RU2238105C1 - Recombinant vaccine for preventing viral hepatitis b (variants) - Google Patents

Abstract

FIELD: biotechnology, gene engineering.

SUBSTANCE: it is suggested to apply recombinant vaccines for preventing viral hepatitis B. Vaccines are elaborated based upon antigens of serotypes adw and ayw obtained out of Pichia angusta yeast strains VKPM Y-2412 and VKM Y-2924D. Recombinant vaccines for preventing viral hepatitis B contain efficient quantity of surface antigen of hepatitis B virus, an adjuvant and physiologically acceptable solvent. Vaccines could contain antigen of either out of one serotype or, at equal quantities, the mixture of serotypes adw and ayw. Vaccines obtained are being highly immunogenic, non toxic and posess no side effects.

EFFECT: higher efficiency.

6 cl, 10 ex

Description

The invention relates to biotechnology, namely to genetic engineering, and relates to recombinant vaccines for the prevention of viral hepatitis B containing in their composition as an active component HBs antigens of serotypes adw and / or ayw isolated from strains of the yeast Pichia angusta (Hansenula polymorpha) VKPM Y - 2412 and VKM Y-2924D.

A vaccine is known for immunization against hepatitis B, comprising a surface antigen of hepatitis B virus containing pre S2 in an effective amount and at least one of physiologically acceptable components selected from the group of carrier, diluent or adjuvant. The producer of the hepatitis B virus surface antigen is the S. cerevisia yeast strain CCC 0098 BP.

RU, Patent No. 2122430, 1995, C 12 N 15/51, A 61 K 39/29.

When implementing the known technical solution is not provided a sufficiently high level of synthesis of antigen, which is an active part of the resulting vaccine.

Known recombinant vaccine for the prevention of viral hepatitis B, containing an effective amount of the surface antigen of hepatitis B virus, obtained by cultivating the yeast strain S. cerevisiae VKPM Y-2203, adjuvant, preservative and a physiologically acceptable carrier.

RF patent No. 2088664, 1996, A 61 K 39/29

When implementing the invention in accordance with the specified patent when using a strain of yeast S. cerevisiae it is impossible to achieve high levels of synthesis of antigen. The methods described so far to achieve a high level of synthesis of HBsAg in yeast cells have suggested the introduction of a large number of copies of the gene encoding it into the cells. The problem is that some of the copies can get mutations, which, in turn, can change the properties of the resulting protein. At the same time, monitoring the occurrence of mutations against the background of a high copy number of the gene is almost impossible. Moreover, a very high level of synthesis of foreign RNA can lead to depletion of some tRNAs in the cell, as a result of which the corresponding codons are translated using other tRNAs. This leads to the appearance in the final product of an impurity of mutant forms of the polypeptide containing amino acid substitutions.

All of the above, in turn, may affect the quality of vaccines received.

The objective of the invention is to expand the arsenal of tools for the prevention of viral hepatitis B, by creating highly immunogenic, non-toxic, non-side-effect vaccines.

The problem was solved by introducing the serotypes ad and / or au serotypes obtained by culturing the Pichia angusta (Hansenula Polymorpha) VKPM Y-2412 and VKM Y-2924D yeast strains as the main active components of the surface antigens of hepatitis B virus.

The yeast producers used for the selection of antigens should possess the following properties: 1) the method for producing the producer should make it possible to control the absence of mutations in the expressed foreign gene; 2) the expression level should not be very high in order to increase the accuracy of translation of foreign mRNA, while the yield of the target protein should be high enough to ensure the economic feasibility of using producers.

According to the invention, one copy of the synthetic DNA sequence encoding HBsAg / ayw or HBsAg / adw is integrated into the genome of the Pichia angusta strain (Hansenula polymorpha). To increase the proportion of antigen in the fraction of soluble cellular proteins, a strain with an impaired MOX gene encoding alcohol oxidase is used. The absence of mutations that could have formed in the gene encoding HBsAg / ayw or HBsAg / adw during its introduction into the yeast genome or during cultivation of the producer strain can be detected by determining the nucleotide sequence of the product of the polymerase chain reaction with primers for this gene.

Yeast Pichia angusta (H. polymorpha) is preferred over S. cerevisiae because it allows higher levels of antigen synthesis to be achieved with simpler methods for producing high density cultures.

The strains of the yeast Pichia angusta (H. polymorpha), which are producers of the hepatitis B virus surface antigen of serotypes ad and ay and vaccines against hepatitis B, are new and are not described in the patent or in the scientific and technical literature.

The invention can be illustrated by the following examples. Moreover, all routine genetic engineering operations are carried out according to standard methods (maniatis) and instructions of companies producing enzymes and kits for manipulating DNA in vitro. For PCR, Vent polymerase (NEB) was used in all cases. Synthesis of oligonucleotides was carried out by Syntol CJSC in Moscow. The following oligonucleotides were used to obtain the ayw serotype antigen:

In accordance with the changes made to the classification of yeast, N. Polymorpha is also designated as Pichia angusta.

Example 1. Obtaining a DNA fragment encoding HBsAg / ayw (fragment A). The reaction mixture containing 1 μM oligonucleotides lu and 8L, oligonucleotides 2u to 8u; 1L to 7L to 10 pM, ThermoPol buffer (10 mM KCI, 20 mM tris-HCI pH 8.8, 10 mM (NH ₄ ) ₂ SO ₄ 2 mM MgSO ₄ , 0.1% Triton X-100, 250 uM dNTP) and 5 units of Vent polymerase activity are incubated for 30 cycles of temperature change. Each cycle includes incubation at 95 ° C for 30 sec, at 50 ° C 30 sec, at 72 ° C 90 sec. After this, the reaction products are separated electrophoretically in a 1% agarose gel, and a fragment of 730 base pairs is isolated. The correspondence of the selected fragment to the desired size is also determined by electrophoresis in 1% agarose gel, comparing its mobility with the mobility of the molecular weight marker bands.

Example 2. Obtaining a fragment of genomic DNA of Pichia angusta (H. polymorpha) containing the promoter of the MOX gene (fragment B).

2-3 μg of cells of the strain Pichia angusta (Hansenula polymorpha) BKM 4-2559 (obtained from soil CC4 38-22-2 Institute of Biochemistry and Physiology of Microorganisms RAS) are suspended in 0.1 ml of a 0.25% sodium dodecyl sulfate solution and incubated in boiling water bath for 5 minutes Cells are pelleted by centrifugation at 10,000 g for 5 minutes. 1 μl of the obtained supernatant is added to 25 μl of a mixture containing 1 μM moxU5 and moxL3 oligonucleotides, ThermoPol buffer (10 mM KCI, 20 mM tris-HCI pH 8.8, 10 mM (NH ₄ ) ₂ SO ₄ , 2 mM MgSO ₄ , 0, 1% Triton X-100, 250 uM dNTP) and 5 units of Vent polymerase activity. The reaction mixture is incubated for 30 cycles of temperature change. Each cycle includes incubation at 95 ° C for 30 sec, at 50 ° C 30 sec, at 72 ° C 90 sec. After this, the reaction products are separated electrophoretically in a 1% agarose gel and a fragment of 850 base pairs is isolated. The correspondence of the selected fragment to the desired size is also determined by electrophoresis in 1% agarose gel, comparing its mobility with the mobility of the molecular weight marker bands.

Example 3. Obtaining a fragment of genomic DNA of Pichia angusta (H. polymorpha) containing a portion of the TRP 3 gene (fragment B).

Suspend 2-3 μg of cells of the strain Pichia angusta (H. polymorpha) VKM 4-2559 in 0.1 ml of a 0.25% solution of sodium dodecyl sulfate and incubate in a boiling water bath for 5 minutes. Cells are pelleted by centrifugation at 10,000 g for 5 minutes. 1 μl of the obtained supernatant is added to a mixture of oligonucleotides trpU5 and trpL3, 1 μM each, ThermoPol buffer (10 mM KCI, 20 mM tris-HCI pH 8.8, 10 mM (NH ₄ ) ₂ SO ₄ , 2 mM MgSO ₄ , 0.1% Triton X-100, 250 uM dNTP) and 5 units of Vent polymerase activity. The reaction mixture is incubated for 30 cycles of temperature change. Each cycle includes incubation at 95 ° C for 30 sec, at 50 ° C 30 sec, at 72 ° C 120 sec, at 50 ° C 30 sec, at 72 ° C 120 sec. After this, the reaction products are separated electrophoretically in a 1% agarose gel and a fragment of 1360 base pairs is isolated. The correspondence of the selected fragment to the desired size is also determined by electrophoresis in 1% agarose gel, comparing its mobility with the mobility of the molecular weight marker bands.

Example 4. Obtaining a DNA fragment of Pichia angusta (H. polymorpha) containing the sequence encoding HBsAg / ayw, fused with the MOX promoter and the TRP 3 gene (fragment D).

The reaction mixture (0.05 ml) containing 1 μM moxU5 and trpL3 oligonucleotides, 1 pg fragments A, B and C, ThermoPol buffer (10 mM KCI, 20 mM tris-HCI pH 8.8, 10 mM (NH ₄ ) ₂ SO ₄ , 2 mM MgSO ₄ 0.1% Triton X-100, 250 uM dNTP) and 5 units of Vent polymerase activity are incubated for 30 cycles of temperature change. Each cycle includes incubation at 95 ° C for 30 seconds, at 50 ° C for 30 seconds, at 72 ° C for 3 minutes. After this, the reaction products are separated electrophoretically in a 1% agarose gel and a fragment of 2.9 thousand base pairs is isolated. The correspondence of the selected fragment to the desired size is also determined by electrophoresis in 1% agarose gel, comparing its mobility with the mobility of the molecular weight marker bands.

Example 5. Integration of the sequence encoding HBsAg / ayw in the genome of Pichia angusta (H. polymorpha).

The strain Pichia angusta (H. polymorpha) DLT (Agaphonov et al., 2002; a collection of strains of the laboratory of molecular genetics of ILV PK MH RF) was grown for 15-20 hours in YNB-D medium containing tryptophan 20 mg / l at 37 ° C in conditions of intensive aeration. 0.5 ml of culture is added to 30 ml of the same medium and incubated under the same conditions for 4 hours. Cells were harvested by centrifugation at 3000 g for 5 min and suspended in 15 ml of T buffer (10 mM tris-HCI pH 7.4; 100 mM CH ₃ COOLi; 0.5 mM EDTA). After incubation for 30 min at 30 ° C, the cells are again pelleted and suspended in 200 μl of buffer T. To 0.05 ml of suspension add 1 μg of fragment G, mix, add 120 μl of 70% PEG 4000 solution, mix again and incubate for 1 hour at 30 ° C.

The transformation mixture is incubated at 45 ° C for 15 minutes, cooled to room temperature and plated on Petri dishes with medium containing 50 mg / ml leucine. The grown colonies of transformants are tested for their ability to grow on a medium containing methanol as the sole carbon source. Clones that are unable to grow on such a medium are selected and tested for their ability to synthesize HBsAg / ayw. The resulting strain of Pichia angusta yeast (H. polymorpha) is a producer of hepatitis B virus surface antigen (HBsAg / ayw). The strain has a registration number VKM Y-2924D, 02/25/2003 and deposited at the Institute of Biochemistry and Physiology of Microorganisms RAS named after G.K.Skryabin.

Example 6. Analysis of the ability of the strain to synthesize HBsAg / ayw. The test strain is seeded in YPD medium (1% yeast extract, 2% peptone and 2% glucose) and incubated at 37 ° C for 15-20 hours under high aeration conditions. The resulting culture was diluted five times with YPM medium (1% yeast extract, 2% peptone and 2% methanol) and incubated under the same conditions for 30-50 hours. Cells from 0.5 ml of culture are collected by centrifugation, suspended in 0.2 ml of distilled water, 0.2 ml of 0.2 M NaOH are added and incubated in a boiling water bath for 8 minutes. The suspension is centrifuged for 10 min at 10,000 g, after which the supernatant is analyzed by electrophoresis and immunoblotting using antibodies to HBsAg / ayw. As a standard, purified HBsAg / ayw protein is used. In the paths corresponding to the strains that synthesize HBsAg / ayw, the band at 24-27 kDa is the same as in the case of purified protein.

Example 7. Determination of the sequence of the HBsAg / ayw gene in the yeast genome.

2-3 μg of the cells of the analyzed strain are suspended in 0.1 ml of a 0.25% sodium dodecyl sulfate solution and incubated in a boiling water bath for 5 minutes. Cells are pelleted by centrifugation at 10,000 g for 5 minutes. 1 μl of the obtained supernatant is added to a mixture containing 1 μM MOX and TRP oligonucleotides, ThermoPol buffer (10 mM KCI, 20 mM tris-HCI pH 8.8, 10 mM (NH ₄ ) ₂ SO ₄ , 2 mM MgSO ₄ , 0.1 % Triton X-100, 250 uM dNTP) and 5 units of Vent polymerase activity. The reaction mixture is incubated for 30 cycles of temperature change. Each cycle includes incubation at 95 ° C for 30 seconds, at 50 ° C for 30 seconds, at 72 ° C for 2 minutes. After this, the reaction products are separated electrophoretically in a 1% agarose gel and a fragment of 800 base pairs is isolated. The fragment is sequenced using primers MOX and TRP. The coincidence of the sequencing results with the original sequence shows the absence of mutations in the HBsAg / ayw gene integrated into the yeast genome.

Example 8. Isolation of HBsAg (ayw) from Pichia angusta (H. polymorpha) cells.

After fermentation of the original strain of yeast Pichia angusta (H. polymorpha) HBsAg is isolated according to the standard method according to the following scheme. Cells from the culture fluid are pelleted by centrifugation at 4000 g in a Beckman centrifuge. The precipitated biomass is resuspended to a concentration of 250 g of wet cells per liter in PBS extraction buffer pH 6.8 with the addition of EDTA, PMSF and Tween-20. Next, the cells are destroyed in a Dyno-Mill mill of the KDL type using glass balls with a diameter of 0.5-0.7 mm. Most of the non-target proteins from the resulting homogenizate are precipitated by the addition of polyethylene glycol (PEG) to a concentration of 4.5%. The precipitated proteins are separated by centrifugation. The resulting supernatant was filtered on Pellicon ultrafilters (500 kDa). In this case, proteins are separated from the pier. weighing less than 500 kiloDaltons, PEG is also removed and the HBs antigen solution is transferred to PBS buffer pH 6.7. At the same time, a concentrated solution occurs, which is essential for the next stage of absorption onto the glass. Planting of HBsAg on macroporous glass (MPS) is carried out in columns of the StreamLine system developed by Pharmacia (Sweden). To remove non-specifically bound proteins, the column is washed with the same PBS buffer. The antigen is eluted with carbonate buffer (KB) pH 9.5. The eluate is concentrated by ultrafiltration and separated by ultracentrifugation on a zonal rotor (Beckman) in a glycerol / sucrose gradient. The obtained fractions were analyzed for HBsAg content using a ROPHA kit and the fractions containing antigen were combined and purified by gel filtration on a Toyopearl HW-65 sorbent (ToyoSoda Corp., Japan). The purity of the thus obtained HBs antigen is determined by SDS-PAGE. If the purity of the product is insufficient (<95%), additional purification is carried out using ion exchange chromatography on a TSK-DEAE sorbent (ToyoSoda Corp., Japan). The output of antigen 25-30 mg with 1 liter of culture fluid.

The resulting HBsAg (ayw) solution was analyzed by a number of methods.

1. Antigenic activity is measured by ROPGA sets (reverse passive hemagglutination test of red blood cells) according to the manufacturer’s methodology (NPO Diagnostic Systems, Nizhny Novgorod) using the industry standard HBsAg activity standard (OSO 42-28-153-88) manufactured by GISK them. L.A. Tarasevich.

2. The purity of recombinant HBsAg is determined by polyacrylamide gel electrophoresis under reducing conditions (SDS-PAGE) stained with silver and Coomassie Brilliant Blue R-250. The purity of the isolated antigen is not <95%.

3. The immunospecificity of the antigen is determined by immunoblotting. Primary antibodies for this purpose were obtained by immunizing rabbits with recombinant HBsAg (manufactured by Combiotech) reconstituted in the presence of 2% SDS and 5% mercaptoethanol. Immunoblot was stained with immune antisera, followed by visualization with specific antibodies to rabbit immunoglobulins conjugated to horseradish peroxidase according to the improved chemiluminescence (ECL) technique (Amersham, UK). Recombinant HBs antigen (ayw) is presented as a 24-27 kDa monomer band and weak dimer and trimer bands.

Obtaining a yeast strain Pichia angusta (H. polymorpha) VKPM Y - 2412, (deposited at the State Research Institute of Genetics. - producer of hepatitis B virus surface antigen (HBsAg / adw) is carried out similarly to examples 1-5 with the only difference that the following oligonucleotides are used in the work :

Isolation of HBsAg / adw is carried out analogously to example 8. Analysis of the isolated HBsAg / adw is carried out by the methods described for the HBsAg / ayw antigen.

Example 9. Analysis of the ability of the strain to synthesize HBsAg / adw. The test strain is seeded in YPD medium (1% yeast extract, 2% peptone and 2% glucose) and incubated at 37 ° C for 15-20 hours under high aeration conditions. The resulting culture was diluted five times with YPM medium (1% yeast extract, 2% peptone and 2% methanol) and incubated under the same conditions for 30-50 hours. Cells from 0.5 ml of culture are collected by centrifugation, suspended in 0.2 ml of distilled water, 0.2 ml of 0.2 M NaOH are added and incubated for 3-4 minutes. The cells are then precipitated, resuspended in a sample buffer and incubated in a boiling water bath for 8 minutes. The suspension is centrifuged for 10 min at 10,000 g, after which the supernatant is analyzed by immunoblotting using antibodies to HBsAg / adw. As a standard, purified HBsAg / adw protein is used.

Example 10. Sequence determination of the HBsAg / adw gene in the yeast genome.

2-3 μg of the cells of the analyzed strain are suspended in 0.1 ml of a 0.25% sodium dodecyl sulfate solution and incubated in a boiling water bath for 5 minutes. Cells are pelleted by centrifugation at 10,000 g for 5 minutes. 1 μl of the obtained supernatant is added to a mixture of 1 μM MOX and TRP oligonucleotides, ThermoPol buffer (10 mM KCI, 20 mM tris-HCI pH 8.8, 10 mM (NH ₄ ) ₂ SO ₄ , 2 mM MgSO ₄ , 0.1% Triton X-100, 250 uM dNTP) and 5 units of Vent polymerase activity. The reaction mixture is incubated for 30 cycles of temperature change. Each cycle includes incubation at 95 ° C for 30 seconds, at 50 ° C for 30 seconds, at 72 ° C for 2 minutes. After this, the reaction products are separated electrophoretically in a 1% agarose gel and a fragment of 800 base pairs is isolated. The fragment is sequenced using primers MOX and TRP and determine the absence of mutations by comparing the results with the original sequence.

The resulting strains have the species names Pichia angusta (Hansenula polymorpha), the names of the strains VKPM Y - 2412 (HBsAg / adw) and VKM Y-2924D (HBsAg / ayw).

Cultural and morphological features of the strains:

rounded cells, small in size;

on agar medium YPD form large round colonies with a pronounced convex middle.

The activity of the strains is not less than 30 mg / l of culture fluid.

Storage at - 70 ° C in the form of a suspension of cells in a sterile (30-50)% glycerol solution.

Genetic features:

auxotroph on leucine, no phages in yeast.

Strains are not zoopathogenic or phytopathogenic.

The method for determining the activity: in the clarified cell homogenizate by enzyme immunoassay.

The method, conditions and composition of the media for the propagation of strains:

incubation by pumping at 30 ° C in a nutrient medium composition: 2% peptone, 1% yeast extract, 2% glucose.

Conditions and composition of the fermentation medium: pumping at 29 ± 1 ° С and pH 5.0-5.5 in a medium containing up to 10-15% glycerol and salt.

Isolated hepatitis B virus antigens of serotypes ayw and adw are used to create hepatitis B vaccines based on them.

In this case, vaccines were obtained in several versions:

1) the composition contains the ayw serotype antigen and there is no preservative;

2) the composition contains an adw serotype antigen and there is no preservative;

3) the composition contains a mixture of antigens of two serotypes ayw and adw in equal proportions and there is no preservative;

4) received variants of compositions, in the composition of which antigens of various serotypes or their mixture may be present with the additional inclusion of preservative - merthiolate in the compositions.

Specific examples of compositions derived from isolated antigens.

1. The composition for the prevention of viral hepatitis B contains:

20 μg of HBsAg / ayw obtained by culturing a strain of the yeast Pichia angusta (H. Polymorpha) VKM Y-2924D,

0.5 mg of aluminum hydroxide gel (in terms of Al ³⁺ ),

up to 1 ml of phosphate-buffered saline (FSB),

represented by 50 mM Na - phosphate buffer, pH 6.8 and 0.13 M NaCl.

2. The composition for the prevention of viral hepatitis B contains:

15 μg HBsAg / adw obtained by culturing a yeast strain

Pichia angusta (H. Polymorpha) VKPM Y - 2412,

0.3 mg of aluminum hydroxide gel (in terms of Al ³⁺ ),

up to 1 ml - FSB (50 mM Na - phosphate buffer, pH 6.8 and 0.13 M NaCl).

3. The composition for the prevention of viral hepatitis B contains:

10 μg HBsAg / ayw obtained by culturing a yeast strain

Pichia angusta (H. Polymorpha) VKM Y-2924D,

0.5 mg of aluminum hydroxide gel (in terms of Al ³⁺ ),

up to 1 ml - FSB (50 mM Na - phosphate buffer pH 6.8 and 0.13 M NaCl).

4. The composition for the prevention of viral hepatitis B contains:

25 μg of HBsAg / ayw obtained by culturing a strain of the yeast Pichia angusta (H. Polymorpha) VKM Y-2924D,

0.6 mg of aluminum hydroxide gel (in terms of Al ³⁺ ),

70 μg of merthiolate, up to 1 ml of FSB (50 mM Na - phosphate buffer, pH 6.8 and 0.13 M NaCl).

5. The composition for the prevention of viral hepatitis B contains:

8 μg of HBsAg / adw obtained by culturing a strain of the yeast Pichia angusta (H. polymorpha) VKPM Y - 2412,

8 μg HBsAg / ayw obtained by culturing a yeast strain

Pichia angusta (H. polymorpha) VKM Y-2924D,

35 mcg thiol,

0.5 mg of aluminum hydroxide gel (in terms of Al ³⁺ ), up to 1 ml of FSB (50 mM Na - phosphate buffer, pH 6.8 and 0.13 M NaCl)

According to the invention, compositions based on the isolated antigens can be obtained both with the introduction of the preservative merthiolate and without it.

The absence of a preservative in the formulations does not affect the quality of the resulting vaccines.

It was established that the absence of a chemical reagent in vaccines makes the compositions more preferable for administration to the children's contingent, reducing the risk of individual allergic reactions.

A vaccine dose of 0.5 ml is prepared for children. The content of ingredients in the finished vaccines can vary within the following limits (in 1 ml of product):

HBsAg 20 ± 5 mcg

Aluminum hydroxide 0.3-0.6 mg (in terms of Al ³⁺ )

Mertiolate 30-70 mcg

The content of HBsAg in the finished vaccines was determined by enzyme-linked immunosorbent assay using AUSZYME MONOCLONAL kits (Abbot Diagnostics, Illinois, USA).

The immunogenicity of the vaccines in the test on Balb / c mice weighing 12-14 g is 0.05 - 0.1 μg per ED 50 (the dose of the vaccine that causes seroconversion in 50% of mice).

It is known that medical personnel of medical institutions belong to the category of special risk of infection, for which viral hepatitis B is an occupational disease, and therefore specific immunoprophylaxis is a necessary measure.

According to the results of preliminary screening, markers of hepatitis B virus infection are observed in a significant part of hospital staff working with patients with hepatitis B: HBsAg - 7.4%; Anti-HBs - 22.6%; Anti-HBc - 37.8%.

In the hospital, for several years, 2872 employees were immunized, the vaccination of which with the vaccine obtained according to the invention was carried out according to the results of preliminary screening in the absence of serum hepatitis B virus infection markers. Immunization was carried out according to the known standard scheme. (0-1-6 months). The level of anti-HBs in blood serum after vaccination was determined individually by the method of quantitative enzyme-linked immunosorbent assay using Anti-HBsEIA kits, manufactured by Hoffman - La Roche Ltd (Switzerland) and an industry standard sample of anti-HBs hepatitis B virus antigen (anti-HBsAg ) (OCO-42-2815488).

According to the results of studies conducted after 3 months in the group vaccinated according to the standard scheme, the geometric mean titers amounted to 1783.5 ± 73.5 ME, i.e. marked high immunological efficacy.

The frequency of seroconversions ranged from 93.8 to 98.5%.

The research results also indicate that in the analysis of the post-vaccination response, the vast majority of the immunized had specific immunity levels of more than 500 IU / L - 75.5%.

As a result of the immunization, no further newly registered cases of the disease or infection with hepatitis B virus were detected in the staff who received the vaccination course.

Claims (6)

1. A recombinant vaccine for the prevention of viral hepatitis B containing an effective amount of a surface antigen of hepatitis B virus (HBsAg), an adjuvant and a physiologically acceptable diluent, characterized in that the recombinant vaccine contains HBsAg / adw obtained by culturing a yeast strain as an antigen of hepatitis B virus Pichia angusta VKPMY-2412. 2. Recombinant vaccine for the prevention of viral hepatitis B according to claim 1, characterized in that it additionally contains merthiolate. 3. Recombinant vaccine for the prevention of viral hepatitis B, containing an effective amount of a surface antigen of hepatitis B virus (HBsAg), an adjuvant and a physiologically acceptable diluent, characterized in that the recombinant vaccine contains HBsAg / ayw obtained by culturing a yeast strain as an antigen of hepatitis B virus Pichia angusta VKM Y-2924D. 4. Recombinant vaccine for the prevention of viral hepatitis B according to claim 3, characterized in that it additionally contains merthiolate. 5. Recombinant vaccine for the prevention of viral hepatitis B, containing an effective amount of the surface antigen of hepatitis B virus (HBsAg), an adjuvant and a physiologically acceptable diluent, characterized in that the recombinant vaccine contains in equal proportions a mixture of antigens HBsAg / adw as hepatitis B virus antigen obtained by cultivating a strain of the yeast Pichia angusta VKPM Y-2412, and HBsAg / ayw obtained by cultivating a strain of the yeast Pichia angusta VKM Y-2924D. 6. Recombinant vaccine for the prevention of viral hepatitis B according to claim 5, characterized in that it further contains merthiolate.