Immunogenic peptides of the virus of foot and mouth disease


C12N15/42 - Foot-and-mouth disease virus

 

The invention relates to biotechnology. Vaccine against FMD virus contains the active substance on the basis of peptides with the sequence of at least eight amino acids, the corresponding part of the sequence of unstructured region of the protein of FMDV, which was selected by immunoreactivity with specific virus antibodies or by immunoreactivity with specific virus T-lymphocytes. The vaccine has long term stability, effective and high security. 3 S. and 6 C.p. f-crystals.

The invention relates to immunogenic peptides of at least 8 amino acids, which are found in unstructured regions of FMDV.

Foot and mouth disease is an acute infectious disease that affects the most important animals producing milk and meat, cattle, pigs, goats and sheep.

The disease is caused by picornaviruses, the FMD virus. This refers to RNA virus with single-stranded RNA with a length of 8.5 So-called with the polarity of plus-strands, which can occur in different serotypes with a large number of subtypes. Animals that have recovered from infection with one serotype remain polnolunie pathways occurs in the throat. Then infected neighboring lymph nodes and the virus enters the blood. Through the blood, the virus spreads to various organs and tissues. Clinical symptoms come in 2-14 days after infection regardless of the dose of virus, the virus strain and route of infection. In a few severe cases, the infection is overcome through 14 days. The virus of foot and mouth disease occurs in older animals only in rare cases fatalities, but has a significant impact on their productivity, growth and good health. In addition, the recovered animals may excrete the virus of foot and mouth disease despite high titers of antibodies and thus infect other animals. The problem are also vaccinated animals that were exposed to infectious virus. These animals can also be infected without showing clinical symptoms. These animals, which, although healthy, but despite this are carriers of the virus, called carriers, and they pose a serious risk of further spread of FMD virus. Isolation of virus in pigs is possible up to one month after infection (Van Bekkum, 1973) [10] in the case of cattle it is even possible for eckert, 1990) [7], which include single-stranded RNA. The capsid nothing is covered and has an icosahedral shape. Proteins 1B-1D are partially on the surface, while protein 1A (RA) is in the middle of the capsid.

Encoded in the N-terminal part of the genome proteins 1A-1D are structured proteins and form an icosahedral capsid. Unstructured proteins 2A-2C and 3A encoded-end and are responsible for viral replication.

The FMD control is difficult because of the simple contagiousness of the virus, its ability to infect many species of animals and due to its multiple antigenic forms.

Vaccination against FMD was carried out in Germany until 1992 with trivalent killed vaccine for subtypes Of a, and C. These vaccines consisting of inactivated viruses, however, are thermally unstable and does not guarantee long lasting immunity (Terpstra and others, 1989) [9]. The danger that comes from vaccines is primarily in the presence of mainactivity virus killed vaccine and release of viruses from relevant sources producing vaccines (Beck and others, 1987) [1].

In the European Union (EU) restrictions on the trade in animals, to Renesse infection, but also to animals immunized with conventional killed vaccines.

For this reason, since carried out intensive research to develop better vaccines against FMD virus. Desirable would be to have the vaccine, which differ in the long-term sustainability, effective and high security. Would be useful, in addition, vaccines or methods, which allow to distinguish vaccinated from infected animals.

Three circumstances must be, in particular, taken into account in the development of vaccines with specific epitopes: 1. Polymorphism of proteins of the pathogen appears primarily in the segments of proteins involved in immune response. In particular, RNA viruses (quasimidi") contain areas with extremely high variability of sequences.

2. It is when the immune response of T-lymphocytes there is a high variability of individuals of the species of the host body. Cell T-helper learns, as a rule, one specific antigenic peptide only in connection with a particular molecule is a class II major histocompatibility complex (MHC) (Schwartz, 1985) [8]. Each individual expresses his own MHC molecules that are encoded by genes with high allelic, ikno different.

3. The fraction of T-lymphocytes show a very heterogeneous effector mechanisms, which, of course, as a rule, corallium with MHC-restriction (Mosmann and others , 1989). For FMD virus in cattle still could find only limited MHC-II functions of T helper (Glass and others, 1989) [3] ; (Glass and others, 1990) [4]; (Glass and others, 1992) [5]; (llen and others, 1991) [2].

To obtain peptide vaccines should be primarily known immunogenic sites of the pathogen, the so-called provisions of the pathogen, which are recognized by the immune system of the natural species of the host body, as well as In - or T-lymphocytes of cattle and pigs. About this till now there is no data.

Finally, it was found that it is possible to obtain a vaccine against the virus of foot and mouth disease on the basis of peptides with the sequence of at least 8 amino acids, the corresponding part of the sequence of unstructured region of the protein of FMDV, which were selected by immunoreactivity with specific virus antibodies or by immunoreactivity with specific virus T-lymphocytes.

Such peptides are preferably 8-35 amino acids, particularly preferably of 8-25 amino acids, in the highest degree tidy to match the parts of the regions on the genome of FMDV, which encode proteins L/L', 1A, 1B, 1C, 2B, 2C, 3A, 3B, 3C, 3D.

To obtain a vaccine against FMD virus for cattle should these peptides correspond to the parts of the regions on the genome of FMDV, which encode proteins 1D, 2B, 2C, 3A, 3B.

It is therefore particularly preferred peptides corresponding to parts of the regions on the genome of FMDV, which encode proteins 2A, 2B, 2C, 3A, 3B, 3C, 3D.

In particular, it is necessary to mention specified in the Protocol sequence peptides.

Especially it is necessary to specify presented in the Protocol sequences of the peptides represented by numbers 6, 8, 10, 12, 15, 16, 17, 18, 19, 20, 21, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 43, 44, 45, 48.

Special mention should be made, in addition, the peptides represented by numbers 12, 13, 14, 22, 33, 37, 40, 41, 42, 45, 46, 47.

In the highest degree important to note peptides marked with numbers 12, 37, 40, 42, 45, 47, 48.

Foods that contain these peptides, can be used for immunization to protect against the virus of foot and mouth disease and for the detection of infection with FMD virus, that is, for diagnostic purposes.

As already mentioned, the peptides according to the invention correspond to areas areas unstructured proteins of FMD virus. These areas define thanks to them and to the FMD virus to T-lymphocytes.

Under immunoreactivity in this regard, understand their ability to react with specific virus antibodies. Confirm the reaction is carried out in this case, the interaction is specific to FMD virus antibody with bound onto the solid phase peptides using enzyme immunoassay, which uses a color reaction.

Another opportunity to confirm the reactivity is evidence of competition binding specific to FMD virus antibody with recombinant viral proteins with the respective peptides.

Under the immunoreactivity mean the ability of the peptides to interact with lymphocytes, which were obtained from virus infected FMD animals vaccinated animals. These lymphocytes after coincubation with the corresponding peptides to show specific reactions: a) additional, depending on the concentration of peptide growth (specific to the antigen peptide proliferation); b) specific peptides additional production of specific substances (pitocin, such as interleukin-2); and differentiation to specific virus cytolytic T-lymphocytes that are able to find out the suitable is the following peptides on the surface.

Specific to FMD antibodies are antibodies that are formed in animals after vaccination or infection with FMD virus and are able to recognize certain patterns of FMD virus and contact with these structures. They can be detected ex vivo, in vitro using a specific virus enzyme immunoassay. Specific to FMD virus antibodies recognize either a virus, certain viral proteins or protein fragments in the form of peptides that are encoded specific to the virus sequences.

Specific to FMD T-lymphocytes can be obtained when mononuclear cells isolated from blood infected with FMD virus or vaccinated animals.

Below is an overview of the possible methods of obtaining the peptides according to the invention. These methods have only to explain the invention but not to limit it in any way.

To obtain mononuclear cells from the blood of pigs (mononuclear cells of peripheral blood, MCPC) of heparinized blood (0.1 mg of heparin in 1 ml of blood) was diluted in the ratio 1:2 phosphate buffered saline (SFR). Out at room temperature with 30 ml is transferred into a 15 ml ficoll-vipaka(1,077 Guti carefully selected by pipette from the interphase between serum and picollo. Underlined cells washed once SFR and twice 20 ml of culture medium for lymphocytes/ 10% amniotic calf serum in the tube with a volume of 50 ml and precipitated by centrifugation (10 min each time, 750 g).

Enrichment of T-lymphocytes using columns with nylon wool This method of enrichment of T-lymphocytes is based on the physical adhesion of b-lymphocytes and part of monocytes with nylon wool. For this purpose nylon wool three times, heated to boiling in distilled water to the mark 5 ml in loosely stoppered syringe with a volume of 10 ml and autoclave (120oC, 20 min). Before use, the column washed twice with 20 ml SPR. For speed control of plum skewer cannula with a diameter of 0.8 mm by subsequent washing with 10 ml of culture medium for lymphocytes wash fluid down to the beginning of the column and then the cannula is sealed with a rubber stopper. Each column is applied to 1108MCPC in 1 ml of medium, to which a rubber stopper for a short time delay to allow pass-containing cells of the liquid. Then gently skewer prevents the spray tube to prevent drying of the column and to avoid contamination when posledna nylon wool MCPC can be suirvey by washing the column impaled through the cannula 20 ml culture nutrient medium for lymphocytes.

Determination of immunoreactivity carried out in a known manner according to the methods described in the following sources:A. , Jonjic, S.,H.-J. Reddehase, M. J. & Koszinowski, U. H. (1987). Monoclonal antibodies reactive to lymphocytes pigs. II. Detection of antigen on sleeping T-lymphocytes with reduced regulation upon activation. J. Immunol. 138, 1852-1857.

A. & Maurer, S. (1994). Expressing porcine T-lymphocyte antigen class II major histocompatibility are active antigen-presenting cells in a mixed culture of cells. Immunobiol., 190, 23-34.

A., Hirt, W., Maurer, S. & Weiland, E. (1994). The difference between the two subpopulations pork cytolytic T-lymphocytes CD8+by the expression of CD5 antigen. Immunology, 81, 578-583.

Pauly, T., Elbers, K.,M., Lengsfeld, So,A. & Thiel, H.-J. (1995). Classic specific to swine fever virus cytolytic T-lymphocytes and identification of the epitope of T-lymphocytes. J. Gen. Virol., 76, 3039-3049.

Summerfleld, A. , Rziha, H.-J. &A. (1996). Functional characterization of porcine extrememusic T-lymphocytes CD4+CD8<). The limited differentiation between the major histocompatibility complex and not limited major histocompatibility complex pork cytolytic T-lymphocytes. Immunology, 88, 238-246.

In the following example describes the measurement is specific to the antigen of the virus proliferation (analysis of proliferation).

MCPC or allocated out of the cell population were sown in tablets titration with a round bottom when the number of cells105cells on the algae (200 μl/cell) at a cell concentration 1106/ml in minimum support-environment. Stimulation was carried out by adding virus or peptides from the coding regions of the genome of FMDV (specific activation). Data added quantities of the virus resulted in the quantities characterizing the multiplicity of infection (MOH), which corresponded to the number of infectious particles. Then the cells were cultured in the closet for incubation. After 5 days was added to each cell placed in a 20 µl environment3H-thymidine, 37 kBq (1 µci), and the culture incubated for a further 18 hours and Then stopped embedding3H-thymidine by freezing the entire plate for titles is sivali on the filtration mats. They were dried in a microwave oven (160 W, about 5 min). Then napravlyali solid scintillation tablet on the filter Mat in a microwave oven (160 W, about 2 min). After cooling scintillator filtration Mat was welded in a transparent case for trial and was measured in a scintillation counter radioactivity of individual crops in the number of disintegrations per minute (counts per minute).

Determination of interleukin-2 from the supernatant of cell culture specific to the virus antigen activated T-lymphocytes (analysis of interleukin-2) For semi-quantitative determination of the content of interleukin-2 (IL-2) in cultures of cells pigs used cell line HT-2 mouse-dependent IL-2. This cell line grows only in the presence of IL-2, the source of which can be human, mouse, and pig. The proliferation of the cell line HT-2 is, thus, a measure of the content of IL-2 in the supernatant of cell culture, which in turn correlates with the production of IL-2 corresponding cell population.

After activation MCPC or allocated out of cell populations were collected after 5 days of appropriate cells of the tablet for the titration of 100 μl containing clethodim speed /log2/ (supernatant 1:1, 1:2, 1:4 and 1:8 in the environment; in each case 100 μl/algae). Finally, add 100 ál of cell suspension with 4103cells HT-2 in the cell so that the final volume was 200 μl/cell. To measure cell proliferation in HT-2 in each case was placed triple the number of cultures. As a matter of comparison used human recombinant IL-2 with a certain number of international units (ME) and was titrated in a few steps. The growth of cells HT-2 was quantitatively assessed by determining DNA synthesis. To this was added after 24 h incubation3H-thymidine (37 kBq/algae) and then cells were incubated for another 18 h in the closet for incubation. Further, the method was similar to the one for measuring the proliferation of lymphocytes.

Measurement of cytolytic activity specific to the antigen of the virus cytolytic T lymphocytes Specific for the antigen of the virus cytolytic T-lymphocytes are formed by at least one-week cocultivation MCPC infected animal or allocated out of cell populations (2105cells/cell) with autologous infected by FMD virus (1-10 MOH) by the epithelial cells of the kidney. Specifiy analysis release51SG. In these analyses the CTLs has acultural from 4 to 8 h with either autologous labeled51SG infected with FMD virus by the epithelial cells of the kidney, or loaded with peptides by epithelial cells of the kidney and then the supernatant of the respective cell cultures was determined resulting from the activity of CTLs chrome. In the "idle" experience for this study used the uninfected cells of the epithelium of the kidney. The specific activity of CTLs calculated according to the following formula: % specific lysis = x - spontaneous lysis/total embedding - spontaneous lysis.

For further analysis of CTL epitopes were also used recombinant virus cowpox - FMD, and cowpox viruses are partial sequences of FMDV and expressed during infection.

Obtaining peptides was carried out in a known manner. For example, have conducted multiple peptide synthesis on a modified robotic device company Tecan.

To obtain Hexapeptide in the reaction vessels load in each case, 30 mg of resin containing 5-(4-aminomethyl-3',5'-dimethoxyphenoxy)valeric acid (loading of 0.4 mmole/g). For microscale other peptides to the original cm the values derived peptides could ask the Manager computing device synth and essential amino acids with 9-fluorenylmethoxycarbonyl (Fmoc) protection was loaded into the collection. The amino acid was dissolved in 0.5 M N-hydroxybenzotriazole (peso) in dimethylformamide (DMF) to a concentration of 0.5 M sparingly Soluble amino acids were treated for 5-10 min in an ultrasonic bath to obtain a clear solution. Necessary to enable 2 M solution dicyclohexylcarbodiimide (DCGK) was obtained using dichloromethane (DHM)/DMF (8:2). Piperidine for removal of Fmoc protection group was diluted to 40% in DMF, and together with the solution DCGK prepare in the synthesizer. Synthesis of peptides was carried out by simple combinations and conducted according to the following Protocol synthesis: 1. The removal of Fmoc-protection with 40% piperidine within 15 minutes

2. Six wash cycles 150 μl of DMF and 0.3 minutes

3. The addition to the reaction vessels 30 ml of reagent combinations (2 M DCGK in DMF).

4. The addition of 60 μl of activated Fmoc-amino acids (acids with Fmoc-protected dissolved in 0.5 M peso/DMF).

5. Keeping this solution for 60 min mix of amino acids.

6. Three cycles of washing in 150 ml of DMF and 0.6 minutes

At the end of the synthesis the resin twice washed with ethyl ether (200 ml) and dried.

For removal obtained at the microscale peptides used a modified reagent (0.75 g kristallicheskoe (1:1) in triperoxonane acid (TFUC). In this case, synthesized accrued links separated from the synthetic block and drain opening was filled with molten wax. Concentrated TFUK while slowly dissolved wax from the drain, and used for removal of the solution with the already derived from the resin peptide could sopivat only underneath the synthesized parts of the tube for purified peptides. In tubes for purified peptides can then be chipped off of the protective groups of the side chains. When this was added to each link 150 μl of acceptor/solution TFUK and incubated for 3 h at room temperature. In tubes with purified peptides were then added with 8-channel spray approximately 1 ml of sulphuric ether/heptane (1:1) and kept for 2 h at -20oC. the precipitate to centrifugation (2000 rpm, 5 min), ether decantation and the precipitate twice resuspendable with ethyl ether (1-2 ml) using ultrasound and re-centrifuged. At the end of the precipitate was dissolved in 1-1,5 ml of tert-butyl alcohol/water (4:1) and subjected to lyophilization.

The selection of sera and the determination of specific antibodies Obtaining sera of cattle and pigs Undiluted blood was incubated at room the bones serum was divided into equal portions and stored at -20oC.

Standard peptide-ELISA
Standard peptide-ELISA (ELISA=enzyme immunosorbent assay) for the detection of specific virus antibodies in the sera of infected or vaccinated animals was performed as described below.

Tablets for ELISA-test (Nunc-Measurement Plate, the firm Maxisorb) were coated with peptides at concentrations of 0.5, 1 and 3 mg in the cell. Peptides were first dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mg/ml From this solution was then prepared by main solution in distilled water with a concentration of 1 mg/ml Diluted in distilled water the main solution of the peptide in the amount of 100 μl were then dried over night at 37oC. After that, the tablet is pre-incubated for 2 h at 37oWith 3% serum albumin bovine (bovine serum albumin, BSA) in SFR to prevent non-specific binding in subsequent stages of incubation. Tablets after each stage of incubation, washed three times SFR-twin, before addition of substrate five times. And used the serum, and the conjugates were diluted in 5% BSA in SFR.

Serum of infected or vaccinated cattle or pigs were used in a concentration of 1:100. Each mesh is knitted with a horseradish peroxidase conjugate, or goat against cattle (dilution of 1: 2.500), or the goat prativasinau (dilution of 1:5.000). Then again incubated for 1 h at 37oC. After several washing steps for the detection of positive samples was added 60 μl of substrate/cell. Substrate served as dissolved nitrate buffer, orthophenylphenol. The reaction of the substrate with horseradish peroxidase as the color reaction was carried out at room temperature in the dark. The reaction was stopped after about 20 min with 2 M sulfuric acid, if the staining was used a positive control was sufficient. The colour intensity was measured in a measuring device for ELISA at 492 nm.

The Biotin-streptavidin ELISA
Because swine serum detect extremely high nonspecific reaction, investigated the possibility of increasing the sensitivity of the measurement system through a modified ELISA test. Was used for this biotinylated peptides.

These biotinylated peptides used in the same concentrations as the peptides in standard-peptide-ELISA. Instead of distilled water was diluted with SFR/0.5% BSA. This solution was made in the amount of 100 μl/cell on streptavidin coated tablets Le incubation for 1 h at room temperature, triple rinse buffer for washing and add 150 ál labeled with horseradish peroxidase goat against cattle or goat antisera prativasinau antisera in the cell (see breeding for the standard peptide-ELISA) were incubated 1 h at room temperature. Again washed three times and added 150 μl of the cell, Azino-di-3-ethylbenzthiazolinesulfonic - substrate solution. Measurement of extinction (optical density) was carried out each time after 15 min and after 1 h at 405 nm in a measuring device for ELISA.

ELISA for the study of competition
Previously carried out ELISA tests, standard-peptide-ELISA and biodistribution-ELISA are, as a rule, for the detection of linear epitopes of b-lymphocytes. Often, however, the corresponding molecules of immunoglobulins are well known linear and conformational epitopes. This kind of epitopes detected under certain circumstances in ELISA tests in the study of competition. For this tablets for ELISA-test (Nunc-Measurement Plate, Maxisorb) first cover overnight with 100 µl of protein solution in a suitable concentration, which sees a positive reaction in the standard-peptide-ELISA. The tablets were then preincubate in accordance with standard peptic tablet titration for, at least 1 h with 100 µg/ml intended for studies of peptides. Then the analysis was performed according to standard peptide-ELISA.

Results
Identification of linear epitopes of b-lymphocytes
For identification of linear epitopes In lymphocytes of FMDV in cattle and pigs peptides (14-Mer and 15-Mer), which were synthesized in accordance with outdoor readable star-shaped arrangement of the chromosomes of the genome of the virus of foot and mouth disease, were studied to determine recognizable whether they are antibodies sera of infected or vaccinated animals.

The study of synthetic peptides of FMD virus on linear epitopes of b-lymphocytes in pigs
Peptides marked with numbers 6, 8, 10, 12, 15, 16, 17, 18, 19, 20, 21, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 43, 44, 45 in the Protocol sequences identified as epitopes of b-lymphocytes pigs.

Identification of linear epitopes In lymphocytes of FMDV in cattle
Peptides marked with numbers 12, 13, 14, 22, 33, 37, 40, 41, 42, 45, 46, 47, 48 in the Protocol sequences identified as linear epitopes of b-lymphocytes of FMDV in cattle.

Identification of conformational epitopes of b-lymphocytes with protein 3D VII 8 peptides which are able to bind serum is specific to FMD virus antibody against the protein 3D. We are talking about the peptides represented by numbers 1, 2, 3, 4, 5, 7, 9, 11 in the Protocol sequence.

The use of linear epitopes of b-lymphocytes to recognize differences between infected by FMD virus and vaccinated animals
In this test investigated the serum infected with different serotypes of FMD virus and vaccinated animals. Served as control serum of uninfected animals and serum of animals that were infected with bovine leukemia (BL).

It was shown that the number 37 peptide from area 2B and number 48 from the field of 3V virus reacted positively with many serums infected by a virus or vaccinated animals. With sera of infected BLV animals or with negative sera, as a rule, the reaction was not observed.

Next, you may find that the serum of animals infected with strain O1K virus, reacted with the largest number of peptides compared to other study groups. Can also be determined from the difference between the positive type On and vaccinated animals. In V. 37, 48, and with the control peptide G1-32, serum of infected animals showed additionally a clear reactivity to the peptides represented by numbers 12, 13, 40, 42, 45, 47, 48.


Claims

1. Vaccine against FMD virus, containing one active substance and additives target, characterized in that it contains as active substance at least one peptide with an amino acid sequence corresponding to part of the sequence of unstructured region of the protein of FMDV, which was selected by immunoreactivity with specific virus antibodies or by immunoreactivity with specific virus T-lymphocytes, in an effective quantity, and the specified sequence selected from the group comprising SEQ ID 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 and 48.

2. Vaccine against FMD virus under item 1, characterized in that the peptides correspond to parts of the regions on the genome of FMDV, which encode proteins L/L', 2B, 2C, 3A, 3B, 3D.

3. The vaccine against the virus of foot and mouth disease on p. 2, characterized in that the peptides correspond to parts of the regions on the genome of the virus of foot and mouth disease, which is lnasty of the unstructured region of the protein of FMDV, who was selected by immunoreactivity with specific virus antibodies or by immunoreactivity with specific virus T-lymphocytes, and the specified sequence selected from the group comprising SEQ ID 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 and 48.

5. The peptide under item 4, characterized in that it corresponds to the parts of the regions on the genome of FMDV, which encode proteins L/L', 2B, 2C, 3A, 3B, 3D.

6. The peptide under item 5, characterized in that it corresponds to the parts of the regions on the genome of FMDV, which encode proteins 2B, 2C, 3A, 3B.

7. The DNA sequence encoding the peptides with amino acid sequence corresponding to part of the sequence of unstructured region of the protein of FMDV, which was selected by immunoreactivity with specific virus antibodies or by immunoreactivity with specific virus T-lymphocytes, and the amino acid sequence selected from the group comprising SEQ ID 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 and 48.

8. The DNA sequence under item 7, is distinguished by the virus, which encode proteins L/L', 2B, 2C, 3A, 3B, 3D.

9. The DNA sequence under item 8, characterized in that it encodes a peptide with the amino acid sequence corresponding to part of the regions on the genome of FMDV, which encode proteins 2B, 2C, 3A, 3B.

 

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FIELD: veterinary virology, biotechnology.

SUBSTANCE: the suggested vaccine contains avirulent and purified antigenic material out of strain A (Georgia) being homologous to infection agent 1999/N1721 obtained in passaged cell culture VNK-21 being the suspension that contains, predominantly, 146S and 75S immunogenic components of foot-and-mouth disease virus. Moreover, the vaccine contains maintenance medium and butyric adjuvant in efficient ratios. The strain has been deposited in collection of FGU VGNKI under registration number - industrial culture strain of foot-and-mouth disease virus A (Georgia) 1999/N1721-DEP of serotype A. As maintenance medium it is necessary to apply serum-free Earle's solution at addition of FGMS, GBCS and antibiotics at pH being 7.4-7.6. Out of butyric adjuvants the vaccine contains butyric adjuvant of the All-Russia Research Institute of Animal Protection (VNIIZZH) or butyric adjuvant of Montanide ISA-70 or Montanide ISA-260 marks by "Seppic" (France). The vaccine provides efficient protection against homologous infection agent circulating in Transcaucasian countries and those of Central Asia, Near and Middle East.

EFFECT: higher efficiency.

11 cl, 1 dwg, 5 ex, 8 tbl

FIELD: veterinary virology, biotechnology.

SUBSTANCE: the suggested vaccine contains avirulent and purified antigenic material out of strain A (Georgia) being homologous to infection agent 1999/N1721-DEP obtained in passaged cell culture VNK-21 being the suspension that contains, predominantly, 146S and 75S immunogenic components of foot-and-mouth disease virus, adjuvants aluminum hydroxide with saponin and maintenance medium in efficient ratios. The vaccine is of high immunogenicity and is capable to provide efficient protection against homologous infection agent circulating in Transcaucasian countries and those of Central Asia, Near and Middle East.

EFFECT: higher efficiency.

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FIELD: biotechnology, virology.

SUBSTANCE: claimed strain is obtained by passaging of epizootic isolate on sensitive hetero- and homologous cell cultures. Virus strain is reproduced in sensitive cell cultures and after incubation for 18-24 h 6.0-7.66 lg TCD50/ml is accumulated. In case of mass infection strain takes cytopathic effect over 5 hours, and retains starting characteristics for 10 passages.

EFFECT: strain of high biological, antigenic and immunogenic activity.

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FIELD: medicine; veterinary.

SUBSTANCE: method of vaccine obtaining involves virus antigen cultivation in BHK-21 suspension cell culture at the temperature of 36-37°C, purification of virus suspension from ballast admixtures, inactivation, concentration of foot-and-mouth disease virus antigen obtained and combination of antigen concentrate with adjuvant. After 6-8 hours of virus antigen cultivation, percentage of dead cells is registered every 2 hours. When the level of dead cells reaches 95% or more, further cultivation is performed for 2-8 hours depending on the virus type. Foot-and-mouth disease vaccine obtained by this method contains antigen material of a-type and/or O-type and/or Asia-1 type foot-and-mouth disease viruses in effective quantity of 146S-component, aluminium hydroxide gel, saponin and maintenance medium.

EFFECT: increased volume of antigen material during foot-and-mouth disease virus cultivation and obtaining harmless immunogenic vaccine.

13 cl, 1 tbl, 14 ex

FIELD: medicine; veterinary science.

SUBSTANCE: method involves preparation of antigen material from avian influenza virus strain thereafter cleaned from ballast impurity, virus inactivation, mixing of antigen material and oil adjuvant with controlling the end product. The avian influenza virus strain is represented with the strain "Primorsky" of avian influenza virus referred to Orthomyxoviridae family, Influenzae virus avicum type of serotype A, subtype H5N2, collection Federal State Institurion VGNKI, serial №129 - "ДЕП". The vaccine made under the given method, contains cleaned and avirulent antigen material from strain " Primorsky" and oil adjuvant in ratio (wt %): antigen material - 30.0-40.0, oil adjuvant - 60.0-70.0.

EFFECT: decreased labour and power inputs in making the vaccine and improved quality of antigen material, high antigen activity of the vaccine and effective protection of susceptible poultry from epizootic virus of subtype H5.

14 cl, 5 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: invention relates to veterinary virology and biotechnology, as well as the foot-and-mouth disease virus Aphtae epizooticae type A Picornaviridae, genus Aphtovirus, which is deposited in the Russian State Centre for Quality and Standardisation of Veterinary Drugs and Feed under registration number A No.2045/Kyrgyzstan/2007 - DEP. The disclosed strain is reproduced in a monolayer swine kidney cell culture, passaged Siberian mountain goat kidney cell cultures (PSGK-30), VHK-21 and IB-RS-2. After 18-24 hours of incubation, virus yield in said cell cultures reaches 6.0-7.5 Ig TCD50/cm3. For multiplicity of infection (1-10 TCD/cell), the strain causes CPD after 5 hours, while preserving the initial characteristics when passaging in cell cutures for 10 passages.

EFFECT: disclosed strain can be used to control antigenic and immunogenic performance of foot-and-mouth disease vaccines and to produce biopreparations for diagnosis and specific prevention of foot-and-mouth disease type A.

6 cl, 1 dwg, 10 tbl, 12 ex

FIELD: chemistry.

SUBSTANCE: polyepitope proteins consisting of two or more protein fragments of the foot-and-mouth disease virus, particularly serotype O/Taiwan/99, connected by linker amino acid sequences, are constructed. The vaccine polyepitope protein can be characterised by general formula VP4(X1 -X2)-GRL-VP1(X3-X4)-GRL-VP1 (X5-X6)GRL-2C(X7-X8)-GRL-3D(X9-X10)-GRL-3D(X11-X12), where GRL is a glycine-rich linker, Xn-Xm are integers denoting the number of amino acid residues of the corresponding foot-and-mouth disease virus protein, VP4, VP1, 2C, 3D are names of foot-and-mouth disease proteins. The invention discloses a nucleotide sequence (NS) which codes peptides included in the polyepitope protein, a recombinant plasmid which facilitates synthesis of a hybrid polyepitope protein in procaryote (E.coli) and eucaryote (plant) cells, and a solvent composition for a foot-and-mouth disease vaccine based on the polyepitope protein.

EFFECT: polyepitope protein has high immunising power and can be considered a potential recombinant vaccine against foot-and-mouth disease.

15 cl, 7 dwg, 2 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: invention refers to a polyepitope vaccine protein applicable for immunisation against murrain virus, nucleic acid coding this protein, recombinant plasmid pA7248-AMV-H-PE for production of said protein in plants and to a vaccine preparation for prevention and protection against infection caused by murrain virus. The polyepitope vaccine protein has the amino acid sequence SEQ ID NO: 1 which contains the amino acid sequence of B-cell epitope of VP4 protein from 21-th to 40-th amino acid starting with N-terminal, the amino acid sequence of B-cell epitope of VP1 protein from 135-th to 160-th amino acid starting with N-terminal, the amino acid sequence of B-cell epitope of VP1 protein from 200-th to 213-th amino acid starting with N-terminal, the amino acid sequence of T-cell epitope of 2C protein from 68-th to 76-th amino acid starting with N-terminal, the amino acid sequence of T-cell epitope of 3D protein from 1-st to 115-th amino acid starting from N-terminal, and the amino acid sequence of T-cell epitope of 3D protein from 421-st to 460-th amino acid of murrain virus serotype O/ Taiwan/99. Nucleic acid coding such protein has the nucleotide sequence SEQ ID NO: 2. Recombinant plasmid pA7248-AMV-H-PE has a physical map presented in dwg. 5.

EFFECT: invention provides creating polyepitope vaccine protein which is applicable for immunisation against murrain virus.

5 cl, 7 dwg, 2 tbl, 6 ex

FIELD: biotechnology.

SUBSTANCE: application of aethonium as the adjuvant for production of sorbed foot-mouth disease vaccine is proposed, where aethonium is used as a 10% aqueous solution which is introduced into the composition of foot-mouth disease vaccines in an amount of 750 mcg per 1 cm3 of the preparation.

EFFECT: invention extends the list of adjuvants for production of foot-mouth disease vaccines.

4 tbl, 3 ex

FIELD: biotechnology.

SUBSTANCE: method of production of vaccine against foot and mouth disease comprises culturing virus antigen in suspension culture of cells BHK-21 at a temperature of 36-37°C, purification of the viral suspension from ballast impurities, inactivation, concentration of the obtained foot and mouth disease virus antigen, and connection of the antigen concentrate with an adjuvant. Purification of the virus suspension from ballast impurities is carried out by adding derived polyguanidines at a final concentration of 0.005-0.01%, or the mixture of chloroform and derivatives of polyguanidines taken in weight ratios of (40-160):1, respectively, at a final concentration of 0.4-0.8. As derivatives of polyguanidines dihydrochloride 1,12 diguanidinohexane or dihydrochloride bis (3-guanidinopropyl) piperazine, or dihydrochloride 3,6-dioxaoctane-1,8-diguanidine, or dihydrochloride 4,9-dioxadodecane-1,2-dibiguanide.

EFFECT: improvement of purification level of virus suspension from ballast impurities and increase in immunogenicity of the target product.

2 cl, 1 tbl, 16 ex

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