Chimeric vaccine antigens against classical swine plague virus

FIELD: medicine, veterinary.

SUBSTANCE: claimed invention relates to field of veterinary and describes chimeric vaccine antigen against classical swine plague virus (CSPV), characterised by the fact that it consists of extracellular segment of glycoprotein E2 of CSPV viral envelop on N-end of chimeric antigen and extracellular domain of swine CD 154 molecule, identified as SEQ ID NO: 2, on C-end of chimeric antigen. Chimeric antigens can be obtained in expression systems, which guarantee proper folding of tertiary structure of chimeric molecules. Vaccine compositions, which contain such chimeric antigens, cause early and strong immune response in vaccinised swine and create complete protection against CSPV. Chimeric antigens like obtained compositions, can be applied in veterinary as vaccines for preventive application in swine.

EFFECT: obtained vaccine compositions prevent transmission of virus from sows to their offspring.

10 cl, 11 dwg, 1 tbl, 5 ex

 

The SCOPE TO WHICH the INVENTION RELATES

The present invention relates to veterinary medicine, in particular to new chimeric antigens, including viral subunit of classical swine fever virus (CSFV), United with proteins capable of stimulating cellular and humoral immune system, causing a strong and early immune response to these viruses in pigs.

BACKGROUND of INVENTION

Suppose that the classical swine fever (CSF), also known as cholera pigs for its highly infectious nature, is the most important disease in pigs, and it is included in the official list of diseases of the world veterinary organization. The etiological agent of this disease, CSF, is a virus of the genus Pestvirus from the family Flaviviridae. It is known that this virus has a lipid membrane, the diameter from 40 to 60 nm and hexagonal symmetry, with single-stranded ribonucleic acid (RNA) as their genetic material (Kummerer et al. (2000). The genetic basis of cytopathogenicity of pestviruses. Vet. Environ. 77:117-128; Moenning et al. (2003)).

CSF is a highly contagious disease in its acute form presents with fever, destruction of capillary blood vessels, necrosis of the internal organs and death. The first clinical symptoms appear after an incubation period of 2 to 6 days, vizualizarea, reduced mobility and loss of appetite, with the deterioration in the following days, and the temperature can reach 42°C. Also developing leukopenia with quantities of white blood cells in the blood less than 8000/mm3. Pigs also develop conjunctivitis, constipation followed by diarrhea, vomiting, loss of coordination, convulsions and paralysis of the muscles in the terminal phase. It is shown in red skin, spreading across the belly, muzzle, ears and inner parts of the limbs. In most fatal cases, histopathological examination of the brain revealed negniy encephalitis with high vascularization (Moenning et al. (2002) Clinical Signs and Epidemiology of Classical Swine Fever. A review of knowledge. Vet. Journal 161:1-10).

CSFV acts as an immunosuppressant during the course of infection (Susa et al. (1992) Pathogenesis of Classical Swine Fever: B-lymphocyte deficiemcy caused by Hog Cholera virus. J. Virol. 66:1171-1175), and the detection of neutralizing antibodies starts at 2 and 3 the week after infection (Laevents et al. (1998)) An experimental infection with a classical swine fever virus introduction in natural outbreaks. Vet. Q. 20: 46-49). The terminal stage of infection is associated with a significant decrease In lymphocytes in the circulating blood as well as in lymphoid tissue (Susa et al. (1992) Pathogenesis of Classical Swine Fever: B-lymphocyte deficiency = MKD caused hog cholera virus. J. Virol. 66: 1171-1175). Most of the pigs that are sick, dying in the time interval from the 10th to the 20th day after infection, with what level of mortality over 95%. Characteristic lesions at CoES autopsy was hemorrhagic diathesis with petechiae in most organ systems. Most constantly these changes occur in the kidneys, bladder and lymphatic ganglia, although they can also appear in the spleen, throat, mucous and serous membranes (Mouwen et al. (1983) Atlas of Veterinary Pathology, Bunge, Utrecht, The Netherlands).

Transplacental infection is another clinical form CoES; in this case, the virus is able to penetrate through the placenta of pregnant sows, infecting fruit. The consequences of this infection may be a miscarriage, stillborn offspring, mummification, malformation, birth of weak pigs and problems associated with the differentiation of organs. Depending on the duration of pregnancy, which was infected, as a result of infection from sows (vertical transmission) can be immune offspring. Piglets remain infected and vermicasting to death, creating a stable center of the spread of CSFV in the herd (Moenning et al., (2003) Clinical Signs and Epidemiology of Classical Swine Fever: a rewiew of new knowledge. Vet. Journal 165: 11-20). Mortality associated with CSF, is an economic problem for the affected countries, adversely affecting the economic and social situation of developing countries. For this reason, the article is anach, with a high density of pigs and a large spread of the virus becomes necessary to use monitoring programs based on vaccination. In developed countries, in which pigs mainly subsidized by governments, such as Europe, the United States and Canada, used the method of eradication through full sanitary slaughter. However, the cost of this method is very high, and such countries are still sensitive to possible epidemics.

The European Union (EU) is considered as a high risk of re-emergency of new CSFV-epizootics due to the high population density of pigs, its policy of revaktsinirovano and its geographical proximity to the Eastern European countries in which CSFV is enzootic disease. One of the problems associated with the threat of new epidemics in this region is the presence of wild boars with endemic infection CSF (Laddomada (2000) Incidence and control of CSF in wild boars in Europe. Vet. Microb, 73:(121-30)). These new epizootics occurred despite the hard control programs that have been implemented in the European Union, which included sanitary destruction of all contagious populations and limiting the export of pork from the affected areas to areas free from the disease(van Oirschot (2003) Vaccinology of Classical Swine Fever: from lab to field. Vet Environ, 96:367-384). So urgent is the need to develop vaccines that induce an immune response, early and safe, protecting against infection and transmission of the virus.

Were developed vaccines against CSFV on the basis of intact virus: the vaccine against the virus, inaktivirovannye crystal violet or formalin (Biront et al. (1998) Classical swine fever related infections. Liess B.M. Ed. Martinus Nijhoff Publishing, Boston: 181-200), vaccine virus, attenuated by passage in the rabbit, for example strain Sinlak (Baibikov et al. EN 2182495) and strain Lapinizied Chinese (Dahle et al. (1995) Assessment of safety and protective value of a cell culture modified strain C vaccine of hog cholera/classical swine fever virus. Berl-Munch. Tieraztl.Wsch, 108:20-25), or vaccine virus, attenuated in tissue cultures derived from rabbit, Guinea pigs and pigs (Kachiku et al. JP 73001484; Terpstra et al. (1990) Development and properties of a cell culture produced for hog cholera based on Chinese strain. Ditsh. Tierarztl.Wsch. 97: 77-79). The use of these types of vaccines pose a risk due to the probability content of the fractions of active virus, vaccinated susceptible animals, will cause new outbreaks of CSF. In addition, in some cases, to obtain a protective immunological response required repeated immunization, because inactivation affects immune and genetic properties of the virus.

In some cases, the use of live vaccines with attenuated virulent the activities they have a high risk that there will be partial attenuation or restore virulence. In some cases, they will form a pathogenic viral particles, which, when entered susceptible animals will make it possible infection, clinical disease and the spread of CSF in herds. These problems cause an even greater risk for pregnant sows, because the virus can infect the fruit, which are vazokonstrikciei, and the affected offspring will spread the disease.

There are vaccines based on strains of CSFV, which were attenyerevan, such as strain C Chinese, strain PAV 250, strain Thierval and strain IFFA/A-49 (Björlund, H.JV. et al. (1998) Molecular characterization of the 3'noncoding region of classical swine fever virus vaccine strains. Virus Genes 16: 307-312, Launais et al. (1978) Hog Cholera Virus: Active immunization of piglets with the Thiverval strain in the presence and absence of calostral passive immunity. Vet. Microbiology 3:31-43). These strains are used only in countries where the disease is enzootic, because they have such a disadvantage that they do not allow to distinguish vaccinated animal and the animal, the affected native virus. Animals vaccinated with these strains show the same responses in serological tests, as infected animals. Specific antibodies anti-CSFV, which are produced when using vaccines based on attenuated in the Rus, interfere with the diagnosis of infection with CSF. Diagnosis is carried out by immunological determination of infectious virus in tonsils and reproduction vaccinal viral strain occurring in the tonsils. For this reason, attenuated strains are unsuitable for use in programs of eradication. Vaccination strain LK-VNIIVVM and additional hyperimmunization purified strain of Shi-Myng made with adjuvant-blockers, is another example. But immunization 40-45 places is impossible when the vaccination campaign, in which hundreds of animals should be vaccinated every day (Balashova et al. RU2183972).

Immunization with these vaccines that contain a virus, prevents the differential diagnosis of infections caused CSFV, and infections caused by other members of the genus Pestvirus that can infect pigs, such as viral diarrhea virus cows (abbreviated WWDC) and the virus border disease of sheep (abbreviated VPB), (Dahle et al. (1991) Clinical Post Mortem and Virologocal Findings after Simultaneous Inoculation of Pig with Hog Cholera and BVD Virus. J. Med. Vet. 38: 764-772).

To avoid the inconvenience of vaccines based on whole virus obtained is suitable for use of the vaccine is completely safe, such as options on the basis of subunits or viral proteins obtained by recombinant. These options must protect the flock about the re-introduction of virus strains, and also allow to distinguish between vaccinated and infected animals simple serological methods. For this purpose we have developed vaccines based on viral subunits. Vaccines containing viral proteins such as glycoprotein E2 envelope of the virus (Bourna et al. (2000) Duration of the onset of the herd immunity induced by E2 subunit vaccine classical Swine Fever virus 18: 1374-1381), are safe because their application does not contain the risk return virulence and does not preclude the diagnosis. These vaccines help to differentiate infected animals and vaccinated animals, because of the antibodies that are produced that react only against viral segment. Thus, they are suitable for the programme of eradication of CSF.

Have developed several recombinant vaccines based on the expression of E2 protein in prokaryotes, and vaccines based on synthetic peptides of this protein (Chen et al. WO 200232453). In these cases, the protein is deglycosylated, so suffer its immunogenicity and protective properties. Other options vaccines using viral vectors for the expression of heterologous gene E2 in eukaryotic cells like a virus pseudoleskeella pigs (Peeterrs et al. (1997). Biologically safe, non-transmissible pseudorabies virus vector vaccine protect pigs against both Aujeszky''s disease and classical swine fever. J. Gen. Virol. 78: 3311-3315), variola virus of swine (Gibbs et al. US62117882) and adenovirus pig is th (Nagy et al. WO 200183737). In these cases, infection with wild-type virus induces the production of neutralizing antibodies against the viral vector of the same serotype. Thus, suffers induction of immune response against CSFV. Also viral vectors based virus pseudoleskeella pigs and variola virus pigs may not apply in countries recognised as being free from these viruses, due to concerns about the legislation. Also was used as a vector, vaccinia virus, but the resolution of the world health organization complicate its application (Meyeers et al. EP 1087014).

Vaccines based on devoid shell deoxyribonucleic acid (DNA) for the expression of E2 protein in myocytes and osteocytes have the inconvenience associated with the fact that in order to induce an immune response requires a higher concentration of DNA, since transfection with the use of devoid of membrane DNA is very inefficient. This vaccine is subjected to strict legislative control, which makes it difficult to apply (Audonnet et al. WO 20152888).

Getting E2-CSFV as antigen in the expression systems, insect cells, baculovirus-mediated became possible alternative (Van Rjin et al. (1990). An experimental marker vaccine and accompanying serological diagnostic test both based on enveloped glycoprotein E2 of classical swine fever virus. Vaccine, 17: 433-440; Kretzdom et al. US 20040028701). In this system the Birmingham recombinant E2 get glycoprotein, increasing the immunogenicity relative to deglycosylation isoforms. Baculovirus additionally inactivated without pathogenic effects on pigs. However, effective protection against infection occurs three weeks after the vaccination, and protection against intrauterine infection is incomplete. Therefore, an important issue in the prevention of disorder is the absence of recombinant vaccines based on the subunits that make possible the carrying out differential diagnosis between vaccinated and infected animals, and is capable of forming early protection after vaccination, eliminating transplacental transmission from pregnant sows to their offspring.

Description of the INVENTION

The present invention solves the above problem. The new vaccine contains chimeric antigens containing viral subunit in combination with molecules that stimulate the immune system, which make possible the development of the early immune response that protects pigs from infection with CSFV. Another advantage of the proposed solution is that it stops the transmission of the virus from infected pregnant sows the seed due to the effect of activation of immune stimulating molecules, which are combined with the viral proteins in the chimeric antigens.

In particular, the invention relates to chimeric antigens against CSF, which have as the main component of the glycoprotein E2 of the shell CSFV. The extracellular segment of glycoprotein E2 is used as an immunogen combined with protein, stimulating the immune system (called in the context of this invention "molecular adjuvant"), to strengthen and early stimulation of the cellular immune response, and the evocation produce higher titers of neutralizing antibodies to CSFV.

In a specific embodiment of the invention a protein that stimulates the immune system, is an interferon alpha or extracellular segment of the CD154 molecule. In a preferred embodiment, interferon alpha or extracellular segment of the CD154 molecule can be obtained from any mammal.

Vaccine antigens of the present invention on the basis of the chimeric proteins ensure the protection of vaccinated pigs from the first week after immunization, when they are controlling infection 105DL50(dose of the virus, which causes death of 50% of the animals infected with CSFV). This protection is mediated by a strong cellular immune response against CSFV, which is directly related to a combination of elements that are combined in a chimeric antigen. There is also a reduction of the time of induction neutrality is their antibodies which appeared in the second week following vaccination. It enhances protection against CSFV vaccinated pigs. Immunized animals do not have symptoms of clinical disease, and the allocation of CSFV from biological fluids could not be obtained either in one day after control of infection the virus.

Chimeric antigens with a molecular adjuvant E2 prevent the vertical transmission of CSFV from sows to the fruits. These proteins induce early protection in pregnant sows, which delays the development of clinical disease and prevents replication of the virus, not only mothers, but also the fruit, after control of infection of 105DL50CSFV.

In a preferred embodiment, chimeric vaccine antigen is characterized by the content mainly amino acid sequence of the extracellular segment (or domain) of E2 CSFV, which is represented in the List of sequences as SEQ ID NO: 1; and the extracellular segment of the molecule CD154 pigs, which is represented as SEQ ID NO: 2. Chimeric vaccine antigen in fact contains such amino acid sequences, but it may also include the extracellular segment of any isolated E2 CSFV.

Another aspect of the present invention is that the Chimera in czisny antigen can be obtained recombinant synthetic means or by chemical conjugation. In a specific embodiment of the invention the variant-based chimeric protein containing 2his (extracellular segment E2, merged with the tail 6 histidines), and molecular adjuvant was obtained in the form of a fused protein. With this purpose, the spacer elements peptide consisting of 4 repeating units Gly4Ser (4G4S), and a molecule that stimulates the immune system, were added on To the end 2his. The inclusion of peptide 4G4S admits a certain degree of relaxation of polypeptide chain. This ensures proper tertiary structural stacking structure of a protein to obtain a fused protein with the same tertiary structure as native. One of the vaccine antigens, the object of the invention is the extracellular domain of CD154 molecule pigs, merged with him To-end, as a molecular adjuvant (2his-D154).

Up to the present time have not been studied obtaining recombinant vaccine candidates against CSFV-mediated expression systems of animals as bioreactors. Despite this, the ability of the breast to Express glycosylated recombinant proteins with the correct laying their tertiary structure, makes it adequate expression system to obtain glycoprotein E2 with high immunogenicity and protective properties. Transient expression system in breast jachnow the animal, mediated adenoviral vectors constitutes a means for obtaining high levels of expression of recombinant proteins, quick and simple way (Toledo et al., WO 2004/034780). This method is very useful for the preparation of recombinant E2 for use in vaccination programmes aimed at the eradication of CSF.

In one embodiment of the invention, the vaccine antigens, the object of the present invention is expressed in the epithelial cells of the mammary gland of genetically modified mammals during lactation and secreted into the milk. Recombinant chimeric molecules get in the milk of transgenic mammals, or by direct transformation of the epithelium of the mammary glands nereshennyh mammals using adenoviral vectors. In another embodiment of the invention chimeric vaccine antigens get in genetically modified yeast cells. Such antigens are produced in the culture medium of the yeast transformed with the use of a chimeric gene and regulatory sequences allowing expression and secretion of the recombinant protein into the culture medium.

Protein native E2 CSFV is exposed on the viral membrane in the form of glycosilated, stabilized miaocheng disulfide with the ides. It defines what the neutralizing and protective antibodies are produced against conformational epitopes presented on homodimer. Vaccine antigens developed in the present invention are expression systems that allow for the correct folding of these recombinant proteins. Configuration genetic constructs ensures that there are no changes in the tertiary structure of fused proteins. Recombinant vaccine antigens are easily cleaned using a simple procedure affinity chromatography on ion metals.

Configuration genetic constructs, the use of expression systems and the relative ease of cleanup procedure ensures that vaccine antigens against CSFV described in this invention are the same as the viral E2 protein, antigenic and immunogenetic properties. Immunization with chimeric molecules obtained in such expression systems, such as Pichia pastoris or mammary gland of the goat, leads to a strong and early immune response. The extracellular domain of E2 forms homodimer that provide conformational epitopes for the formation of neutralizing and protective antibodies. The segment of CD154 acts as a molecular adjuvant that stimulates the immune system of the vaccinated pigs, causes cellular immune response, to whom that protects animals from CSFV with next after the first vaccination week. The combination of both molecules in the chimeric protein, which contains a peptide spacer elements, ensures the correct folding of each molecule. Applied expression system allowed to Express recombinant proteins in the glycosylated isoforms. It also helps to obtain molecules with the correct tertiary structure.

Another aspect of the present invention is a vaccine composition with the ability to cause protects the immune response against CSFV, which are characterized by a content of chimeric antigens described previously, containing the extracellular domain of glycoprotein E2 and molecular adjuvant. Such vaccine compositions can be administered to animals of the system by or through the mucous membranes to prevent CSF and avoid material and economic losses which are caused by infection with CSFV swine herds.

BRIEF DESCRIPTION of DRAWINGS

Figure 1.Analysis by SDS-electrophoresis in SDS page in reducing conditions the expression 2his in cells RK-15, transductional adenoviral vectors Ad-E2his-sec. (A) SDS-electrophoresis in SDS page, lane 1: culture medium from transduced cells, lane 2: culture medium from untreated cells, MWM: molecular weight marker. (C) Immunogenetical 2his using Western blot turns with the use of monoclonal antibodies, on rublennyh on his-tag tail, track 1: cultural medium of transduced cells, lane 2: culture medium from untreated cells, lane 3: positive control for his-tag tail, MWM: molecular weight marker. (C) Immunogenetical 2his using Western blot turns using polyclonal serum obtained from CSFV infected pigs, lane 1: culture medium from untreated cells, lane 2: culture medium from transduced with Ad-E2his cells, MWM: molecular weight marker.

Figure 2.The analysis of the conditions of expression 2his and E2his-CD154 in cell RK-15, transduction adenoviral vectors Ad-E2his-sec and Ad-E2hisCD154-sec. Proteins in the culture medium sephirot using SDS-electrophoresis in SDS page in non conditions. Immunogenetical interesting molecules was carried out using Western blot turns with the use of monoclonal antibodies (Mab) against protein E2 CSFV (Mab-1G6). Lane 1: culture medium from transduced by the vector Ad-E2his-sec cells, lane 2: culture medium from transduced by the vector Ad-E2hisCD154-sec cells, MWM: molecular weight marker.

Figure 3.The kinetics of expression of 2his in the milk of goats, transduction vector Ad-E2his-sec. Proteins from samples of whey corresponding to each day milking, separated using SDS-electrophoresis in SDS page in Sevostyanova the General conditions. Immunogenetical interesting molecules was investigated using Western blot turns using Mab-1G6. Track RK: positive control 2his, expressed in the culture medium of cells RK-15, transduction vector Ad-E2his-sec, track-samples milk serum from goats, not exposed to processing, tracks 1-8: samples of milk serum from goats, transduction vector Ad-E2his-sec, corresponding to each of the 8 days of milking, following adenoviral transduction.

Figure 4.The kinetics of expression of 2his-CD154 in the milk of goats, transduction vector Ad-E2hisCD154-sec. Proteins from samples of whey corresponding to each day milking, separated using SDS-electrophoresis in SDS page in non conditions. Immunogenetical interesting molecules was investigated using Western blot turns using Mab-1G6. Tracks 1-5: samples of milk serum from goats, transduction vector Ad-E2hisCD154-sec, corresponding to each of the 5 days of milking, following adenoviral transduction, track-samples milk serum from goats, not exposed to processing, path RK: positive control 2his-CD154 expressed in the culture medium of cells RK-15, transduction vector Ad-E2hisCD154-sec.

Figure 5.Analysis of purity and identification 2his subjected to SDS-electrophoresis in SDS page Neuve in ostanavlivaya conditions. Protein expressively in the milk of goats, transduction vector Ad-E2his-sec, and purification was carried out using affinity chromatography with metal ions. (A) SDS-electrophoresis in SDS page various stages of purification. (C) Immunogenicity using Western blot turns using Mab-1G6. Lane 1: positive control 2his, expressed in the culture medium of cells RK-15, transduction vector Ad-E2his-sec, track 2: samples of milk serum from goats, not exposed to processing, lane 3: sample breast serum from goats, transduction vector Ad-E2his-sec, taken as source material for chromatography, lane 4: material not contacting the matrix, lane 5: wash 20 mm imidazole, lane 6: washing with 50 mm imidazole, lane 7: elution with 200 mm imidazole.

6.Comparison of antigenic recognition of two isoforms of vaccine antigen E2his antibodies present in the serum of pigs infected with virulent strain of CSFV. E2his, purified from the milk of goats, transformirovannykh adenoviral vector Ad-E2his-sec, investigated by means of electrophoresis and Western blot turns in reducing conditions (monomer) and non conditions (glycosilated). (A) SDS-electrophoresis in SDS page. (C) Western blot turns using polyclonal serum CSF from infected pigs, track 1: E2his, all goty electrophoresis in non conditions track 2: E2his, thrown electrophoresis in reducing conditions.

7. Kinetics of neutralizing antibodies obtained in two groups of pigs vaccinated with one dose of vaccine antigen E2his, the antibody titers were determined using test neutralization, based on the peroxidase. Group a was inoculated with a dose of 30 mcg on each animal and group In a dose of 50 micrograms per each animal. Both groups were subjected to control infection three weeks after vaccination dose of virus CSFV 105DL50. The results are shown as geometric mean of the respective titles.

Fig. Lymphoproliferative analysis with lymphocytes isolated from pigs on day 5 after immunization with antigens E2-CD154 (groups D and E) and 2his (group F). The results are expressed as stimulation index (SI)defined as the ratio between the values of bills per minute (cpm) of stimulated cultures and levels cpm of control cultures not exposed to stimulation. Lymphoproliferative response, which was defined SI≥2 were considered positive. Was assessed proliferation in cultures treated with CSFV as well as inhibition of proliferation in cultures treated with CSFV and Mab against domain CD4 pigs.

Fig.9. Study of the antiviral activity of the cells RK-15 with the use of serum, recip is authorized from pigs, vaccinated with antigens E2-CD154 (groups D and E) and 2his (group F). The results are expressed as geometric mean of the respective titles.

Figure 10.Kinetics of neutralizing antibodies obtained in two groups of pigs vaccinated with antigens E2-CD154 (group N) and 2his (group I), with a dose of 50 μg per animal. Antibody titers were determined by neutralization test based on the peroxidase. The results are shown as geometric mean of the respective titles.

11. The analysis of the conditions of expression E2his and E2his-CD154 in cell GMGE, transduced lentivirusnye vectors Lv-E2his and Lv-E2his-CD154. Proteins in the culture medium were separated by the method of SDS-electrophoresis in SDS page in non 4 conditions. Immunogenetical interesting molecules was performed Western blot analysis using Mab against CSFV E2 (αE2-1G6). Lane 1: culture medium of the cells transduced by the vector E2his-CD154; lane 2: culture medium of the cells transduced by the vector E2his; lane 3: culture medium from untreated cells; lane 4: positive control, E2his, expressed in the culture medium of cells RK-15, transduced by the vector Ad-E2his-sec.

EXAMPLES

Example 1: Amplification plots of the gene encoding the extracellular domains CSFV E2 and CD154 pigs and cloning plasmid pMOS-2his-CD154

The portion of a gene encoding the extracellular domain of E2 of 363 amino acids, amplified by reverse transcription and polymerase chain reaction (RT-PCR) viral genome Cuban variant of CSFV, strain, "Margarita", the access number AJ704817 in the database of the National Center for Biotechnology Information (National Center for Biotechnology Information, NCBI). 3'-oligonucleotide includes a sequence encoding a "tail" of 6 histidine residues, to enable easy purification of the antigen.

The coding sequence for the extracellular domain of CD154 pigs of 210 amino acids get by chemical synthesis using as reference gene CD154 pigs Sus scrofa (NCBI access number AB040443). In the 5'-end coding sequence of this molecule include a section that encodes the peptide of the four repeating units of Gly-Gly-Gly-Gly-Ser (4G4S). When subclavian in plasmid pMOS-BLUE (Amersham, USA) synthesized sequence (4G4S-CD154) is inserted immediately after the "tail" of the 6 histidines in the coding sequence 2his. Get plasmid pMOS 2Ehis-CD154.

Example 2: Genetic construction molecules E2his and E2his-CD154 with secretion signals for mammalian cell

The sequence corresponding to E2his and the obtained RT-PCR, inserted by site Bgl II EcoR V plasmid pAEC-SPT (Herrera et al. (2000) Biochem. Biophys. Res. Commun. 279:548-551). Thus the floor of the up vector pE2his-sec, containing the coding sequence for E2his, which is preceded by a signal secretion of tissue plasminogen activator person (htPA) and which is under the transcriptional control pretannage promoter of human cytomegalovirus (PCMV).

The sequence corresponding to E2his-CD154 and subcloned in the vector pMOS-BLUE insert on the sites for the restriction endonucleases Bgl II-Sal I in plasmid pAEC-SPT. Thereby receive vector pE2his D154-sec containing the coding sequence for E2his-D154, which is preceded by a signal secretion htPA and which is under the transcriptional control of the PCMV.

Example 3: the Formation of recombinant adenoviral vectors containing coding sequences for E2his and E2his-CD154 with secretion signals for mammalian cell

Replication defective adenoviral vectors (Ad-ΔE1, ΔE3) are obtained as described in the instructions to the AdEasy system (AdEasytm-Vector System Quantum Biotechnology, EE.UU). Plasmid pAdTrack-CMV used as a carry vector. The coding sequence for E2his signal secretion htPA (E2his-sec) cut out from the plasmid E2his-sec by enzymatic digestion with restriction endonucleases Nco I and EcoR V and insert by restriction site EcoR V in the vector pAdTrack. Receive recombinant pAdT-E2his-sec with secretively option E2his under the transcriptional control of the PCMV.

Encoding posledovatelnostj secreted E2his-CD154 cut out from the plasmid E2his-CD154-sec by enzymatic digestion with restriction endonucleases Nco I and Sal I and inserted into the restriction sites Kpn I-Xho I in the vector pAdTrack. Receive recombinant pAdT-E2hisCD154-sec with E2his-CD154sec under the transcriptional control of the PCMV.

Carrying adenovirus vectors pAdT-E2his-sec and pAdT-E2hisCD154-sec linearized by enzymatic digestion with the restriction enzyme Pme I to formation of recombinant adenoviral genomes.

Each of the linear vectors introduced by electroporation with a vector pAdEASY-1 in Escherichia coli strain BJ5183. Recombinant genomes vector pAd-E2his-sec and pAd-E2hisCD154-sec obtained by homologues recombination. One of them contains the coding sequence for molecules E2his-sec, and the other contains the coding sequence for molecules E2his-CD154-sec. In both cases, they remain under the transcriptional control of the PCMV.

Recombinant adenoviral genomes then digested with the endonuclease Pac I and transferout in complementary cell line SOME A and get infectious virions. Formed two adenoviral vector Ad-E2his-sec and Ad-E2his-CD154-sec. Vectors independently multiply in the cell line SOME A to achieve a titer of 1×1012colony forming units/ml (CFU/ml) and purified twice by centrifugation in CsCl gradient. The vectors then they are removed by dialysis against buffer storage (10 mm Tris, pH 8.0, 2 mm MgCl2, 4% sucrose) and stored at -70°C. the Ability of adenoviral vectors Ad-E2his-sec and Ad-E2hisCD154-sec to transform mammalian cells and to mediate e is cpressey and secretion of molecules E2his and E2his-CD154 in the cultural medium was confirmed by tests on the infectivity of the cells of the pig line RC. Samples of proteins present in the environment of the cultivation of infected cells, were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate in non conditions and analyzed by Western blot turns with monoclonal antibodies against CSFV E2 (αE2-1G6) (figure 1 and 2).

Analysis of molecular mass glycoproteins E2his and E2his-CD154 confirmed that they were consistent with dimeric and trimeric isoforms. On track 1 figure 2 shows two bands corresponding to dimeric (180 kDa) and the trimeric isoform E2-CD154 (270 kDa).

Example 4: In situ transduction of the epithelium of the mammary gland of the goat to get E2his and E2his-CD154 in milk

For the transformation of the epithelium of the mammary gland expression cassettes E2his and E2his-CD154 used recombinant adenoviral vector Ad-E2his-sec and Ad-E2hisCD154-sec. In both cases, the vectors are introduced into the mammary gland of goats producing milk, direct infusion into the udder through the channel of the nipple. Adenoviral vectors infect secretory epithelial cells that form the epithelium of the breast, allowing the expression of recombinant proteins.

Are goats on the second month of lactation, with a capacity of about 1 liter per day. For injection of adenoviral vectors females first are milking to remove milk from the udder, then isoosmotic saline solution vlive is carried out in tanks directly through the channel of the nipple with a soft massaging the udder to ensure complete washing of the breast. The salt solution is removed by thorough milking udder, and the process repeated twice. Subsequently, introduced adenoviral inoculate with a titer of 109CFU/ml in saline solution containing 36 mm EGTA. The volume flow of the drug varies depending on the capacity of the udder and guaranteed fills. After infusion of the udder massage to facilitate homogeneous distribution of inoculum in the gland and achievements of the secretory epithelial cells of the alveoli. After 24 hours adenoviral inoculum is removed by milking. In order to remove residual adenoviral vectors from the tank or duct mammary glands washed again by infusion of saline solution.

Twenty-four hours after this by manually milking begin to collect milk from the transformed animals. Daily produce two milking with a 12-hour intervals. Collected milk maintained at -70°C. the Kinetics of expression of recombinant proteins E2his and E2his-CD154 in milk analyze for each nativeimage sample (figure 3 and 4). It is proved that the molecular size of the recombinant proteins correspond to dimeric and trimeric isoforms. For E2his was obtained, the average expression of 1.03 g/l 2-8 days after inoculation, with an average output with 5.22 g of one animal. For recombinant molecules E2his-CD154 was obtained, the average expression of 0.73 g/l, with the front output 3.04 from g from one animal.

Example 5: identification of antigens E2his and E2his-CD154 from the milk of goats

Samples from each day milking containing recombinant vaccine antigens E2his and E2his-CD154, respectively, mixed and centrifuged at 15000g for 30 minutes at 4°C. the Soluble phase (milk serum) sephirot and fat phase drop. The collected serum diluted with buffer to separate milk (10 mm Tris-HCl, 10 mm l2, pH 8.0) at a ratio of 1:4. The mixture is cooled on ice for 30 minutes and centrifuged at 15000g for 30 minutes at 4°C. Supernatant and precipitation analyze SDS-electrophoresis in SDS page and Western blot turns. It was determined that the greatest number of such recombinant proteins present in the soluble phase, and precipitation contain casein.

Fractions of the serum containing the interest of recombinant antigens (E2his and E2his-CD154), lighten sequential filtration through a membrane with pores of 0.8 μm and 0.4 μm (Millipore) and then put on HC column (Amersham, USA)filled with Ni-NTA-agarose resin (Qiagen, USA). It is washed in two stages, first with 100 mm phosphate buffer with 20 mm imidazole, pH 7,2, then 100 mm phosphate, 50 mm imidazole, pH of 7.2. After washing of interest elute protein with 100 mm phosphate buffer with 200 mm imidazole, pH of 7.2. The peak corresponding to the pure fractions, translated by dialysis in 10 mm phosphate buffer, pH 7,2 (figure 5).

The procedure of isdi E2his and E2his-CD154 goat milk is the same for both vaccine antigens. Two proteins to be obtained with a purity level exceeding 90%. Output E2his is 70%, for E2his-CD154 the resulting output is 58%. Purified proteins analyze SDS-electrophoresis in SDS page and Western blot turns in order to detect the formation of protein aggregates. Can be determined that the dimeric isoforms (homodimer) E2his obtained in milk, now recognized by polyclonal serum from pigs infected with CSFV, which indicates that this specific conformation increases molecular antigenicity (6).

Example 6. Construction of expression vectors in the methylotrophic yeast Pichia pastoris

Vector pPS10 for expression in P. pastoris digested by the restriction enzyme Nae I to insert interested in the coding sequence at the 3'-end signal secretion of invertase sucrose Saccharomyces cerevisiae (Suc2). Encoding the E2 sequence, amplified by PCR, inserted at the restriction site Nae I in plasmid pPS10. The sequence encoding E2his-CD154, cut out from the plasmid pMOS E2his-CD154 by enzymatic digestion with restriction endonucleases Sma I-EcoR V and insert by restriction site Nae I in plasmid pPS10. Thus, receive plasmids pPS-E2his and pPS-E2his-CD154. Coding sequences for both molecules are connected with a signal secretion Suc2 from S. cerevisiae and remain under the transcriptional control of the promoter of the enzyme and the alcohol oxidase (AOX1) of the yeast P. pastoris.

Recombinant plasmids linearized the restriction enzyme Pvu II and inserted by electroporation in electrocompetent cells of P. pastoris, strain MP36. So, get several clones of P. pastoris, strain MP36, stably transformed with plasmids pPS-E2his and pPS-E2his-CD154. This strain is a mutant, auxotrophic for histidine, therefore, the recombinant yeast acquire His+phenotype that allows its auxotrophic selection.

Recombinant yeast, first identified in the dot-blotting, analyze also a blot on the Southern to determine the nature of integration, which occurs when replacing the AOX1 gene of P. pastoris with getting Muts-His phenotype (low concentration of methanol). Genetic replacement of AOX1 occurs by recombination between the 5'-region of AOX1 promoter and 3'-AOX1 region, present in the genome of the yeast, and others, present in the plasmid, resulting in the destruction of the site, the coding gene AOX1. Recombinant yeast with Mutsphenotype support the production of the enzyme alcohol oxidase with AOX2 gene, but their growth rate in methanol is low. The integration of the Mut phenotype+-His can also be obtained by the replacement.

Sequence encoding options E2his and E2his-CD154, remain under the regulatory control of the AOX1 promoter, induced by methanol. P. pastoris secretes proteins in low to the ncentratio, and the conditions for its cultivation does not require additional proteins, so it can be expected that the secretory heterologous protein is a large part of the total protein in the medium (up to more than 80%). Production of recombinant protein is carried out in a 5-liter fermenters. The induction of expression carried out by adding methanol to the culture for 5 days, and recombinant proteins get into the culture medium in the fermentation. E2his secreted into the environment of the cultivation of recombinant yeast-level 0,143 mg/ml In the case E2his-CD154 obtained the level of 0.122 mg/ml

Example 7: identification of antigens E2his and E2his-CD154 from the environment of the cultivation of Pichia pastoris

The product of fermentation centrifuged at 10000g for 30 minutes at 4°C to separate the biomass from the liquid phase. The cultural medium was filtered through a membrane with pores of 0.8 μm and 0.2 μm (Millipore) and applied to XK16 column (Amersham, USA)filled with Ni-NTA agarose matrix (Qiagen, USA). Lavage is carried out with 100 mm phosphate buffer with 30 mm imidazole, pH of 7.2, and a protein of interest elute 100 mm phosphate buffer with 200 mm imidazole, pH of 7.2. Pure faction dialysis transferred in 10 mm phosphate buffer. Allotment E2his and E2-CD154 from the supernatant of the fermentation of genetically transformed P. pastoris is the same for both vaccine antigens. Two proteins were obtained with a purity level of 95%. Output E2hi was 83%, if E2his-CD154 the yield was 78%.

Example 8: Test of immunity in pigs vaccinated secretively option E2his

This test uses twenty-four healthy pigs weighing about 20 kg, with negative against CSFV serology owned not vaccinated and free from CSF to the flock. Pigs are placed into groups of 8 animals each, and placed in three separate experimental rooms (a, b and C) with water and food in an unlimited amount.

Animals from group a and group b are subjected to immunization vaccine composition containing an antigen E2his, in the form of a single dose of 30 μg (group a) and 50 μg (group b) to the animal group are subjected to immunization With placebo. The antigen is prepared as an emulsion of water in oil, and 2 ml intramuscularly in the neck. Placebo consists of adjuvant and phosphate salt solution in the ratio 1:1 (volume/volume) and injected in the same conditions. On the third week after immunization to control the infection all animals 105DL50the homologous strain of CSFV, "Margarita", by intramuscular injection.

The introduction of the drug vaccines E2his does not cause adverse reactions, judging by the fact that there were changes in normal clinical settings. Titers of neutralizing antibodies exceeding 1:50 (considered immunity) get vaccinated groups from the second week immunis the tion. After the third week credits increase to 1:1600-1:6400 (Fig.7), but between the vaccinated groups (a and b) differences in the immune response is not observed. In vaccinated pigs do not develop pyrexia or clinical symptoms, and lymphocytes are not viruses after control of infection days. However, in the placebo group developed all clinical symptoms, including fever, hemorrhage and negniy encephalitis. In this group of viral isolates are obtained from the fourth day after control of infection until the day of slaughter. Here it is demonstrated that weaned pigs vaccinated with drug E2his taken in a dose of 30 µg according to the proposed scheme of vaccination remain protected from clinical symptoms and infection with CSFV.

Example 9: a Test of hereditary immunity in pregnant sows vaccinated secretively antigen E2his

Take ten sows, serologically negative relative to CSFV, free from CSFV and no vaccination history (within the previous 3 years) herd. After weaning from the breast induce estrous cycle hormonal treatment, and in three days all sows are fertilized. Fertilization takes place simultaneously with immunization. Take a group of 5 sows and injected intramuscular them what nycla in the neck with 2 ml of the vaccine, referred to in example 7 (group b). The remaining 5 sows taken as negative control and injected 2 ml of placebo composed of adjuvant and saline in a ratio of 1:1 (volume/volume). After 21 days, the vaccinated group revaccinated. Pregnant sows examined by measuring the clinical triad (temperature, heart rate and breathing rate) and weekly blood sampling for Hematology and detection of neutralizing antibodies against CSFV. Two months later produce control infection in pregnant sows 105DL50the homologous strain of CSFV "Margarita" by intramuscular injection. At 3 and 5 days after control of infection produce virus isolation from peripheral blood lymphocytes to detect the presence of CSFV. Two weeks after infection control sows score and retrieve the results for the morphological and pathological analysis and test for virus isolation. In the course of the experiment, sows have unlimited access to feed and water.

The vaccine proved to be safe for all pregnant sows, cases of miscarriage or clinical disorders after immunization was not. Vaccinated animals developed specific neutralizing antibodies against CSFV with titers ranging from 1:50 to 1:51200. Sows from the HAC is ionirovanii group remained completely healthy after control of infection. None of these animals showed no fever, leukopenia, thrombocytosis, or any other clinical symptom of CSF.

Morphometric and postmortem analysis can determine that the fruits of vaccinated sows have normal size, and they are not present histopathological abnormalities. CSFV was not isolated from leukocytes in blood samples of females, taken after control of infection, either from the blood or organs of goals fruit.

Sows in the placebo group after control of infection had a fever and leukopenia. One of the sows had suffered a miscarriage on day 8 after control of infection and was filled on day 9 after control of infection. Pathological characteristics such as small size and moumifizirovania, enlargement of the spleen, multiple petechial hemorrhages in the kidneys and bladder and negniy encephalitis was observed in fetuses from sows slaughtered at 2 week after control of infection, and the aborted fetuses. CSFV was isolated from blood and from all organs from fetuses of this group. Vaccination of pigs with the preparation of the vaccine E2his prevented the transmission of CSFV from sows the seed.

Example 10: Testing early immunity in pigs vaccinated with the preparation of the vaccine E2his-CD154

Take four groups of 6 pigs each (in the same in the circumstances, in example 8) and apply to them the composition of the vaccine with the following amounts of antigen: 50 µg E2his-CD154 (group D), 80 µg E2his-CD154 (group E), 50 µg E2his (group F). The group G is taken as a placebo. Antigens prepared in the form of emulsion, water-in-oil" and 2 ml vaccinated by intramuscular injection, the placebo group vaccinated with adjuvant without proteins. Vaccines used as single dose. On day 8 after immunization conduct control infected animals 105DL50the CSFV strain "Margarita" by intramuscular injection. Clinical signs recorded daily during the experiment and weekly take blood for hematological analysis and determination of neutralizing antibodies. In addition, blood samples taken at 1, 3, 5 and 7 days post-vaccination, is used to assess the cellular immune response in lymphocyte proliferation test and "antiviral activity of serum".

After vaccination was observed in normal clinical signs, and was not observed adverse reactions at the vaccination site. It was detected an increased number of lymphocytes in cultures of lymphocytes from animals vaccinated with the antigen E2-CD154 (groups D and E) and increased activity of mitogen phytohemagglutinin in the test lymphocyte proliferation. This response was blocked by monoclonal antibodies against domain of CD4, which indicated which indicates that that immune response was mediated by T-helper lymphocytes. Lymphocytes from samples from animals of groups F and G (placebo) did not meet this test to mitogen stimulation or CSFV (Fig).

3, 5 and 7 days post-vaccination antigen E2-CD154 in samples from groups D and E had a high titer of interferon alpha. However, during the experiment interferon was not detected in animals vaccinated with the antigen E2his (group F) and in the placebo group (G). The test antiviral activity against virus transmissible gastroenteritis performed on cells RK-15. In groups D and E was obtained the title of antiviral activity 1:512; however, anti-virus protection was not detected in samples from pigs immunized E2his, or the placebo group (Fig.9). These experiments determined that the antigen E2 associated with the CD154 molecule enhances the cellular immune response to CSFV, which relates to immunostimulatory activity CD154.

Example 11: a Comparison of the kinetics of neutralizing antibodies in pigs vaccinated with a single dose of vaccine compositions containing E2his and E2his-CD154

Take three groups of 6 pigs, weighing approximately 20 kg, serologically negative against CSFV, from the flock, in which the previous 3 years no cases CoES or vaccination history. Animals provide water and daily food in neogenic nom volume.

Each animal Vaccinium 50 mcg E2his-CD154 in group H, 50 µg E2his in group I and group J is taken as a placebo. Antigens prepared in the form of emulsion, water-in-oil" and 2 ml vaccinated by intramuscular injection, the placebo group vaccinated with adjuvant without proteins. Enter a single dose, and within 5 weeks after immunization measure the level of neutralizing antibodies using a test based on the neutralization of hydrogen peroxide (NPLA).

Neutralizing antibodies are detected starting from the second week of immunization, titers exceeding 1:50 (considered as the immune system) in the groups vaccinated with E2-CD154 and E2his (H and I). During the tests was not detected antibodies in animals of group a placebo. In the second week after immunization titers of neutralizing antibodies in animals from group N (E2 antigen-CD154) were higher than the titers in the group immunized with the antigen E2his. These results suggest a higher stimulation of the humoral response in animals of group N (figure 10).

It was concluded that the composition of the vaccine E2his-CD154 at a dose level of 50 mg is safe, immunogenic and induces early humoral response in vaccinated pigs compared with the composition of the vaccine E2his.

Example 12: a Test of hereditary immunity in pregnant sows vaccinated with the vaccine composition E2his-CD154

Otber who have ten sows of the same health status and origin, that were used in example 8. After weaning from the breast hormonal treatment to induce estrous cycle, and in three days all sows are fertilized. At the same time a group of 5 pigs subjected to immunization 2 ml of vaccine composition E2his-CD154 (80 μg/animal; the composition used in example 10 for group E), using intramuscular immunization behind the ear, on the neck. A group of 5 remaining pigs subjected to immunization adjuvant as a placebo.

Pregnant sows examined by measuring the clinical triad (temperature, heart rate and respiratory rate), weekly take blood for Hematology and detection of neutralizing antibodies against CSFV. On the 2nd month of pregnancy to control the infection of animals 105DL50of CSFV strain "Margarita". The presence of virus testing in blood collected at 3 and 5 days after control of infection. Two weeks sows were slaughtered, and the fruit extract for Virology, morphological and pathological analysis. During the experiment, the animals have unrestricted access to feed and water.

After immunization was not observed cases of miscarriages or other clinical manifestations of CSF. Thus, the composition of the vaccine E2his-CD154 after a single immunization was harmless to pregnant animals. Vaccinated animals viraat the Wali of specific neutralizing antibodies against CSFV in titers from 1:50 to 1:16000.

After control of infection in the group of vaccinated sows were observed temperature increase, radiation or thrombocytosis. In this group the fruits had a normal size, and there was no histopathological changes, judging by the results of morphometry and postmortem analysis. CSFV was not detected in white blood cells or in blood samples taken from vaccinated mares after control of infection, either in the body or the blood of their fruits.

Sows in the placebo group had a fever, leukopenia, and loss of appetite after control of infection. The fruits of this group was small and showed histopathological changes compatible with CoES, such as enlargement of the spleen, petechial haemorrhages in the kidneys and bladder, bowel necrosis, multiple hemorrhages in internal organs and negniy encephalitis. CSFV was isolated from all organs and blood of fetuses of this group. Vaccination of pregnant sows drug vaccine E2his-CD154 conducted on a tested scheme, prevented the transmission of CSFV from sows the seed.

Example 13: Genetic construction pL-E2his and pL-E2his-CD154 lentiviral transfer plasmids

The sequence corresponding to E2his received by way of real-time PCR was subclinically in areas Bd1 II-EcoR V plasmids pAEC-SPT (Herrera et a. (2000) Biochem. Byophys Res. Commun. 279:548-551) to obtain the vector E2his-sec. Then this vector was digested with restriction enzymes Nco I-Sma I with a selection gene segment E2, which is preceded by a signal secretion htPA. The resulting strip was processed fragment of polymerase I maple to get the blunt end of the 5' ledge formed by the endonuclease Nco I. the Extracted strip embedded in the site EcoR V vector pBSM-αSIc under the transcriptional control of αSl-casein promoter Buffalo (αSlcP)specific to the mammary glands. Received plasmid pBSM-αSlc-E2his.

To create a vector pL-E2his from pLenti6/V5-GW/lacZ (ViraPowerTM Lentiviral Expression System) remove the cassette, the expression of lacZ by splitting Cla I and Kpn I, and in this place (vector pL) built a synthetic DNA fragment containing the Central polypurine area, flanked by two multicontinuum sites. In the website of Wamn I in the direction 3' poliprinolov plot (vector pLW) was additionally built postregulatory element forest Groundhog (woodchuck post-regulatory element, WPRE). Finally, the fragment containing the promoter αS1 and gene E2his, amplified by PCR from the vector pBSM-αS1c-E2his and cloned sites Sal I-Xho I between paliperidone land and the WPRE sequence, receiving vechor pL-E2his.

Synthesized sequence (4G4S-CD154) was built just behind the tail section, consisting of 6 histidines in the coding sequence of alnost E2his vector pL-E2his. Received vector pL-E2his-CD154 containing the coding sequence for E2his-CD154, which was preceded by a signal secretion htPA, and kohara was under the transcriptional control of a tissue-specific promoter αSlc.

Example 14: the Creation of recombinant lentiviruses vector Lv-E2his and Lv-E2his-CDl54 using transfection using polyethylenimine in cells T

Langewiese particles were obtained using cotransfection plasmid vectors (pL-E2his and pL-E2his-CD154, independently) and assistive devices for the packaging of lentiviral transfer plasmids (pLI, pL2, pLP-VSVG, ViraPower™ Lentiviral Expression System, Invitrogen-Life Technologies, USA) in cells T using polyethylenimine (RE) method. Viral supernatant was collected after incubation carried out for 48 hours at 37°C in 5% CO2. The purification steps included centrifugation (2000 rpm, 10 min, 4°C) and filtration (0.45 µm filters coated with PVDF (polyvinylidene fluoride)). The concentration antivirusnik particles was increased by ultracentrifugation (25000 rpm, 90 min, 4°C) supernatant. Viral particles in the pellets suspended in DMEM. The titration was carried out by PCR in real time, collecting about 108units transduction (T)/ml.

The ability of the lentiviral transfer vector-based Lv-E2his and Lv-E2his-CD154 to transformation of mammalian cells and the ability to oborudovatj the expression and secretion of molecules E2his E2his-CDl54 confirmed by the assessment of transduction in the line of epithelial cells of the mammary gland of goats (GMGE). These cells were sown with a density of about 2.5×105cells/cm2in DMEM, supplemented with 10% fetal bovine serum, epidermal growth factor (10 ng/ml) and insulin (10 μg/ml). Samples of proteins in the culture medium of infected cells, were separated by the method of SDS-electrophoresis in SDS page in nereguliruemyi conditions and were analyzed by Western blot analysis using Mab against CSFV E2 (αE2-lG6) (11).

Example 15: production of transgenic cows to get E2his and E2his-CDl54 in milk, and purification of antigens

Transgenic animals were obtained by subzonal injection of lentiviral transfer vector-based (Lv-E2his and Lv-E2his-CD154, independently) in perivitelline space in vitro Mature oocytes with subsequent in vitro fertilization, cultivation of embryos and embryo transfer to synchronized recipients. Bovine oocyte-Cumulus complexes were collected by aspiration of ovarian follicles obtained from slaughtered animals, and brought to maturation in vitro for 22 hours at a temperature of 39°C in 5% CO2. Injection of oocytes, Cumulus cells were completely removed by shaking in the presence of hyaluronidase before subsoiling viral injection and 18 hours in vitro fertilization. Embryos were cultured in modified synthetic oviduct fluid supplemented with 10% (v/v) of the estrous cow serum when the temperature 39°C in a humid atmosphere, consisting of 5% CO25% of O2and 90% N2.

As recipients used heifers with normal sexual cycle in age from 22 to 36 months during dioestrus. Seven days were transplanted blastocyte non-surgical way. Recipients were tested 28 days after the transfer of embryos by ultrasound in the presence of fertilized eggs and rectal polerowanie 42, 60 and 90 days of pregnancy. Heifers-founders (F0) were identified by analysis of genomic DNA derived from blood and tissue. Samples of milk of transgenic females received as a result of natural and induced (by hand or hormonal) lactation. Then from the milk of transgenic cows spent cleaning antigens E2his and E2his-CD154, using the same procedure as described in example 5. Received two proteins with a purity level of 90%.

Example 16: an Experiment for the protection of pigs orally inoculated with recombinant adenoviruses Ad-E2his-sec and Ad-E2hisCD154-sec

The study used twenty-four healthy pigs weighing about 20 kg, with negative serology for CSFV, which belonged to the herd unvaccinated and free from CSF of animals. Pigs were divided into groups of 8 animals each and were kept in three separate experimental rooms (K, L and M) with free access to food and water.

Animals of group was Communitylevel antigen Ad-E2his-sec, group L were immunized with antigen Ad-E2hisCD154-sec, and from group M were immunized with placebo. Each pig received a single oral dose of 2 ml containing the titer of the virus, equal to 5x106plaque-forming units (p.f.u.)/ml under the same conditions was given a placebo consisting of a solution of phosphate. Were administered a single dose and measured the levels of neutralizing antibodies in serum using analysis of neutralization of bound peroxidase (NPLA) within 5 weeks after immunization. Three weeks after immunization, all animals were provoked by intramuscular injection of strain "Margarita" homologous CSFV 105DL50dose.

Inoculation of recombinant adenovirus did not provoke a negative reaction, because it was not observed any changes in normal clinical settings. Titers of neutralizing antibodies exceeding 1/50 (considered to be protective), was observed in the vaccinated groups after the second immunization week. After the third week titers increased to 1/800-1/2500 (table 1). Animals in the placebo group during the experiment was not registered with any of the antibodies. In the second week after immunization titers of neutralizing antibodies in animals of group L (AdE2-CD154) were higher titers of groups immunized AdE2his. These results confirm a higher level of stimulation of humoral resp is that in animals of group L. The immunized pigs did not develop fever or clinical signs of disease and were not received viral isolates from lymphocytes in the following days after provocation. However, in the placebo group appeared all clinical symptoms, including fever, haemorrhage and negniy encephalitis. Animals in this group died one week after the provocation, and blood samples were unsuitable for testing NPLA.

Table 1
Titers of neutralizing antibodies in pigs after immunization with recombinant adenoviruses through the mucous membranes.
Weeks/GMT*GroupGroup LGroup M
120030025
250070010
3 (provocation)800120012
4 16002000NT
518002500NT
GMT*: mean geometric titer
NT: not tested

1. Chimeric vaccine antigen against classical swine fever virus (CSFV), characterized by: a) extracellular segment of glycoprotein E2 viral envelope CSFV at the N end of chimeric antigen; and (b) the extracellular domain of the molecule CD 154 pigs, identified as SEQ ID NO: 2, With the end of the chimeric antigen.

2. Chimeric vaccine antigen according to claim 1, in which the extracellular segment of the glycoprotein E2 of CSFV identified as SEQ ID NO: 1, which corresponds to the Cuban viral strain "Margarita".

3. Chimeric vaccine antigen according to claim 1, obtained by recombinant.

4. Chimeric vaccine antigen according to claim 1, obtained the source from the milk of genetically modified mammals.

5. Chimeric vaccine antigen according to claim 4, obtained the source from milk nereshennyh mammals through direct genetic transformation of the mammary gland.

6. Chimeric vaccine antigen according to claim 5, where a direct genetic Tr is spermatia breast cancer is performed using adenoviral vectors.

7. Chimeric vaccine antigen according to claim 4, obtained the source from the milk of transgenic mammals.

8. Chimeric vaccine antigen according to claim 3, obtained the source from genetically modified yeast.

9. Vaccine composition capable of inducing a protective immune response against CSFV, characterized by a content of chimeric antigens described in claims 1 to 8 and adjuvants or excipients.

10. Vaccine composition according to claim 9, which may be introduced animal system by or through the mucous membranes.



 

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3 dwg, 6 ex

FIELD: medicine, veterinary.

SUBSTANCE: claimed invention relates to field of veterinary and describes chimeric vaccine antigen against classical swine plague virus (CSPV), characterised by the fact that it consists of extracellular segment of glycoprotein E2 of CSPV viral envelop on N-end of chimeric antigen and extracellular domain of swine CD 154 molecule, identified as SEQ ID NO: 2, on C-end of chimeric antigen. Chimeric antigens can be obtained in expression systems, which guarantee proper folding of tertiary structure of chimeric molecules. Vaccine compositions, which contain such chimeric antigens, cause early and strong immune response in vaccinised swine and create complete protection against CSPV. Chimeric antigens like obtained compositions, can be applied in veterinary as vaccines for preventive application in swine.

EFFECT: obtained vaccine compositions prevent transmission of virus from sows to their offspring.

10 cl, 11 dwg, 1 tbl, 5 ex

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