Method of obtaining peroral vaccine against rabies virus

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to field of biotechnology of veterinary medications. Vaccine against rabies virus represents allantoic fluid of chicken embryos. Fluid contains glycoprotein of rabies virus, as well as mixture if recombinant adenoviruses of birds, which carry gene of surface glycoprotein of rabies virus, one of which contains secreted form of surface glycoprotein of rabies virus, and second - membrane-bound one. Interaction of glycoproteins with animal organism is reached by peroral introduction of medication.

EFFECT: invention can be applied in veterinary.

3 dwg, 1 tbl, 6 ex

 

Rabies is an acute infectious disease of zoonotic origin. Cases of rabies in wild and domestic animals are regularly recorded in many countries, posing a serious threat to the population. In veterinary practice is widespread live and inactivated televisionnye vaccines vaccine strains of rabies virus (Rabies, 2nd edition. Alan .Jackson, William H.Wunner, 2007). Traditional vaccines against rabies derived from nervous tissue of infected animals. The use of such vaccines often leads to undesirable side effects (polyneuritis, encephalomyelitis), associated with the presence of impurities myelin in preparation (J. Postgrad Med. 1970 16(3): 132-4). Less reactogenicity have a vaccine based on the neural tissue of newborn animals (mice, lambs), which is associated with poor development of the myelin sheath in the nervous tissue of these animals. However, these vaccines are unsafe. The first vaccine against rabies, based not on nervous tissue vaccines were produced in duck embryos (Mil Med. 1964; 129:960-5). These vaccines were soon replaced by the modern culture vaccines based on diploid human cells, Vero cells (Dev Biol Stand. 1981; 50:173-82), cells of the kidneys of newborn hamsters (Appl Environ. 1973 Dec; 26(6):858-62), chicken embryo fibroblasts (Monogr Ser World Health Organ. 1966; 23:124-31). Despite effective is Yunosti use of live attenuated strains of rabies virus for vaccination, their use is associated with risk of release to the environment of a live infectious virus, because it does not exclude the possibility of reversion to a virulent form.

Currently, the widely used culture vaccine containing attenuated strains of rabies virus, inactivated various chemical agents (phenol, beta-propiolactone) or physical stress (UV radiation) (Jpn J Med Sci Biol. 1953; 6(6):577-86). However, the receipt of such vaccines is associated with the need to work directly with a live pathogen. In addition, the process of attenuative virulent strain with the purpose of reception of a preparation suitable for mass immunization, is a tedious and a chore, requiring the expenditure of a large amount of time and money.

Advances in the field of cloning and gene expression has led to the development of recombinant vaccines against rabies, which is easy to manufacture, stable in the environment and induce intense immunity. The use of recombinant virus eliminates the ingress of the external environment potentially dangerous genome vaccine rabies virus. The most studied and promising for use in the vaccination of rabies are recombinant vaccines based on cowpox virus (Vaccine. 2009 27; 27(51):7198-201) and adenoviruses (Virology. 2065-20; 356(1-2):147-54).

Recombinant viral vaccine against rabies on the basis of vaccinia virus expressing the glycoprotein of rabies virus used to immunize animals in the wild (J. Wildl. Dis., 1998, 34, 752-763). However, human contact with the cowpox virus may be accompanied by serious side effects that were shown during the mass vaccination of soldiers of the American army (Comp Immunol Environ Infect Dis, 2003, 26, 423-430).

Recombinant adenoviruses in comparison with other vector systems are highly efficient expression of the target transgene in different types of cells, the security of the vector to humans and animals, the accumulation of recombinant viruses in cells producing high titer, the induction of both humoral and cellular immune response to the transgene product, great packing capacity vector (Curr Top Environ Immunol. 2004, 273, 335-57). Recombinant adenoviruses as vaccine can be used for both parenteral and oral administration.

The known method of oral immunization of mice with recombinant replication-defective adenovirus 5 (adenovirus) and 68 serotype (chimpanzee adenovirus), bearing glycoprotein gene of rabies virus (U.S. Pat. 2004019603).

The disadvantages of the described method applies the high cost of the process poluchenierazreshenija for immunization of the preparation of recombinant adenovirus using cell lines of human rights.

The most secure among modern vaccines are split split vaccine containing virus particles - isolated surface and internal proteins. Manufactured vaccine by cleavage of viral particles using organic solvents or detergents, also viral components can be obtained in the laboratory using genetic engineering technology (J Immunol. 1996 15; 156(10):3579-82). Split vaccines are characterized by a significantly lower risk of adverse reactions, in connection with the removal of reactive lipids that make up the shell of the virus. Since the production of neutralizing antibodies against rabies is the surface glycoprotein, this structural component is used to create reiseplaner vaccines. The use of genetic engineering techniques to obtain isolated glycoprotein rabies virus, excluding work with the pathogen, increase safety received vaccines.

A method of obtaining the rabies vaccine, containing insulated surface glycoprotein of rabies virus, expressed in eukaryotic cells (US. Pat. 9411112). The coding DNA sequence of the glycoprotein of rabies virus is produced by reaction of reverse transcription on the matrix RNA virus or artificially synthesized, and then she can is to be embedded in commercial expressing vector. For expression of the glycoprotein of rabies virus in cells of S. cerevisiae developed commercial plasmid vector pYES2 (hivitrogen, San Diego, CA). Receiving glycoprotein of rabies virus in insect cells is provided baculovirus expression system (Virology. 1989; 173(2):390-9), for example, a commercial set Mahvash (bivitrogen, Sail Diego, CA). To obtain the glycoprotein of rabies virus in mammalian cells (cells of the Chinese hamster ovary) can be used plasmid vector pcDNA I bivitrogen, San Diego, CA). High level expression of the gene of interest is provided by the presence of promoter sequences, enhancers, polyadenylation signal. Thus, the receiving surface glycoprotein of rabies virus in prokaryotic and eukaryotic expression systems, however, the use of eukaryotic cells is preferred because they provide the necessary post-translational modification that leads to the formation of functionally active product.

Recombinant glycoprotein of rabies virus is administered orally to the animals as bait with attractive taste and smell in the amount of from 100 to 300 micrograms. Oral immunization as described glycoprotein of rabies virus, once in the body of animals associated with the epithelium of the oral cavity and pharynx, which then leads to the development of mucosal immunity.

The method described as the closest to the claimed chosen for the prototype.

However, the shortcomings of the prototype is rapid degradation in the body due to the action of proteolytic enzymes and cells of the immune system, the short half-life of a foreign protein in the body (approximately 2-3 hours), the need for ingestion of large doses of vaccines to achieve a positive effect; effective oral immunization with isolated viral protein requires the use of the adjuvant; the high cost of the process to obtain preparative amounts of the recombinant glycoprotein of rabies virus associated with the need for the cultivation of eukaryotic cells to obtain preparative amounts of recombinant protein.

The purpose of the proposed invention to provide a multicomponent recombinant vaccine accumulated in allantoine fluid of chicken embryos, a single oral immunization of animals against rabies virus.

The essence of the invention is the use of a mixture of recombinant glycoprotein of rabies virus and adenovirus birds CELO expressing membranosvazanny and secreterial forms of the glycoprotein of rabies virus accumulated in allantoine fluid of chicken embryos. Recombinationally birds CELO, expressing secreterial the form of the glycoprotein of rabies virus ensures the presence of this component directly in vaccine preparation, and recombinant adenovirus birds CELO expressing membranosvazanny glycoprotein of rabies virus allows efficient and prolonged expression of the target gene in the body of immunized animals, which leads to the formation of potent humoral and cytotoxic immune response against rabies.

As a carrier for the active ingredient of the vaccine and the adjuvant used recombinant adenovirus birds CELO. Vector CELO provides a high level of expression of a target gene in chicken embryo cells (Protein Expr Purif. 2009; 65(1): 100-7), which leads to the accumulation of recombinant glycoprotein of rabies virus in allantoine fluid in concentrations sufficient for immunization (up to 100 µg/ml). Vector CELO able to deliver reporter genes and genes of interest in mammalian in vivo and provides a prolonged expression (30 days), which provides an extension of the period of circulation of the antigen in the body (Mol Gen Mikrobiol Virusol. 2008; (4):26-30). This system viral gene delivery is safe for mammals, because the CELO virus is unable to replicate in cells of the data owners. The main advantage of adeno is yushnogo vector CELO is the ability to obtain preparative amounts of recombinant CELO virus in chicken embryos. The number of recombinant adenovirus CELO derived from one chicken embryo, is more than 1012viral particles. The titer of virus in allantoine fluid is 5×108PFU/ml Recombinant glycoprotein of rabies virus and adenovirus particles present in allantoine fluid, a relatively stable during the time required for immunization, therefore, an additional stabilization of the drug is not required.

To solve the problems associated with limited efficiency transduction of mammalian cells by vectors based on adenovirus birds, the developed approaches in the genetic modification process of Penton (fiber). These approaches include the construction of "chimeric" filerow (replacement globular domain with the appropriate domain of alternative serotypes of adenoviruses) or the introduction of various heterologous receptor-binding sequences on the C-end or HI-loop 3 globular domain of the fibre (J Virol., 2007 81(18); 9641-9652.). These modified vectors are able to provide efficient delivery of genetic information into mammalian cells normally resistant to infection by adenoviruses.

Through the ability to interact with receptors of innate immunity adenoviral vector can act as an adjuvant (J. irol., 2002, R-135).

Example 1

The rabies virus vaccine strain TC-80 propagated in cell cultures of kidney saiga. The titer of the resulting virus is determined in accordance with the recommendations of the who Laboratory techniques in rabies. 4thedition. WHO, Geneva, 1996). The titer of the virus is 108PFU/ml

The standard strain of rabies virus CVS-24 for infection with a lethal dose of the virus obtained from the brain tissue of infected newborn mice (J Exp Med. 1977 1; 145(6):1617-22).

Example 2

Gene glycoprotein G is obtained by amplification of cDNA synthesized by RT-PCR (Reverse Tpanscription System "Invitrogene" No. 12236-014, USA) on the matrix RNA isolated from rabies virus vaccine strain TC-80 using TRIZOL ("Invitrogene" No. 15596-018, USA). For PCR using oligonucleotides flanking the complete glycoprotein gene 5'-ggatccaggaaagatggttcctcaggctctcctgtttg and 5'-gctgcagcaaggggaggtgatcttcagacttggatcgt, selected according to the gene sequence of the glycoprotein of rabies virus (gene bank AB518487.1). The DNA fragment carrying the gene for glycoprotein G of rabies virus (sequence No. 1), clone into plasmid vector pGEM-T Easy Vector (Promega, A1360, USA). Gene glycoprotein G of rabies virus subcloning comprising expressing cassette consisting of the CMV promoter with enhancer and polyadenylation site BGH in vector pCBEdlRV containing a fragment of the genome of adenovirus CELO with a deletion of the not essential for virus replication about the Asti. Recombinant adenovirus CELO-glRb carrying the glycoprotein gene of rabies virus, produced by the method of homologous recombination in LMH cells. Soluble form of glycoprotein G of rabies virus (ΔglRb) is produced by site-specific mutagenesis using the oligonucleotides: 5'-aggagcatgcaaactcaag, 5'-ggtggcggccgctcaatgcac, making a substitution in amino acid sequence at position 417: Pro417 stop codon. Variant gene glycoprotein G of rabies virus with a deletion of the transmembrane domain (sequence No. 2) subcloning comprising expressing cassette consisting of the CMV promoter with enhancer and polyadenylation site BGH in vector pCBEdlRV containing a fragment of the genome of adenovirus CELO with a deletion of the not essential for virus replication region. Recombinant adenovirus CELO-ΔglRb carrying the glycoprotein gene of rabies virus with a deletion of the receive method of homologous recombination in LMH cells. Schematic representation of the genomic DNA of recombinant adenovirus birds CELO-glRb carrying the gene for glycoprotein G of rabies virus presented on figure 1A

Plasmid construction, carrying a full-sized genome of adenovirus birds CELO-HIRGD with modifications and structural proteins capsid (fiber) and expressing the cassette with the gene of the glycoprotein of rabies virus, produced by the method of homologous recombination in E. coli strain BJ5183 between dps is amidami pCELO-HIRGD, and Shuttle vector pCShCMV-glRb, bearing expressing cassette (CMV promoter, transgene, the polyadenylation signal), planbureau sequences CELO. Shuttle vector pCShCMV-glRb linearized the restriction enzyme AscI. Plasmid pCELO-HIRGD linearized by PacI. Shuttle vector with plasmid pCELO-HIRGD transformed into cells of E. coli strain BJ5183. The preparation of recombinant clones was confirmed by PCR, restriction mapping and sequencing. Schematic representation of the genomic DNA of recombinant adenovirus birds CELO-HIRGD-glRb, with modifications and structural proteins of the capsid, which carries the gene for glycoprotein G of rabies virus presented on figure 1B.

Example 3

Gene expression of glycoprotein G of rabies virus in chicken embryos infected with adenovirus CELO-ΔglRb, CELO-glRb, CELO-HIRGD-glRb, determined by the method of Western blot turns. Chicken embryos infect recombinant adenovirus CELO-ΔglRb, CELO-glRb, CELO-HIRGD-glRb. As a control use adenovirus CELO wild type. 72 hours after infection selected allantoin liquid which is analyzed by the method of Western blot turns. In the reaction using monoclonal antibodies to rabies virus strain TC-80. Specific interaction of complex antigen-antibody reveal individuum immunoperoxidase conjugate (Goat-antimouse IgG-HRP, Amersham, 1:5000). Coloring, make use of a Hemi is eminiscences of a detection system (ECL-plus Detection System, Amersham). The result of Western blot turns presented in figure 2.

The result of the analysis shows specific interaction of antibodies and recombinant protein corresponding to the electrophoretic mobility of the glycoprotein G of the virus.

Example 4

The obtained culture viral drug CELO-glRb infect chicken SPF embryos, at the rate of 100 μl of the culture of the drug on the embryo. At 72 h after infection selected allantoin liquid. Similarly receive recombinant adenovirus birds CELO-HIRGD-glRb and recombinant adenovirus birds, bearing comprising expressing the gene cassettes of glycoprotein G of rabies virus without transmembrane domain - CELO-ΔglRb. For immunization allantoine the recombinant CELO virus-glRb and CELO-HIRGD-glRb mixed in a 1:1 ratio with recombinant adenovirus birds CELO-ΔglRb. The resulting combination is used for oral immunization of mice in the amount of 1 ml per animal.

Example 5

The first group outbred mice orally once imposed a mixture of recombinant adenovirus CELO-glRb/CELO-ΔglRb as allantoinase of the drug. The second group is administered a mixture of recombinant adenovirus CELO-HIRGD-glRb/CELO-ΔglRb. A third group of mice twice subjected to immunization cultural inactivated vaccine, containing the th strain of rabies virus TC-80. As a negative control group of mice once injected adenovirus CELO wild type. 14 days after immunization mice intracerebral enter 100 BILLION50virus CVS-24. In the group of mice immunized once with recombinant adenovirus CELO-glRb and CELO-HIRGD-glRb, survived 91% and 90% of animals, respectively. When using standard culture inactivated vaccine only 82% of mice were protected from subsequent infection with a lethal dose of rabies virus strain CVS-24 (table).

The level of protection against rabies virus of mice immunized with recombinant adenoviruses birds CELO-glRb and CELO-HIRGD-glRb, with modifications and structural proteins of the capsid
The group of animalsThe number of animals infected with a lethal dose of rabies virusThe number of animals that died from rabies virusThe number of surviving animalsSurvival, %
Immunization vaccine strain of rabies virus TC-80112982
1111091
Immunization with a mixture of adenovirus CELO-HIRGD-glRb/CELO-ΔglRb101990
Immunization with adenovirus CELO wt (negative control)111100

Thus, it is shown that the maximum level of protection of mice from vnutricerepnogo infection with rabies virus strain CVS-24 - 91% - detected after a single immunization with recombinant adenoviruses birds CELO-glRb and CELO-HIRGD-glRb oral.

Example 6

Specific antibodies to rabies virus in the serum of mice immunized with recombinant vaccine CELO-glRb or CELO-HIRGD-glRb, determined by indirect enzyme immunoassay.

Sensitization panels conduct normal and specific antigens that contribute diluted in the working dilution in 0.01 M phosphate-buffered saline (FSB), pH 7,2-7,4 in odd and even rows, respectively. Panel incubated over night at 4°C.

Available adsorption sites of polistina is and blocking with 1% BSA, in the volume of 0.15 ml /well, inquira for 30 minutes at 37°C. After further washing plates FSB with tween-20 (FSS T) subjects serum contribute to the wells of the panels in a dilution of from 1:3 to 1:729 1% BSA in FSB. Plates are incubated for 40 minutes at 37°C, followed by three times washing in the FSB-T, make a working dilution (1:1000) antimisting peroxidase conjugate and incubated for 40 minutes at 37°C.

After that, the plate is washed six times the FSB-T and make a solution of a chromogenic substrate (ABTS). Records of the hold after 30 minutes incubation at room temperature.

Accounting reactions carried out photometrically at a wavelength of 405 nm. The reaction is considered positive and specific, if the optical density of the chromogenic substrate solution into the wells, which are pre-incubated test serum, and in the holes, which made a positive control serum, 2 or more times greater than the mean optical density of the substrate in the hole, which made the control (negative) normal serum.

The result of the titration of specific antibodies to the rabies virus in the serum of mice immunized with recombinant CELO virus-glRb presented on figure 5. The antibody titer in the serum of mice immunized with recombinant adenovirus CELO-glRb and CELO-HIRGD-glRb correlates with the titer of antibodies detectionin in the blood serum of mice after immunization culture vaccine TC-80. Recombinant CELO virus-glRb and CELO-HIRGD-glRb when administered to mice induce the synthesis of high levels of specific antibodies against the glycoprotein G of rabies virus.

Thus, the resulting vaccine containing a mixture of the glycoprotein of rabies virus, and recombinant adenoviruses birds, one of which (CELO-glRb) expresses secreterial the shape of the surface glycoprotein of rabies virus, and the second (CELO-glRb or CELO-HIRGD-glRb) - membranosvazanny, single oral administration which leads to effective protection against a lethal dose of rabies virus.

The method of obtaining oral vaccine against rabies virus in animals, including the production of recombinant glycoprotein of rabies virus for oral immunization, characterized in that get allantoin fluid of chicken embryos containing the recombinant glycoprotein of rabies virus, its producer - recombinant adenovirus birds CELO, as well as recombinant adenovirus birds CELO, which is the producer of membrane-associated glycoprotein of rabies virus, or recombinant adenovirus birds CELO, with modifications and structural proteins of the capsid and which is the producer of membrane-associated glycoprotein of rabies virus.



 

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