretic fractionation was performed on 12% SDS-PAGE gel and transferred to PVDF membrane. A - RecBoHV-1 membrane; primary antibodies used: sera from animals strongly reactive to BoHV-1, BoHV-5 or both viruses (1 - Mab 1240, 3 - rabbits negative for BoHV-1 and 5, 5 - Bay 3, 6 - Mix 1, 7 - Mix 5, 8 - aBoHV-1, 9 - P1, 10 - P5, 11 - PN, 12 - Pab 1, 13 - P1 and 14 - P5). B - BoHV-1 membranes; primary antibody used: sera from animals strongly reactive to BoHV-1, BoHV-5 or both viruses (1 - Mab 1240, 3 - rabbits negative for BoHV-1 and 5, 5 - Bay 3, 6 - Mix 1, 7 - Mix 5, 8 - aBoHV-1, 9 - P1, 10 - P5, 11 - PN, 12 - Pab 1, 13 - P1 and 14 - Bay 3, 15 - P1, 16 - Pab1, 17 - PN, 18 - PN, 19 - P5 and 20 - P5). Kaleidoscope molecular weight marker (BioRad).
Figura 6- Ensaio de Western da proteína recombinante RecPBoHV-5 (10μg) e do vírus purificado BoHV-5 (δμρ). O fracionamento eletroforético foi realizado em gel de SDS-PAGE 12% e transferido para membrana de PVDF. A - Membrana RecBoHV-5; anticorpos primários utilizados: soros de animais fortemente reativos para BoHV-1 , para BoHV-5 ou para ambos os vírus (1 - Mab 1240, 3 -.soros de coelhos negativos para BoHV-1 e 5, 5 - Baia 3, 6 - Mix 1 , 7 - Mix 5, 8 - aBoHV-1 , 9 - P1 , 10 - P5, 11 - PN, 12 - Pab 1 , 13 - P1 e 14 - P5). B - Membrana BoHV-5; anticorpo primário utilizado: soros de animais fortemente reativos para BoHV-1 , para BoHV-5 ou para ambos os vírus (1 - Mab 1240, 3 -.soros de coelhos negativos para BoHV-1 e 5, 5 - Baia 3, 6 - Mix 1, 7 - Mix 5, 8 - aBoHV-1 , 9 - P1 , 10 - P5, 11 - PN, 12 - Pab 1 , 13 - P1 e 14 - P5, 15 - PN, 16 - Pab1). Marcador de peso molecular Kaleidoscope (BioRad). Figura 7 - Ensaio de Western da proteína recombinante (A) do vírus purificado BoHV-1 ^g). e (B) RecPBoHV-1 (^g). O fracionamento eletroforético foi realizado em gel de SDS-PAGE 12% e transferido para membrana de PVDF. (1) Pool coelhos 1 e 2 cont. - Zero; (2) Pool coelhos 1 e 2 cont. - Prime; (3) Pool coelhos 1 e 2 cont. - Boostl ; (4) Pool coelhos 1 e 2 cont. -Boost2; (5) Pool coelhos 1 e 2 RecBoHV-1 - Zero; (6) Pool coelhos 1 e 2 RecBoHV-1 - Prime; (7) Pool coelhos 1 e 2 RecBoHV-1 - Boostl ; (8) Pool coelhos 1 e 2 RecBoHV-1 - Boost2; (9) Pool coelhos 1 e 2 RecBoHV-5 - Zero; (10) Pool coelhos 1 e 2 RecBoHV-5 - Prime; (11) Pool coelhos 1 e 2 RecBoHV-5 - Boostl ; (12) Pool coelhos 1 e 2 RecBoHV-5 - Boost2; (13) Pool coelhos 1 e 2 RecBoHV-1 e 5 - Zero; (14) Pool coelhos 1 e 2 RecBoHV-1 e 5 - Prime; (15) Pool coelhos 1 e 2 RecBoHV-1 e 5 - Boostl ; (16) Pool coelhos 1 e 2 RecBoHV-1 e 5 - Boost2; (17) Pool de soro bovino neg. p/ BoHV-1 e 5; (18) Pool de soro bovino > BoHV- 1 ; (19) Pool de soro bovino > BoHV-5; (20) Pool de soro bovino pos. p/ BoHV- 1 e 5. Figure 6- Western assay of RecPBoHV-5 recombinant protein (10μg) and purified BoHV-5 virus (δμρ). Electrophoretic fractionation was performed on 12% SDS-PAGE gel and transferred to PVDF membrane. A - RecBoHV-5 membrane; primary antibodies used: sera from animals strongly reactive to BoHV-1, BoHV-5 or both viruses (1 - Mab 1240, 3 - rabbits negative for BoHV-1 and 5, 5 - Bay 3, 6 - Mix 1, 7 - Mix 5, 8 - aBoHV-1, 9 - P1, 10 - P5, 11 - PN, 12 - Pab 1, 13 - P1 and 14 - P5). B - BoHV-5 membrane; primary antibody used: animal sera strongly reactive to BoHV-1, BoHV-5 or both viruses (1 - Mab 1240, 3 - BoHV-1 and 5, 5 - negative rabbit serum - Bay 3, 6 - Mix 1, 7 - Mix 5 , 8 - aBoHV-1, 9 - P1, 10 - P5, 11 - PN, 12 - Pab 1, 13 - P1 and 14 - P5, 15 - PN, 16 - Pab1). Kaleidoscope molecular weight marker (BioRad). Figure 7 - Western blot of purified BoHV-1 (g) recombinant protein (A) assay. and (B) RecPBoHV-1 (µg). Electrophoretic fractionation was performed on 12% SDS-PAGE gel and transferred to PVDF membrane. (1) Pool rabbits 1 and 2 cont. - zero; (2) Pool rabbits 1 and 2 cont. - Prime; (3) Pool rabbits 1 and 2 cont. - Boostl; (4) Pool rabbits 1 and 2 cont. -Boost2; (5) Pool rabbits 1 and 2 RecBoHV-1 - Zero; (6) Pool rabbits 1 and 2 RecBoHV-1 - Prime; (7) Pool rabbits 1 and 2 RecBoHV-1 - Boostl; (8) Pool rabbits 1 and 2 RecBoHV-1 - Boost2; (9) Pool rabbits 1 and 2 RecBoHV-5 - Zero; (10) Pool rabbits 1 and 2 RecBoHV-5 - Prime; (11) Pool rabbits 1 and 2 RecBoHV-5 - Boostl; (12) Pool rabbits 1 and 2 RecBoHV-5 - Boost2; (13) Pool rabbits 1 and 2 RecBoHV-1 and 5 - Zero; (14) Pool rabbits 1 and 2 RecBoHV-1 and 5 - Prime; (15) Pool rabbits 1 and 2 RecBoHV-1 and 5 - Boostl; (16) Pool rabbits 1 and 2 RecBoHV-1 and 5 - Boost2; (17) Neg. for BoHV-1 and 5; (18) Bovine serum pool>BoHV-1; (19) Bovine serum pool>BoHV-5; (20) Pool of bovine serum pos. for BoHV-1 and 5.
Figura 8 - Ensaio de Western da proteína recombinante RecPBoHV-5 (10μg) e do vírus purificado BoHV-5 ^g). O fracionamento eletroforético foi realizado em gel de SDS-PAGE 12% e transferido para membrana de PVDF. (1) Pool coelhos 1 e 2 cont. - Zero; (2) Pool coelhos 1 e 2 cont. - Prime; (3) Pool coelhos Figure 8 - Western assay of RecPBoHV-5 recombinant protein (10μg) and purified BoHV-5µg virus). Electrophoretic fractionation was performed on 12% SDS-PAGE gel and transferred to PVDF membrane. (1) Pool rabbits 1 and 2 cont. - zero; (2) Pool rabbits 1 and 2 cont. - Prime; (3) Pool rabbits
1 e 2 cont. - Boostl ; (4) Pool coelhos 1 e 2 cont. -Boost2; (5) Pool coelhos 1 e1 and 2 cont. - Boostl; (4) Pool rabbits 1 and 2 cont. -Boost2; (5) Pool rabbits 1 and
2 RecBoHV-1 - Zero; (6) Pool coelhos 1 e 2 RecBoHV-1 - Prime; (7) Pool coelhos 1 e 2 RecBoHV-1 - Boostl ; (8) Pool coelhos 1 e 2 RecBoHV-1 - Boost2; (9) Pool coelhos 1 e 2 RecBoHV-5 - Zero; (10) Pool coelhos 1 e 2 RecBoHV-5 - Prime; (11) Pool coelhos 1 e 2 RecBoHV-5 - Boostl ; (12) Pool coelhos 1 e 2 RecBoHV-5 - Boost2; (13) Pool coelhos 1 e 2 RecBoHV-1 e 5 - Zero; (14) Pool coelhos 1 e 2 RecBoHV-1 e 5 - Prime; (15) Pool coelhos 1 e 2 RecBoHV-1 e 5 - Boostl ; (16) Pool coelhos 1 e 2 RecBoHV-1 e 5 - Boost2; (17) Pool de soro bovino neg. p/ BoHV-1 e 5; (18) Pool de soro bovino > BoHV- 1 ; (19) Pool de soro bovino > BoHV-5; (20) Pool de soro bovino pos. p/ BoHV- 1 e 5. 2 RecBoHV-1 - Zero; (6) Pool rabbits 1 and 2 RecBoHV-1 - Prime; (7) Pool rabbits 1 and 2 RecBoHV-1 - Boostl; (8) Pool rabbits 1 and 2 RecBoHV-1 - Boost2; (9) Pool rabbits 1 and 2 RecBoHV-5 - Zero; (10) Pool rabbits 1 and 2 RecBoHV-5 - Prime; (11) Pool rabbits 1 and 2 RecBoHV-5 - Boostl; (12) Pool rabbits 1 and 2 RecBoHV-5 - Boost2; (13) Pool rabbits 1 and 2 RecBoHV-1 and 5 - Zero; (14) Pool rabbits 1 and 2 RecBoHV-1 and 5 - Prime; (15) Pool rabbits 1 and 2 RecBoHV-1 and 5 - Boostl; (16) Pool rabbits 1 and 2 RecBoHV-1 and 5 - Boost2; (17) Neg. for BoHV-1 and 5; (18) Cattle serum pool> BoHV- 1 ; (19) Bovine serum pool>BoHV-5; (20) Pool of bovine serum pos. for BoHV-1 and 5.
Figura 9 - Ensaio de ELISA (anti-lgG total de coelho e bovino) utilizando vírus purificado BoHV-1 (AgV-BoHV-1) e proteína recombinante (Ag-RecBoHV-1) na Figure 9 - ELISA (rabbit and bovine total anti-IgG) assay using purified BoHV-1 virus (AgV-BoHV-1) and recombinant protein (Ag-RecBoHV-1) on
5 concentração de 250ng/poço. As amostras, soros provenientes de coelhos da raça Nova Zelândia inoculados com as proteínas recombinantes, foram analisadas por meio de um sistema dose-reforço composto de 03 doses intervaladas com 20 dias. A, C, E = AgV-BoHV-1 X pool de soros de coelhos (dose inicial, reforço 1 e reforço 2 respectivamente); G = AgV-BoHV-1 X pool o de soros de bovinos. B, D, F = Ag-RecBoHV-1 X pool de soros de coelhos (dose inicial, reforço 1 e reforço 2 respectivamente); H = Ag-RecBoHV-1 X pool de soros de bovinos. Como controle do ensaio foi utilizado amostras de uma soroteca bovina previamente testada. Pelas análises estatísticas em (A) bXa- P= 0,01 ; aXc/d - P>0,05; em (B) aXb/c/d- P=0,002; em (C) aXb- P=0,05, aXc/d-5 concentration of 250ng / well. The samples, sera from New Zealand rabbits inoculated with the recombinant proteins, were analyzed by a dose-boosting system composed of three 20-day interval doses. A, C, E = AgV-BoHV-1 X rabbit sera pool (starting dose, booster 1 and booster 2 respectively); G = AgV-BoHV-1 X pool of bovine sera. B, D, F = Ag-RecBoHV-1 X rabbit sera pool (starting dose, booster 1 and booster 2 respectively); H = Ag-RecBoHV-1 X pool of bovine sera. As control of the assay, samples from a previously tested bovine library were used. For the statistical analyzes in (A) bXa-P = 0.01; aXc / d - P> 0.05; in (B) aXb / c / d- P = 0.002; at (C) aXb-P = 0.05, aXc / d-
5 P<0,0001 e dXa/b/c- P<0,0001 ; em (D) bXd- P=0,003; aXb/c- P>0,05; aXd- P=0,003; em (E) aXb/c/d- P<0,0001 , bXc/d- P<0,0001 e cXd- P<0,0001 ; em (F) aXb/c- P<0,0001 , bXd- p<0,0001; em (G) aXb/c- P<0,0001 e bXc- P<0,0001 ; em (H) aXb/c- P<0,0001. 5 P <0.0001 and dXa / b / c- P <0.0001; in (D) bXd-P = 0.003; aXb / c-P> 0.05; aXd-P = 0.003; in (E) aXb / c / d- P <0.0001, bXc / d- P <0.0001 and cXd-P <0.0001; in (F) aXb / c-P <0.0001, bXd-p <0.0001; in (G) aXb / c-P <0.0001 and bXc-P <0.0001; in (H) aXb / c- P <0.0001.
Figura 10 - Ensaio de ELISA (anti-lgG total de coelho e bovino) utilizando vírus 3 purificado BoHV-5 (AgV-BoHV-5) e proteína recombinante (Ag-RecBoHV-5) na concentração de 250ng/poço. As amostras, soros provenientes de coelhos da raça Nova Zelândia inoculados com as proteínas recombinantes, foram analisadas por meio de um sistema dose-reforço composto de 03 doses intervaladas com 20 dias. A, C, E = AgV-BoHV-5 X pool de soros de coelhos 5 (dose inicial, reforço 1 e reforço 2 respectivamente); G = AgV-BoHV-5 X pool de soros de bovinos. B, D, F = Ag-RecBoHV-5 X pool de soros de coelhos (dose inicial, reforço 1 e reforço 2 respectivamente); H = Ag-RecBoHV-5 X pool de soros de bovinos. Como controle do ensaio foram utilizadas amostras de uma soroteca bovina previamente testada. Pelas análises estatísticas em (A) ) aXc- P<0,05; em (C) aXb- P>0,05, aXc/d- P<0,0001, cXb- P<0,0001 , dXa/b/c- P<0,0001 ; em (D) aXb- P>0,05, aXc/d- P<0,0001 , cXd- P>0,05; em (E) aXb/d- P<0,0001 , bXd- P<0,0001 ; em (F) aXb/c/d- P=0,0001 ; em (G) aXb/c- P<0,0001 e em (H) aXb/c- P=0,0003. Figure 10 - ELISA (rabbit and bovine total anti-IgG) assay using purified BoHV-5 virus 3 (AgV-BoHV-5) and recombinant protein (Ag-RecBoHV-5) at a concentration of 250ng / well. The samples, sera from New Zealand rabbits inoculated with the recombinant proteins, were analyzed by a dose-boosting system composed of three 20-day interval doses. A, C, E = AgV-BoHV-5 X rabbit sera pool 5 (starting dose, booster 1 and booster 2 respectively); G = AgV-BoHV-5 X bovine serum pool. B, D, F = Ag-RecBoHV-5 X rabbit sera pool (starting dose, booster 1 and booster 2 respectively); H = Ag-RecBoHV-5 X bovine serum pool. As control of the assay, samples from a previously tested bovine library were used. For the statistical analyzes in (A)) aXc-P <0.05; in (C) aXb-P> 0.05, aXc / d-P <0.0001, cXb-P <0.0001, dXa / b / c-P <0.0001; in (D) aXb-P> 0.05, aXc / d-P <0.0001, cXd-P>0.05; at (E) aXb / d- P <0.0001, bXd-P <0.0001; in (F) aXb / c / d- P = 0.0001; at (G) aXb / c- P <0.0001 and in (H) aXb / c- P = 0.0003.
Figura 11 - Células VERO infectadas com o Vírus Ankara Modificado recombinante expressando a proteína recombinante multiepitopo BoHV-1. 12A - visualização dos clones em microscópio ótico e 12B - visualização dos clones em microscópio de fluorescência.  Figure 11 - VERO cells infected with recombinant Modified Ankara Virus expressing BoHV-1 multiepitope recombinant protein. 12A - visualization of clones under optical microscope and 12B - visualization of clones under fluorescence microscope.
Descrição Detalhada da Invenção Detailed Description of the Invention
A presente tecnologia descreve a produção de uma vacina contendo proteínas recombinantes compreendendo 04 epitopos das principais proteínas antigênicas de cada um dos dois herpesvírus bovinos, BoHV-1 e BoHV-5, produzidas em sistema procarioto e/ou em sistema eucarioto utilizando vírus recombinante gerado na plataforma MVA (Vírus Ankara Modificado). Especificamente, cada proteína recombinante contém 04 epitopos produzidos de forma linear, uma com os epitopos de BoHV-1 e a outra com os epitopos de BoHV-5, e os epitopos ligados com o uso de alças flexíveis de glicina e serina para unir as regiões desejadas e permitir a conformação terciária adequada de cada fragmento, uma abordagem semelhante à utilizada em 2002 por Livingston e colaboradores (LIVINGSTON, B.; CRIMI, C; NEWMAN, M.; HIGASHIMOTO, Y.; APPELLA, E.; SIDNEY, J.; SETTE, A. A rational strategy to design multiepitope immunogens based on multiple Th lymphocyte epitopes. J Immunol 168 (2002) p. 5499-5506), resultando em duas proteínas multiepitopo ruminantes contra os virus BoHV-1 e BoHV-5 para o controle da infecção, disseminação virai e redução do prejuízo económico causado por duas doenças bovinas de grande importância para o setor pecuário mundial: Rinotraqueíte Infecciosa bovina e Encefalite bovina. A tecnologia poderá ser melhor compreendida, de forma não limitante, através dos exemplos que se seguem.  The present technology describes the production of a recombinant protein-containing vaccine comprising 04 epitopes of the main antigenic proteins of each of the two bovine herpesviruses, BoHV-1 and BoHV-5, produced in prokaryotic and / or eukaryotic system using recombinant virus generated in MVA (Modified Ankara Virus) platform. Specifically, each recombinant protein contains 04 linearly produced epitopes, one with BoHV-1 epitopes and the other with BoHV-5 epitopes, and epitopes linked using flexible glycine and serine loops to join the regions. allow appropriate tertiary conformation of each fragment, an approach similar to that used in 2002 by Livingston and colleagues (LIVINGSTON, B.; CRIMI, C; NEWMAN, M .; HIGASHIMOTO, Y .; APPELLA, E .; SIDNEY, J SETTE, A. A rational strategy for multiepitope design immunogens based on multiple Th lymphocyte epitopes (J Immunol 168 (2002), p. 5499-5506), resulting in two ruminant multiepitope proteins against BoHV-1 and BoHV-5 viruses. infection control, viral dissemination and reduction of economic damage caused by two major bovine diseases in the world livestock sector: Bovine Infectious Rhinotracheitis and Bovine Encephalitis. Technology may be better understood, but not limited to, by the following examples.
Exemplo 1: Análises in silico dos genes codificadores das proteínas alvos, construção dos genes sintéticos e clonagem nos vetores de expressão As análises in silico foram o foco inicial do trabalho, onde foram avaliados os genes codificadores das proteínas alvos e suas possíveis regiões reconhecidas por células T e B. Example 1: In silico analysis of targeting protein coding genes, synthetic gene construction, and expression vector cloning In silico analyzes were the initial focus of the work, where the genes encoding the target proteins and their possible regions recognized by T and B cells were evaluated.
O primeiro passo para a identificação dos epitopos imuno-dominantes das The first step in identifying the immunodominant epitopes of the
5 proteínas e subsequente construção da proteína multiepitopo foi a obtenção da sequência de nucleotídeos de cada proteína para os dois vírus. Este processo foi realizado com a utilização do banco de dados Genbank (http://www.ncbi.nlm.nih.gov/genbank/). Proteínas de BoHV-1 : Genoma total de BoHV-1.1 utilizado: AJ004801.1 ; Proteínas de BoHV-5: Genoma total de5 proteins and subsequent construction of the multiepitope protein was obtaining the nucleotide sequence of each protein for the two viruses. This process was performed using the Genbank database (http://www.ncbi.nlm.nih.gov/genbank/). BoHV-1 Proteins: BoHV-1.1 total genome used: AJ004801.1; BoHV-5 Proteins: Total Genome of
) BoHV-5 utilizado: AY261359.1 (linhagem SV507/99 - amostra brasileira). ) BoHV-5 used: AY261359.1 (SV507 / 99 strain - Brazilian sample).
• Glicoproteína B de BoHV-1 :  • BoHV-1 glycoprotein B:
o Localização: Gene UL27. Entre as bases 55394 e 58192.  o Location: Gene UL27. Between bases 55394 and 58192.
• Glicoproteína B de BoHV-5:  • BoHV-5 glycoprotein B:
Localização: Gene UL27. Entre as bases 56661 e 59504.  Location: Gene UL27. Between bases 56661 and 59504.
5 · Glicoproteína D de BoHV-1 : 5 · BoHV-1 Glycoprotein D:
o Localização: Gene US6. Entre as bases 118896 e 120149.  o Location: Gene US6. Between bases 118896 and 120149.
• Glicoproteína D de BoHV-5:  • BoHV-5 Glycoprotein D:
o Localização: Gene US6. Entre as bases 12 29 e 122382.  o Location: Gene US6. Between bases 12 29 and 122382.
• VP8 de BoHV-1 :  • BoHV-1 VP8:
) o Localização: Gene UL47. Entre as bases 11963 e 14182.  ) o Location: Gene UL47. Between bases 11963 and 14182.
• VP8 de BoHV-5:  • BoHV-5 VP8:
o Localização: Gene UL47. Entre as bases 12808 e 15033.  o Location: Gene UL47. Between bases 12808 and 15033.
Primeiramente foi realizada uma busca na literatura por regiões sabidamente imunogênicas nas proteínas gB, gD e VP8 de BoHV-1 e BoHV-5. > Um estudo complementar de possíveis regiões alvo de células T foi realizado para as proteínas que ainda não foram mapeadas neste sentido na literatura. Este estudo foi realizado com o auxílio da plataforma SYFPEITHI: Epitope prediction http://www.syfpeithi.de/Scripts/MHCServer.dll/EpitopePrediction.htm). Foram identificadas as seguintes regiões imunogenicamente relevantes ) (Tabelas 1 , 2 e 3): Tabela 1 - Epitopos imunogenicamente relevantes de gB de BoHV-1First, a literature search for known immunogenic regions on BoHV-1 and BoHV-5 gB, gD and VP8 proteins was performed. > A complementary study of possible T cell target regions has been performed for proteins that have not been mapped to this effect in the literature. This study was performed with the help of the SYFPEITHI: Epitope prediction platform http://www.syfpeithi.de/Scripts/MHCServer.dll/EpitopePrediction.htm). The following immunogenically relevant regions were identified (Tables 1, 2 and 3): Table 1 - Immunogenically relevant BoHV-1 gB epitopes
Sequência de aminoácidos Posição Célula ReativaAmino Acid Sequence Reactive Cell Position
DPWEAPLKPARLSAPGVRGWHT 239 - 260 Célula TDPWEAPLKPARLSAPGVRGWHT 239 - 260 T Cell
FRFTARSLSATFVSDSHTFALQNVPLSFRFTARSLSATFVSDSHTFALQNVPLS
DC V 395 - 424 Célula TDC V 395 - 424 T Cell
VSDSHTFALQNVPLSDCV IEEAEAAVEVSDSHTFALQNVPLSDCV IEEAEAAVE
R V Y 407 - 436 Célula TR V Y 407 - 436 T Cell
HREHTSYSPERFQQIEGYYKR 319-339 Célula THREHTSYSPERFQQIEGYYKR 319-339 T Cell
LQNVPLSDCV IEEAEAAVERV 415-435 Célula TLQNVPLSDCV IEEAEAAVERV 415-435 T Cell
SAPGVRGWHT TDDVYTALGSAG 251 - 272 Célula BSAPGVRGWHT TDDVYTALGSAG 251 - 272 Cell B
GYYKR DMATGRRLKEPV SRNFL 335 - 356 Célula BGYYKR DMATGRRLKEPV SRNFL 335 - 356 Cell B
QQIEGYYKR DMATGRRLKEPV 331 -351 Célula BQQIEGYYKR DMATGRRLKEPV 331 -351 Cell B
YLQELARSNGTLEGLFAAAAP 475-495 Célula BYLQELARSNGTLEGLFAAAAP 475-495 Cell B
EGLFAAAAP KPGPRRARRAAP 487 - 507 Célula B EGLFAAAAP KPGPRRARRAAP 487 - 507 Cell B
Para gB de BoHV-5, os epitopos importantes foram determinados por comparação com a sequência de BoHV-1: For BoHV-5 gB, important epitopes were determined by comparison with the BoHV-1 sequence:
Tabela 2 - Epitopos imunogenicamente relevantes de gB de BoHV-5 Table 2 - Immunogenically relevant BoHV-5 gB epitopes
Figure imgf000014_0001
Figure imgf000014_0001
Tabela 3 - Epitopos imunogenicamente relevantes de gD de BoHV-1  Table 3 - Immunogenically relevant BoHV-1 gD epitopes
Figure imgf000014_0002
Figure imgf000014_0002
Devido à escassez de dados na literatura, a determinação das regiões trabalhadas para as proteínas gD de BoHV-5 e VP8 de ambos os vírus foi feita exclusivamente pela ferramenta de predição de epitopos SYFPEITHI. As sequências determinadas acima foram analisadas no programa e os resultados e as sequências selecionadas são apresentadas nas tabelas 4 e 5, respectivamente: Due to the scarcity of data in the literature, the determination of the worked regions for the BoHV-5 and VP8 gD proteins of both viruses was made exclusively by the SYFPEITHI epitope prediction tool. The sequences determined above were analyzed in the program and the results and the selected sequences are shown in tables 4 and 5, respectively:
Tabela 4 - Epitopos imunogenicamente relevantes de gD de BoHV-5 Table 4 - Immunogenically relevant epitopes of BoHV-5 gD
Figure imgf000015_0001
Figure imgf000015_0001
Para VP8, as regiões são as mesmas para BoHV-1 e 5, identificadas pelo programa SYFPEITHI:  For VP8, the regions are the same for BoHV-1 and 5, identified by the SYFPEITHI program:
Tabela 5 - Epitopos imunogenicamente relevantes de VP8 de BoHV-1 e BoHV-5 Table 5 - BoHV-1 and BoHV-5 VP8 immunogenically relevant epitopes
Figure imgf000015_0002
Figure imgf000015_0002
Como uma forma de confirmar a exposição destes epitopos na estrutura tridimensional da proteína nativa e, desta forma, aumentar a possibilidade de seleção de um epitopo imunogênico, foram realizadas análises dos perfis de hidrofobicidade de cada proteína, através do programa DAS (http://www.sbc.su.se/~miklos/DAS/) da plataforma ExPASy (http://ca.expasy.org), de forma a evitar a escolha de regiões hidrofóbicas, provavelmente ligadas a áreas de ancoragem à membrana plasmática ou a regiões de baixa exposição no espaço. Desta forma, fez-se a escolha de regiões hidrofílicas, e partiu-se para a finalização do desenho dos genes sintéticos.  As a way to confirm the exposure of these epitopes to the three-dimensional structure of the native protein and thus increase the possibility of selection of an immunogenic epitope, analyzes of the hydrophobicity profiles of each protein were performed using the DAS program (http: // www.sbc.su.se/~miklos/DAS/) from the ExPASy platform (http://ca.expasy.org) to avoid choosing hydrophobic regions, probably linked to plasma membrane anchor areas or regions of low exposure in space. Thus, the choice of hydrophilic regions was made, and we proceeded to the finalization of the design of synthetic genes.
Posteriormente às ditas análises, foram selecionadas as seguintes regiões de cada proteína, Tabela 6:  After these analyzes, the following regions of each protein were selected, Table 6:
Tabela 6 - Regiões selecionadas de cada proteína virai para inserção no gene sintético Table 6 - Selected regions of each viral protein for insertion into the synthetic gene
Proteína Espécie Sequência de aminoácidos Posição BoHV-1 HREHTSYSPERFQQIEGYYKRDMATGRRLKEPVSRNFL 319-356 gB Protein Species Amino Acid Sequence Position BoHV-1 HREHTSYSPERFQQIEGYYKRDMATGRRLKEPVSRNFL 319-356 gB
BoHV-5 HREHTSYSPERFQQIEGYYKRDMATGRRLKEPVSRNFL 326-363  BoHV-5 HREHTSYSPERFQQIEGYYKRDMATGRRLKEPVSRNFL 326-363
EAVRRHARAYNATVI 92-106 EAVRRHARAYNATVI 92-106
BoHV-1 BoHV-1
IMAAPARLVEGQ 161-172 gD  IMAAPARLVEGQ 161-172 gD
IADPQVGRTLWGAVRRNARTYNATVIWYKIESGCA 82-116 IADPQVGRTLWGAVRRNARTYNATVIWYKIESGCA 82-116
BoHV-5 BoHV-5
FAYPTDDELGLV AAPARLAEGQYRRALYIDG 151-182 FAYPTDDELGLV AAPARLAEGQYRRALYIDG 151-182
BoHV-1 DEDTSEDENVYDYIDGDSSD 62-81BoHV-1 DEDTSEDENVYDYIDGDSSD 62-81
VP8 VP8
BoHV-5 DEDTSEDENVYDYIDGDSSD 62-81  BoHV-5 DEDTSEDENVYDYIDGDSSD 62-81
Na impossibilidade de selecionar uma mesma região antigênica para ambas as células T e B para a proteína gD, fez-se necessária a seleção de duas regiões distintas para que a cobertura imune fosse adequada. Devido à falta de dados disponíveis na literatura acerca das características imunogênicas de regiões específicas da proteína gD de BoHV-5, optou-se por selecionar a região correspondente à selecionada para gD de BoHV-1 , porém com uma área maior de cobertura para incluir áreas interessantes para o reconhecimento imune. In the impossibility of selecting the same antigenic region for both T and B cells for the gD protein, it was necessary to select two distinct regions so that the immune coverage was adequate. Due to the lack of data available in the literature about the immunogenic characteristics of specific regions of BoHV-5 gD protein, we chose to select the region corresponding to that selected for BoHV-1 gD, but with a larger coverage area to include areas. interesting for immune recognition.
Para a proteína VP8 de ambos os vírus, apenas epitopos de células T foram inseridos nos genes sintéticos. Tal opção justifica-se pelo fato de tratar- se de uma proteína do tegumento virai, não disponível em grande escala para reconhecimento extracelular, e que isso não dispõe de estudos a este respeito na literatura. Portanto, a introdução ou mesmo a busca por epitopos de células B foi descartada.  For VP8 protein from both viruses, only T cell epitopes were inserted into the synthetic genes. This option is justified by the fact that it is a viral tegument protein, not available on a large scale for extracellular recognition, and that this has no studies in this regard in the literature. Therefore, the introduction or even search for B cell epitopes has been ruled out.
Em sequência o trabalho se voltou para o desenho de um gene sintético a ser expresso em linhagens de E. coli. Para tanto, as sequências obtidas no item anterior foram unidas e interpostas por alças flexíveis compostas pelos aminoácidos Glicina (G) e Serina (S). Tal conector (GSGSG) se faz extremamente útil de modo a minimizar a interação entre as sequências de aminoácidos e permitir o dobramento terciário adequado de cada segmento. A escolha de Glicina e Serina é justificada pela característica relativamente inerte deste par, composto de aminoácidos essencialmente neutros. Os sítios de restrição não foram sido inseridos no gene sintético produzido, mas, através da PCR utilizando iniciadores ancorados por sequencias relativas aos sítios requeridos. Também foi analisada a presença de códons raros para a bactéria Escherichia coli em construção na presente tecnologia, de modo a saber se as proteínas provenientes dos genes sintéticos seriam de alguma forma tóxica aos vetores de expressão. Then the work turned to the design of a synthetic gene to be expressed in E. coli strains. For this, the sequences obtained in the previous item were joined and interposed by flexible loops composed of the amino acids Glycine (G) and Serine (S). Such a connector (GSGSG) is extremely useful in minimizing the interaction between amino acid sequences and allowing proper tertiary folding of each segment. The choice of Glycine and Serine is justified by the relatively inert characteristic of this pair, composed of essentially neutral amino acids. Restriction sites were not inserted into the synthetic gene produced, but by PCR using primers anchored by sequences relative to the required sites. The presence of rare codons for the bacterium Escherichia coli under construction in the present technology was also analyzed, in order to know if the proteins coming from the synthetic genes would be in any way toxic to the expression vectors.
A semelhança entre BoHV-1 e BoHV-5 em suas proteínas gB e VP8 possibilitou a utilização dos mesmos fragmentos para ambos os vírus. Só há diferença entre as proteínas nos fragmentos da proteína gD. Este fato aumenta a possibilidade de uma proteção cruzada das proteínas. A inoculação de ambas as proteínas num mesmo animal possivelmente potencializá a imunidade contra cada um dos vírus.  The similarity between BoHV-1 and BoHV-5 in their gB and VP8 proteins allowed the use of the same fragments for both viruses. There is only difference between proteins in gD protein fragments. This fact increases the possibility of protein cross-protection. Inoculation of both proteins in the same animal may enhance immunity against each virus.
Estudo da amplificação dos genes sintéticos Study of synthetic gene amplification
Os genes sintéticos contendo os fragmentos codificadores das proteínas de BoHV-1 e BoHV-5 foram produzidos, e, com uso de um sistema de PCR desenvolvido foi adicionada à extremidade 5' de cada iniciador um sítio de restrição para as endonucleases BamHI e Hindlll (Promega, EUA) - Para os iniciadores senso e antisenso, respectivamente - bem como duas bases extremas para facilitar a ligação das enzimas à fita de DNA. Após, foram clonados em vetor comercial pQE 30, que tem como característica principal a fusão da sequencia de interesse a uma outra codificadora de uma cauda de histidina para facilitar a purificação pós-expressão. Como forma de confirmar a presença e integridade dos insertos e dos cassetes desenhados, os clones selecionados a partir do experimento acima foram submetidos a seqiienciamento, pelo método didesoxi (Sanger et. ai, 1977) utilizando seqijenciador automático (Megabace) com iniciadores que flanqueiam o inserto ou o vetor.  Synthetic genes containing the BoHV-1 and BoHV-5 protein coding fragments were produced, and using a developed PCR system a restriction site for the BamHI and HindIII endonucleases (at the 5 'end of each primer) was added to each primer. Promega, USA) - For sense and antisense primers, respectively - as well as two extreme bases to facilitate the binding of enzymes to the DNA strand. Then, they were cloned into a commercial vector pQE 30, which has as its main feature the fusion of the sequence of interest to another histidine tail coder to facilitate post expression purification. In order to confirm the presence and integrity of the inserts and designed cassettes, the clones selected from the above experiment were subjected to sequencing by the didesoxi method (Sanger et al., 1977) using automatic sequencer (Megabace) with primers flanking. Insert or the vector.
Exemplo 2- Expressão das proteínas recombínantes heterologas em vetor procarioto Example 2- Expression of Heterologous Recombinant Proteins in Prokaryote Vector
Análise da clonagem em vetor pQE 30  PQE 30 Vector Cloning Analysis
As análises da clonagem e expressão das proteínas multiepitopo, incluindo o peso molecular esperado, foram feitas também com o auxílio da plataforma Expasy e do programa Serial Cloner 2.0, e o alinhamento das sequências obtidas com os epitopos desenhados foram obtidos através do uso da ferramenta on-line MultAlin (http://multalin.toulouse.inra.fr/multalin/ multalin.html; Corpet, 1988). A análise da presença de códons raros para Escherichia coli foi feita através do programa on-line "Rare codon calculator (RaCC) da Universidade da Califórina, Los Angeles (UCLA), disponibilizado no servidor do National Institutes of Health (NIH) (http://nihserver.mbi.ucla.edu/ RACC/). Cloning and expression analyzes of multiepitope proteins, including expected molecular weight, were also performed with the aid of the Expasy platform and the Serial Cloner 2.0 program, and the alignment of the sequences obtained with the drawn epitopes were obtained using the online tool MultAlin (http://multalin.toulouse.inra.fr/multalin/ multalin.html; Corpet, 1988). Analysis of the presence of rare codons for Escherichia coli was performed using the University of California, Los Angeles (UCLA) Rare codon calculator (RaCC) online program, available from the National Institutes of Health (NIH) server (http: //nihserver.mbi.ucla.edu/ RACC /).
Transformação das bactérias de expressão Transformation of Expression Bacteria
Bactérias Escherichia coli da linhagem M15 foram transformadas com os plasmídeos pQE clonados com os insertos de interesse - os cassetes gênicos. Os clones escolhidos foram crescidos e estocados ou seguiram para triagem.  M15 strain Escherichia coli bacteria were transformed with pQE plasmids cloned with the inserts of interest - the gene cassettes. The chosen clones were grown and stored or screened.
Triagem dos clones Clone Screening
Para escolher o melhor clone de cada vírus, que seguiria para a expressão da proteína em larga escala, foi realizada uma indução de 5h para análise do perfil de expressão de cada clone em SDS-PAGE. A partir dos perfis de expressão, selecionamos apenas um clone referente a cada proteína para seguir para os experimentos de localização e purificação. Para a proteína de BoHV-1, o clone com melhor expressão foi o 1421 , enquanto para a proteína de BoHV-5, o clone 5511.  To choose the best clone of each virus, which would follow for large-scale protein expression, a 5h induction was performed to analyze the expression profile of each clone in SDS-PAGE. From the expression profiles, we selected only one clone for each protein to follow for the localization and purification experiments. For BoHV-1 protein, the best expressing clone was 1421, while for BoHV-5 protein, clone 5511.
Determinação da localização da proteína recombinante Determination of recombinant protein localization
Para a determinação do tipo de purificação adequado à produção das proteínas, primeiro definiu-se sua localização, se na porção solúvel ou insolúvel. Para tanto, seguimos as instruções do fabricante pQE (Qiagen, Alemanha). A localização das proteínas foi verificada na fração insolúvel, provavelmente em agregados denominados corpúsculos de inclusão. As proteínas foram expressadas na fração insolúvel e utilizou-se agente desnaturante (Guanidina-HCI) para sua solubilização.  To determine the type of purification suitable for protein production, its location was first defined whether in the soluble or insoluble portion. To do so, we follow the pQE manufacturer's instructions (Qiagen, Germany). Protein localization was verified in the insoluble fraction, probably in aggregates called inclusion corpuscles. Proteins were expressed in the insoluble fraction and denaturing agent (Guanidine-HCI) was used for its solubilization.
Exemplo 3- Purificação desnaturante das proteínas recombinantes por cromatografia de afinidade Após a determinação do tipo de purificação à qual as proteínas seriam submetidas, procedeu-se a purificação propriamente dita. O princípio deste processo baseia-se na afinidade da cauda de histidina, inserida nas proteínas pela clonagem do gene sintético ao vetor pQE 30, à resina contendo o ácido nitrilo-tri-acético (NTA) e carregada com íons Ni2+ (Ni-NTA). Esta alta afinidade faz com que as proteínas fiquem imobilizadas no suporte e separada do extrato protéico total, no princípio de cromatografia por afinidade por íon metálico imobilizado (IMAC) já demonstrado na literatura. A lavagem da coluna com tampões de pH básico remove as demais proteínas, mas não as que possuem cauda de histidina. Desta forma, essas proteínas são retiradas, purificadas, em tampões de pH ácido, que rompe as interações iónicas estabelecidas entre as proteínas e a coluna. A lavagem com 10ml do tampão de pH 4,5 resultou na obtenção de 10 eluatos diferentes, de 1 a 10, de 1 ml cada, os quais foram retirados sequencialmente e analisados em SDS-PAGE 12% é apresentada na figura 1 (1A - RecBoHV- ; 1B - RecBoHV-5). Conseguiu-se com sucesso a purificação das proteínas multiepitopo projetadas. Na análise dos eluatos da purificação observou-se a expressão de duas bandas de perfil extremamente assemelhado para cada uma das proteínas dos dois diferentes vírus. Ao contrário de proteínas bacterianas contaminantes, as proteínas "extras" acompanharam o perfil de expressão das proteínas "alvo", estando presentes, embora em menores quantidades, em quase todos os eluatos onde as bandas principais são encontradas. Este fato corrobora a hipótese de se tratarem da mesma proteína, pois dificilmente uma proteína contaminante, mesmo que contenha uma sequência de histidinas, obteria tão similar perfil de expressão, e, como o desenho do cassete gênico incluiu alças flexíveis (GSGSG), as proteínas expressas podem sofrer diferenças no dobramento durante a sua expressão, produzindo mais de uma "população". Example 3- Denaturing Purification of Recombinant Proteins by Affinity Chromatography After determining the type of purification to which the proteins would be subjected, the purification itself was performed. The principle of this process is based on the affinity of the histidine tail, inserted into proteins by cloning the synthetic gene to vector pQE 30, to the resin containing nitrile tri-acetic acid (NTA) and charged with Ni 2+ ions (Ni- NTA). This high affinity causes the proteins to be immobilized on the support and separated from the total protein extract, in the principle of immobilized metal ion affinity chromatography (IMAC) already demonstrated in the literature. Washing the column with basic pH buffers removes the other proteins, but not those with histidine tail. Thus, these proteins are removed, purified, in acid pH buffers, which disrupts the ionic interactions established between the proteins and the column. Washing with 10 ml of pH 4.5 buffer resulted in 10 different 1 to 10 eluates of 1 ml each, which were sequentially removed and analyzed in 12% SDS-PAGE is shown in Figure 1 (1A - RecBoHV-; 1B - RecBoHV-5). Purification of the engineered multiepitope proteins was successfully achieved. In the analysis of the purification eluates, we observed the expression of two extremely similar profile bands for each of the proteins of the two different viruses. Unlike contaminating bacterial proteins, the "extra" proteins followed the expression profile of the "target" proteins and are present, albeit in smaller amounts, in almost all eluates where the major bands are found. This corroborates the hypothesis that they are the same protein, since hardly a contaminating protein, even if it contains a sequence of histidines, would obtain such a similar expression profile, and, as the gene cassette design included flexible loops (GSGSG), the proteins can express differences in folding during expression, producing more than one "population".
Exemplo 4 - Ensaio de toxicidade celular das proteínas recombinantes em culturas ex-vivo de PBMC bovino Example 4 - Cell Toxicity Assay of Recombinant Proteins in Bovine PBMC Ex vivo Cultures
Obtenção e cultivo das PBMC Collection and cultivation of PBMC
Para verificar se as proteínas produzidas no sistema procarioto induziam alguma citotoxicidade em PBMC (células mononucleares do sangue periférico) bovino e/ou produção exacerbada de óxido nítrico, foram realizadas em culturas ex-vivo análise da viabilidade celular e da produção de óxido nítrico - NO, após o tratamento das culturas com as proteínas RecPBoHV-1 , RecPBoHV-5 e RecPBoHV-1 + RecPBoHV-5. To check if proteins produced in the prokaryote system induced any cytotoxicity in PBMC (peripheral blood mononuclear cells) Nitric oxide production and / or exacerbated nitric oxide production, analysis of cell viability and nitric oxide production (NO) were performed in ex vivo cultures after treatment with RecPBoHV-1, RecPBoHV-5 and RecPBoHV-1 + proteins. RecPBoHV-5.
Foi utilizado o meio de cultura RPMI 1640 com L-glutamina (Sigma). O conteúdo de cada frasco foi reconstituído em 1 L de água ultra pura e adicionado bicarbonato de sódio (Merck), HEPES 10mM (N-2 hidroxietilpiperazina, N-2 ácido etanosulfônico) (Sigma). O pH da solução foi ajustado para 7,1 e foi denominado de RPMI não suplementado. O meio usado nas culturas foi suplementado com 10% de soro fetal bovino (Cultilab), 100 U/mL de penicilina e 100Mg/ml_ de estreptomicina (Sigma), e foi denominado de RPMI suplementado. A solução foi filtrada com membrana de poro 0,22 μηη e conservada a 4°C.  RPMI 1640 culture medium with L-glutamine (Sigma) was used. The contents of each vial were reconstituted in 1 L of ultra pure water and sodium bicarbonate (Merck), 10mM HEPES (N-2 hydroxyethylpiperazine, N-2 ethanesulfonic acid) (Sigma) added. The pH of the solution was adjusted to 7.1 and was called non-supplemented RPMI. The medium used in the cultures was supplemented with 10% fetal bovine serum (Cultilab), 100 U / mL penicillin and 100 Mg / mL streptomycin (Sigma), and was called supplemented RPMI. The solution was filtered with 0.22 μηη pore membrane and stored at 4 ° C.
A coleta do sangue bovino foi realizada na Escola de Medicina Veterinária da Universidade Federal de Minas Gerais (UFMG) em Belo Horizonte. Os animais foram contidos, em seguida o sangue foi coletado totalizando um volume final de aproximadamente 28ml_ de sangue de cada animal em tubos de coleta contendo heparina. O PBMC foi purificado em solução de Fico!! (GE Healthcare) na proporção de 2:1 (sangue: Ficoll).  The collection of bovine blood was performed at the School of Veterinary Medicine of the Federal University of Minas Gerais (UFMG) in Belo Horizonte. The animals were contained, then the blood was collected totaling a final volume of approximately 28 ml of blood from each animal in heparin-containing collection tubes. PBMC was purified in Fico solution !! (GE Healthcare) at a ratio of 2: 1 (blood: Ficoll).
As células (PBMC) provenientes dos bovinos foram incubadas na presença das proteínas recombinantes (RecPBoHV-1, RecPBoHV-5 e RecPBoHV-1 + RecPBoHV-5) e na ausência destas, para uso como controle. As PMBC foram ressuspendidas em meio de cultura após contagem, sendo distribuídas em placas para cultivo celular de 96 poços a uma concentração de 1 ,5x105 celulas/poço. As PBMC foram estimuladas ou não com as proteínas recombinantes em quadruplicata (100ng/poço) por 24, 48 e 72 horas a 37°C em atmosfera de 5% CO2. Ao final de cada tempo de cultivo, as amostras foram avaliadas quanto à viabilidade celular e produção de óxido nítrico. Cells (PBMC) from cattle were incubated in the presence of recombinant proteins (RecPBoHV-1, RecPBoHV-5 and RecPBoHV-1 + RecPBoHV-5) and in their absence for control. The PMBCs were resuspended in culture medium after counting and distributed into 96-well cell culture plates at a concentration of 1.5x10 5 cells / well. PBMC were stimulated or not with the quadruplicate recombinant proteins (100ng / well) for 24, 48 and 72 hours at 37 ° C in a 5% CO 2 atmosphere. At the end of each culture time, samples were evaluated for cell viability and nitric oxide production.
Viabilidade celular e produção de óxido nítrico Cell viability and nitric oxide production
Para o ensaio da viabilidade celular, foi utilizado o método colorimétrico de MTT, 3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazólio, (Sigma). Concentração final igual a 5 mg/mL. Para o ensaio de dosagem de óxido nítrico foram utilizadas duas soluções: solução A - 3ml_ de ácido fosfórico - 5% (H3PO4), 47ml_ de água destilada e 1g de sulfanilamida. Solução B - 0,05g de alfa- naphytil-ethilenodiane (NEED) e 25ml_ de água destilada. For cell viability assay, the MTT colorimetric method 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium (Sigma) was used. Final concentration equal to 5 mg / mL. For the nitric oxide assay, Two solutions were used: solution A - 3 ml phosphoric acid - 5% (H 3 PO 4 ), 47 ml distilled water and 1 g sulfanilamide. Solution B - 0.05g of alpha-naphytil-ethylenodiane (NEED) and 25ml of distilled water.
Após o período de incubação com as proteínas multiepitopo RecPBoHV- 1 , RecPBoHV-5 e RecPBoHV-1 + RecPBoHV-5 das células, 24, 48 e 72 horas a 37°C em estufa de CO2, foram adicionados 20μΙ_ da solução de MTT (5mg/ml_) ao abrigo da luz e incubadas por 4 horas a 37°C em estufa de C02. Ao término da incubação foram retirados 150μΙ_ do conteúdo de cada poço e adicionados 150μΙ_ de isopropanol (0,04 M HCI), células e solução foram exaustivamente misturados por pipetagem e, em seguida, a leitura das placas foi realizada em espectrofotômetro (ASYS Hitech GmbH) (□ = 595 nm). Observou-se que a estimulação com as proteínas recombinantes, avaliadas nos 3 diferentes tempos de incubação, não alterou significativamente a viabilidade celular, tendo esta sido mantida para todos os tempos e diferentes proteínas em torno de 87% (Figura 2), comprovando a sua segurança para uso em sistemas biológicos. Following the incubation period with RecPBoHV-1, RecPBoHV-5 and RecPBoHV-1 + RecPBoHV-5 multiepitope proteins from the cells, 24, 48 and 72 hours at 37 ° C in a CO2 oven, 20μΙ_ of MTT solution ( Light and incubated for 4 hours at 37 ° C in a C0 2 oven . At the end of the incubation, 150μΙ_ of the contents of each well were removed and 150μΙ_ of isopropanol (0.04 M HCI), cells and The solution was thoroughly mixed by pipetting and then the plates were read on a spectrophotometer (ASYS Hitech GmbH) (□ = 595 nm). It was observed that stimulation with recombinant proteins, evaluated at the 3 different incubation times, did not significantly change cell viability, which was maintained for all time and different proteins around 87% (Figure 2), proving their safety for use in biological systems.
A dosagem da concentração de NO nos sobrenadantes, recolhidos após 24, 48 e 72 horas foi realizada em placas de 96 poços, adicionando-se 100μΙ_ da amostra, 50μΙ_ da solução A e 50μΙ_ da solução B. As amostras foram incubadas à temperatura ambiente por 10 minutos e em seguida foi determinada em espectrofotômetro (absorbância 540nm - ASYS Hitech GmbH). Os resultados obtidos em μΜ foram determinados a partir de uma curva padrão realizada com nitrito de sódio (Sigma) em concentrações de 100 a 0,097μΜ. As dosagens e a curva padrão foram feitas em triplicata.  Measurement of the NO concentration in supernatants collected after 24, 48 and 72 hours was performed in 96-well plates by adding 100μΙ_ of sample, 50μΙ_ of solution A and 50μΙ_ of solution B. Samples were incubated at room temperature for 10 minutes and then determined by spectrophotometer (absorbance 540nm - ASYS Hitech GmbH). Results obtained in μΜ were determined from a standard curve performed with sodium nitrite (Sigma) at concentrations of 100 to 0.097μΜ. Dosages and standard curve were made in triplicate.
Neste estudo, não se observou diferença significativa na produção de óxido nítrico por PBMC bovino na presença das proteínas recombinantes BoHV-1 e BoHV-5 em nenhum dos tempos avaliados, 24, 48 e 72 horas em relação aos seus respectivos grupos controles (Figura 3).  In this study, no significant difference was observed in nitric oxide production by bovine PBMC in the presence of recombinant BoHV-1 and BoHV-5 proteins at any of the evaluated times, 24, 48 and 72 hours in relation to their respective control groups (Figure 3 ).
Exemplo 5- Imunização de coelhos da raça Nova Zelândia com as moléculas vacinais e estudo da resposta imune induzida. Example 5- Immunization of New Zealand Rabbits with Vaccine Molecules and Study of Induced Immune Response.
Obtenção, manutenção e eutanásia dos animais Os coelhos foram adquiridos junto à Fazenda Experimental Professor Hélio Viana, pertencente à Universidade Federal de Minas Gerais - UFMG, no município de Igarapé (MG). Esta parte experimental foi submetida ao CETEA- UFMG, sob o número 239/11. Collection, maintenance and euthanasia of animals The rabbits were purchased from Professor Hélio Viana Experimental Farm, belonging to the Federal University of Minas Gerais - UFMG, in the municipality of Igarapé (MG). This experimental part was submitted to CETEA-UFMG, under number 239/11.
As inoculações e procedimentos experimentais envolvendo os coelhos foram realizados no biotério da Escola de Veterinária (Universidade Federal de Minas Gerais), que conta com ventilação e temperatura controladas. Os coelhos foram mantidos em gaiolas individuais e receberam ração e água filtrada à vontade.  The inoculations and experimental procedures involving the rabbits were carried out in the animal room of the Veterinary School (Federal University of Minas Gerais), which has controlled ventilation and temperature. The rabbits were kept in individual cages and fed ration and filtered water at will.
O sangue dos coelhos foi coletado na veia marginal da orelha, com auxílio de algodão embebido em xilol para promover vasodilatação local, no início do experimento, após cada imunização e ao final foi realizada punção cardíaca (sob controle farmacológico - Quetamina (20 a 40mg/kg) e Xilazina (3 a 4 mg/Kg), via intravenosa).  Rabbit blood was collected in the marginal vein of the ear, with the aid of xylol-soaked cotton to promote local vasodilation, at the beginning of the experiment, after each immunization and at the end cardiac puncture (under pharmacological control - Ketamine (20 to 40mg / kg). kg) and Xylazine (3 to 4 mg / kg) intravenously).
Inoculação dos animais Animal Inoculation
As proteínas purificadas foram preparadas com o adjuvante hidróxido de alumínio (70% de proteína, 5% de hidróxido de alumínio e 25% de emulsigen) e inoculadas em coelhos da raça Nova Zelândia (sistema dose-reforço composto de 03 doses intervaladas com 20 dias), seguindo o apresentado na Figura 4.  The purified proteins were prepared with aluminum hydroxide adjuvant (70% protein, 5% aluminum hydroxide and 25% emulsigen) and inoculated into New Zealand rabbits (dose-boosting system consisting of three 20-day interval doses). ), as shown in Figure 4.
Ensaios de Western Blotting - reconhecimento por soros bovinos naturalmente infectados pelo BoHV-1 e/ou BoHV-5 Western Blotting Assays - Recognition for BoHV-1 and / or BoHV-5 naturally infected bovine sera
Para os ensaios de Western foram utilizadas as proteínas recombinantes produzidas e proteínas totais dos vírus BoHV-1 e BoHV-5 purificados, como controle comparativo. Foram preparados géis de SDS-PAGE 12% de canaleta única. Um gel foi aplicado com 180μΙ_ da proteína recombinante RecBoHV-1 (10μg) ou com a proteína RecBoHV-5 e o outro com 180μΙ_ do vírus purificado BoHV-1 ^g) ou BoHV-5. Após a corrida dos géis a 100v por 1 hora e 30 minutos foram transferidos para membranas de PVDF. Foram utilizados como anticorpos primários, soros provenientes de uma soroteca bovina contendo soros de animais fortemente reativos para BoHV-1 , para BoHV-5 ou para ambos os vírus, animais estes naturalmente infectados. Utilizou-se como anticorpo secundário / revelador, soro anti-lgG total bovino comercial (Sigma) total Os soros MIX 1 (Pool de soros policlonais anti-BoHV-1 ) e MIX 5 (Pool de soros policlonais anti-BoHV-5) reconheceram tanto o vírus BoHV-1 quanto a proteína recombinante RecPBoHV-1 (Figura 5). Os soros MIX 5 (Pool de soros policlonais anti-BoHV-5), a-BoHV-1 (Soro policlonal anti-BoHV-1) e P1 (Pool de positivos para BoHV-1) reconheceram tanto o vírus BoHV-5 quanto a proteína recombinante RecPBoHV-5 (Figura 6). For Western assays, recombinant proteins produced and BoHV-1 and BoHV-5 purified total proteins were used as a comparative control. 12% single channel SDS-PAGE gels were prepared. One gel was applied with 180μΙ_ of RecBoHV-1 recombinant protein (10μg) or RecBoHV-5 protein and the other with 180μΙ_ of BoHV-1µg purified virus or BoHV-5. After the 100v gels were run for 1 hour and 30 minutes they were transferred to PVDF membranes. Serum from a bovine library containing sera from animals strongly reactive to BoHV-1, BoHV-5 or both viruses, which were naturally infected, were used as primary antibodies. It was used as secondary antibody / developer, total commercial bovine total anti-IgG serum (Sigma) total MIX 1 (Anti-BoHV-1 Polyclonal Serum Pool) and MIX 5 (Polyclonal Anti-BoHV-5 Serum Pool) sera recognized both BoHV virus -1 for RecPBoHV-1 recombinant protein (Figure 5). MIX 5 (Anti-BoHV-5 Polyclonal Serum Pool), a-BoHV-1 (Anti-BoHV-1 Polyclonal Serum), and P1 (BoHV-1 Positive Pool) sera recognized both BoHV-5 and RecPBoHV-5 recombinant protein (Figure 6).
Ensaios de Western Blotting - reconhecimento por soros dos coelhos Nova Zelândia inoculados com as proteínas recombinantes produzidas. Western Blotting Assays - Sera recognition of New Zealand rabbits inoculated with the produced recombinant proteins.
Para os ensaios de Western foram preparados géis de SDS-PAGE 12% de canaleta única. Um gel foi aplicado com 180μΙ_ da proteína recombinante RecBoHV-1 (1( g) ou com a proteína RecBoHV-5 e o outro com 180μΙ_ do vírus purificado BoHV-1 ^g) ou BoHV-5. Após a corrida dos géis a 100v por 1 hora e 30 minutos foram transferidos para membranas de PVDF. Foram utilizados como anticorpos primários soros de coelhos da raça Nova Zelândia inoculados com as proteínas recombinantes (RecBoHV-1 , RecBoHV-5 ou ambas) utilizando um sistema dose-reforço composto de 03 doses intervaladas com 20 dias. Como controle dos ensaios foi utilizada uma soroteca bovina previamente testada pelos métodos de soroneutralização virai e por um ELISA comercial para BoHV-1 , contendo soros de animais fortemente reativos para BoHV-1 , 5, ambos os vírus ou negativos para ambos. Utilizou-se como anticorpo secundário / revelador, soro anti-lgG total bovino ou soro anti-lgG total de coelho comerciais (Sigma) (Figura 7 e Figura 8).  For Western assays 12% single channel SDS-PAGE gels were prepared. One gel was applied with 180μΙ_ of RecBoHV-1 recombinant protein (1 (g) or RecBoHV-5 protein and the other with 180μΙ_ of BoHV-1µg purified virus) or BoHV-5. After the 100v gels were run for 1 hour and 30 minutes they were transferred to PVDF membranes. Serum antibodies from New Zealand rabbits inoculated with the recombinant proteins (RecBoHV-1, RecBoHV-5 or both) were used as primary antibodies using a dose-boosting system composed of three 20-day interval doses. As control of the assays a bovine seroteca previously tested by the viral seroneutralization methods and by a commercial BoHV-1 ELISA containing sera from animals strongly reactive to BoHV-1, 5, both viruses or negative for both was used. Secondary antibody / developer was either total bovine anti-IgG serum or commercial rabbit anti-IgG total serum (Sigma) (Figure 7 and Figure 8).
Os ensaios de Western Blot confirmaram a reatividade das proteínas produzidas, tendo as mesmas sido reconhecidas tanto pelo soro dos coelhos inoculados quanto pela soroteca bovina proveniente de animais naturalmente infectados, utilizada como controle.  Western blot assays confirmed the reactivity of the proteins produced and were recognized by both the inoculated rabbit serum and the bovine seroteca from naturally infected animals used as controls.
Ensaio imunoenzimático - ELISA ELISA - enzyme immunoassay
Foi desenvolvido e padronizado um ELISA indireto para análise da reatividade dos soros dos coelhos (Nova Zelândia) frente às proteínas recombinantes. Como controle dos ensaios foi utilizada uma soroteca bovina previamente testada pelos métodos de soroneutralização virai e por um ELISA comercial para BoHV-1 , contendo soros de animais fortemente reativos para BoHV-1 , 5, ambos os vírus ou negativos para ambos. An indirect ELISA was developed and standardized to analyze the reactivity of rabbit sera (New Zealand) against recombinant proteins. As control of the assays a bovine soroteca was used previously tested by viral seroneutralization methods and a commercial BoHV-1 ELISA containing sera from animals strongly reactive to BoHV-1, 5, both virus-negative or both.
Antígeno virai total - BoHV-1/BoHV-5 e AgRecBoHV-1/BoHV-5 Total Viral Antigen - BoHV-1 / BoHV-5 and AgRecBoHV-1 / BoHV-5
Para os ensaios de ELISA, placas foram sensibilizadas com 250ng/poço do antígeno recombinante produzido ou com proteínas totais dos vírus purificados. Como anticorpo primário foram utilizados soros provenientes de coelhos da raça Nova Zelândia inoculados com a proteínas recombinantes (RecBoHV-1 , RecBoHV-5 ou ambas) utilizando um sistema dose-reforço composto de 03 doses intervaladas com 20 dias. Como controle dos ensaios foi utilizada uma soroteca bovina previamente testada pelos métodos de soroneutralização virai e por um ELISA comercial para BoHV-1 , contendo soros de animais fortemente reativos para BoHV-1 , 5, ambos os vírus ou negativos para ambos. Em seguida, foram utilizados como anticorpos secundários o anti- IgG bovino e anti-lgG de coelho (Figuras 9 e Figura 10).  For ELISA assays, plaques were sensitized with 250 ng / well of the recombinant antigen produced or with total purified virus proteins. Serum from New Zealand rabbits inoculated with recombinant proteins (RecBoHV-1, RecBoHV-5 or both) was used as the primary antibody using a dose-boosting system consisting of three 20-day interval doses. As a control of the assays, a bovine library previously tested by the viral seroneutralization methods and by a commercial BoHV-1 ELISA containing sera from animals highly reactive to BoHV-1, 5, both virus or negative for both, was used. Next, the anti-bovine IgG and anti-rabbit IgG were used as secondary antibodies (Figures 9 and Figure 10).
No teste imunoenzimático, ELISA, observou-se a produção de anticorpos específicos pelos animais imunizados que reconheceram tanto as proteínas virais quanto as proteínas recombinantes, comprovando a imunogenicidade das proteínas recombinantes produzidas e o seu potencial para uso vacinai.  In the enzyme-linked immunosorbent assay, ELISA, the production of specific antibodies was observed by immunized animals that recognized both viral and recombinant proteins, proving the immunogenicity of the recombinant proteins produced and their potential for vaccine use.
Análise estatística Statistical analysis
As análises estatísticas foram realizadas com o programa PRISM4, utilizando o método ANOVA one way com pós-teste Tukey para análises paramétricas ou Kruskal-Wallis para análises não paramétricas. Statistical analyzes were performed using the PRISM4 program, using the one-way ANOVA method with Tukey posttest for parametric analysis or Kruskal-Wallis for nonparametric analysis.
Exemplo 6 - Construção dos vírus recombinantes Vaccinia Ankara Modificado (MVA) contendo os cassetes gênicos para a expressão de Rec-BoHV-1 / BoHV-5 Example 6 - Construction of Modified Vaccinia Ankara Recombinant Virus (MVA) Containing Rec-BoHV-1 / BoHV-5 Expression Genetic Cassettes
O Vírus Ankara Modificado The Modified Ankara Virus
O Vírus Ankara Modificado (MVA), é uma linhagem do vírus Vaccinia que foi atenuado pela passagem seriada por mais de 500 vezes em fibroblastos embrionados de frango. Seu nome se deve a que o vírus parental foi isolado de uma lesão de um cavalo em Ankara, na Turquia (BLANCHARD, T. J., ALCAMI, A., PA AYIOTA, A., SMITH, G. L. Modified vaccinia vírus Ankara undergoes limited replication in human cells and lacks several immunomodulatory proteins: implications for use as a human vaccine.Journal of General Virology.v.79, p.1 159- 1167, 1998). Durante sua atenuação, o MVA sofreu perdas genômicas, totalizando aproximadamente 30kb, resultando assim na perda total de 26 ORFs junto com a fragmentação de mais 21 ORFs, além de numerosas mutações de pequena escala. O MVA foi usado nos estágios finais da campanha de erradicação da varíola, sendo administrado de forma intracutânea, subcutânea e intramuscular em mais de 120000 pessoas no sul da Alemanha e Turquia, e nenhum efeito colateral significativo foi relatado. O MVA é um candidato promissor para uso vacinai, devido a seu restrito espectro de hospedeiros, imunogenicidade e avirulência em modelos animais e seu excelente prognóstico de segurança como vacina para varíola. Modified Ankara Virus (MVA) is a strain of Vaccinia virus that has been attenuated by serial passage for more than 500 times in embryonic chicken fibroblasts. Its name is because the parental virus was isolated from a horse injury in Ankara, Turkey (BLANCHARD, TJ, ALCAMI, A., PA AYIOTA, A., SMITH, GL. Modified vaccinia virus Ankara undergoes limited replication in human cells and lacks several immunomodulatory proteins: implications for use as a human vaccine. Journal of General Virology.v.79, p.1 159-1167, 1998). During its attenuation, MVA suffered genomic losses totaling approximately 30kb, resulting in a total loss of 26 ORFs along with the fragmentation of 21 more ORFs, as well as numerous small-scale mutations. MVA was used in the final stages of the smallpox eradication campaign and was administered intracutaneously, subcutaneously and intramuscularly to over 120,000 people in southern Germany and Turkey, and no significant side effects were reported. MVA is a promising candidate for vaccine use due to its restricted host spectrum, immunogenicity and avirulence in animal models and its excellent safety prognosis as a smallpox vaccine.
Clonagem em vetor pLW 44 PLW vector cloning 44
Os vetores pQE 30 contendo os cassetes gênicos produzidos foram usados como molde em uma reação utilizando iniciadores contendo sítio de restrição para as enzimas SAL I e PST I para a subclonagem no vetor pLW44. O plasmídeo pLW44 possui 5028 pb, os promotores p11 e mH5, flanqueados por regiões gênicas do vírus MVA, uma região codificadora para a proteína GFP {Green fluorescent protein) e os sítios para restrição enzimática de Sma I, Sal I e Pst I. Os cassetes gênicos foram inseridos na região controlada pelo promotor mH5, entre os sítios de restrição das enzimas Sal I e Pst I. O promotor p1 1 (promotor tardio) controla a produção de GFP. Os dois promotores p11 e mH5 estão sob o controle de ativadores transcricionais precoces e/ou tardios do vírus MVA. Desta forma, após as células Vero serem infectadas pelo vírus MVA, ativadores transcricionais presentes juntamente com o vírion e/ou produzidos após infecção virai se associarão aos promotores levando à produção e expressão de GFP e das proteínas recombinantes multiepitopo de BoHV-1 e BoHV-5. A confirmação da clonagem das colónias recombinantes contendo os fragmentos gênicos trabalhados foi feita por PCR a partir das culturas de bactérias, utilizando-se os iniciadores PST-R e SAL - F. Subsequente sequenciamento confirmou a correta clonagem e posicionamento dos cassetes gênicos. The pQE 30 vectors containing the gene cassettes produced were used as a template in a reaction using restriction site primers for the enzymes SAL I and PST I for subcloning into the pLW44 vector. Plasmid pLW44 has 5028 bp, the p11 and mH5 promoters flanked by MVA virus gene regions, a coding region for GFP protein (Green fluorescent protein) and the enzymatic restriction sites of Sma I, Sal I and Pst I. gene cassettes have been inserted into the region controlled by the mH5 promoter between the restriction sites of the Sal I and Pst I enzymes. The p11 promoter (late promoter) controls GFP production. Both p11 and mH5 promoters are under the control of early and / or late MVA virus transcriptional activators. Thus, after Vero cells are infected with the MVA virus, transcriptional activators present together with the virion and / or produced after viral infection will associate with promoters leading to the production and expression of GFP and BoHV-1 and BoHV-multiepitope recombinant proteins. 5 Confirmation of cloning of recombinant colonies containing the worked gene fragments was done by PCR at from bacterial cultures using the primers PST-R and SAL - F. Subsequent sequencing confirmed the correct cloning and positioning of the gene cassettes.
Recombinação homóloga Year Recombination
O método utilizado para geração dos MVA recombinantes foi realizado por meio de recombinação homóloga. As células susceptíveis foram infectadas com o MVA "selvagem" e transfectadas com plasmídios de transferência contendo os cassetes gênicos construídos (pLW44) sob o controle de um promotor de VACV (vírus Vaccinia), o gene marcador selecionável e flancos correspondentes a regiões não essenciais do genoma do MVA. A transfeção do plasmídio de transferência em células previamente infectadas pelo vírus MVA permite que a recombinação homóloga entre o plasmídio e o genoma do vírus ocorra naturalmente em taxas relativamente altas (- 0,1%). Os vírus recombinantes expressando as proteínas de interesse foram selecionados em passagens sucessivas em sistema de cultivo celular, tendo sido clonados biologicamente, utilizando como marcação a fluorescência da GFP (proteína verde fluorescente) selecionados de diversas maneiras, dependendo do marcador de seleção utilizado (Figura 11).  The method used for the generation of recombinant MVA was performed by homologous recombination. Susceptible cells were infected with "wild" MVA and transfected with transfer plasmids containing the constructed gene cassettes (pLW44) under the control of a VACV (Vaccinia virus) promoter, the selectable marker gene, and flanks corresponding to nonessential regions of the MVA genome. Transfer plasmid transfection in cells previously infected with the MVA virus allows homologous recombination between the plasmid and virus genome to occur naturally at relatively high rates (-0.1%). Recombinant viruses expressing the proteins of interest were selected in successive passages in a cell culture system and were cloned biologically using the fluorescence labeling of the selected green fluorescent protein (GFP) as a marker, depending on the selection marker used (Figure 11 ).

Claims

REIVINDICAÇÕES
1- Proteínas Recombinantes caracterizadas por compreenderem as sequências representadas por SEQ ID N°1 e SEQ ID N°2 e/ou suas análogas, homólogas e heterólogas. Recombinant proteins comprising the sequences represented by SEQ ID NO: 1 and SEQ ID NO: 2 and / or their homologous and heterologous analogs thereof.
2- Polinucleotídeos caracterizados por compreenderem as sequências representadas por SEQ ID N°3 e SEQ ID N°4 e/ou seus análogos, homólogos e heterólogos.  2. Polynucleotides comprising the sequences represented by SEQ ID NO: 3 and SEQ ID NO: 4 and / or their analogs, homologs and heterologues.
3- Vacina contra Herpesvirus Bovino caracterizada por compreender a proteína recombinante representada pela sequência SEQ ID N°1 e/ou a proteína recombinante representada pela sequência SEQ ID N°2 e/ou o polinucleotídeo (SEQ ID N°3) que codifica a proteína representada pela sequência SEQ ID N°1 e/ou o polinucleotídeo (SEQ ID N°4) que codifica a proteína representada pela sequência SEQ ID N°2 e/ou seus análogos, homólogos, heterólogos e excipientes farmacologicamente e farmacêuticamente aceitáveis.  Bovine Herpesvirus vaccine comprising the recombinant protein represented by the sequence SEQ ID NO: 1 and / or the recombinant protein represented by the sequence SEQ ID NO: 2 and / or the polynucleotide (SEQ ID NO: 3) encoding the protein. represented by the sequence SEQ ID NO: 1 and / or the polynucleotide (SEQ ID NO: 4) encoding the protein represented by the sequence SEQ ID NO: 2 and / or its pharmaceutically and pharmaceutically acceptable analogs, homologues, heterologues and excipients.
4- Vacina contra Herpesvirus Bovino de acordo com a reivindicação 3 caracterizada pelas proteínas recombinantes representadas por SEQ ID N°1 e SEQ ID N° 2 poderem ser produzidas em vetores procariotos, eucariotos e/ou utilizando vírus recombinantes produzidos para a expressão em sistema eucarioto.  Bovine Herpesvirus vaccine according to Claim 3, characterized in that the recombinant proteins represented by SEQ ID NO: 1 and SEQ ID NO: 2 can be produced in prokaryotic vectors, eukaryotes and / or using recombinant viruses produced for expression in the eukaryotic system. .
5- Uso da vacina contra Herpesvirus Bovino de acordo com as reivindicações 3 e 4 caracterizado por ser preferencialmente utilizada no controle e prevenção da Rinotraqueíte Infecciosa bovina e Encefalite bovina.  Use of the bovine herpesvirus vaccine according to claims 3 and 4, characterized in that it is preferably used for the control and prevention of Bovine Infectious Rhinotracheitis and Bovine Encephalitis.
6- Uso da vacina contra Herpesvirus Bovino de acordo com as reivindicações 3 e 4, caracterizado por ser na imunização de bovinos e/ou outros ruminantes.  Use of the bovine herpesvirus vaccine according to claims 3 and 4, characterized in that it is for the immunization of cattle and / or other ruminants.
7- Uso da vacina contra Herpesvirus Bovino de acordo com as reivindicações 3 e 4, caracterizada por ser no controle e prevenção da infecção por BoHV-1 e/ou BoHV-5 em bovinos e/ou outros ruminantes.  Use of the bovine Herpesvirus vaccine according to claims 3 and 4, characterized in that it is in the control and prevention of BoHV-1 and / or BoHV-5 infection in cattle and / or other ruminants.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106405093A (en) * 2016-08-31 2017-02-15 北京市农林科学院 Detection kit for detecting infectious bovine rhinotracheitis virus antibody

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993011792A1 (en) * 1991-12-11 1993-06-24 University Of Saskatchewan Recombinant bovine herpesvirus type 1 polypeptides and vaccines
WO2001034184A2 (en) * 1999-11-05 2001-05-17 The Board Of Regents Of The University Of Nebraska Methods and compositions for protection against bovine herpesvirus 1
WO2004026265A2 (en) * 2002-09-23 2004-04-01 Macrogenics, Inc. Compositions and methods for treatment of herpesvirus infections

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993011792A1 (en) * 1991-12-11 1993-06-24 University Of Saskatchewan Recombinant bovine herpesvirus type 1 polypeptides and vaccines
WO2001034184A2 (en) * 1999-11-05 2001-05-17 The Board Of Regents Of The University Of Nebraska Methods and compositions for protection against bovine herpesvirus 1
WO2004026265A2 (en) * 2002-09-23 2004-04-01 Macrogenics, Inc. Compositions and methods for treatment of herpesvirus infections

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ABDELMAGID ET AL.: 'Fine Mapping of Bovine Herpesvirus-1 (BHV-1) Glycoprotein D (gD) Neutralizing Epitopes by Type- Specific Monoclonal Antibodies and Sequence Comparison with BHV-5 gD' VIROLOGY vol. 206, 1995, pages 242 - 253 *
GAO ET AL.: 'Mapping T and B lymphocyte epitopes of bovine herpesvirus-I glycoprotein B' JOURNAL OF GENERAL VIROLOGY vol. 80, 1999, pages 2699 - 2704 *
LIVINGSTON ET AL.: 'A Rational Strategy to Design Multiepitope Immunogens Based on Multiple Th Lymphocyte Epitopes' JOURNAL OF IMMUNOL. vol. 168, 2002, pages 5499 - 5506 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106405093A (en) * 2016-08-31 2017-02-15 北京市农林科学院 Detection kit for detecting infectious bovine rhinotracheitis virus antibody

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