RU2233672C1 - Mixed vaccine for immunoprophylaxis of viral hepatitis b and d, tetanus and diphtheria - Google Patents

Abstract

FIELD: biotechnology, medicine, vaccines.

SUBSTANCE: invention relates to a mixed vaccine comprising some antigens in a single preparation. New mixed vaccine comprises the following components: hepatitis B virus surface antigen (HBsAg/ayw) prepared by culturing the yeast strain Pichia angusta VKM Y-2924 D and/or hepatitis B virus surface antigen (HBsAg/adw) prepared by culturing the yeast strain H. polymorpha VKPM Y-2412 in the amount 5-10 mcg; diphtheria anatoxin, 5-10 Lf; tetanus anatoxin, 5-7.5 EC; aluminum hydroxide (Al³⁺) gel, 0.35-0.55 mg; merthiolate, 15-35 mcg, and 0.9% NaCl, up to 0.5 ml. Hepatitis B virus antigens (HBsAg/ayw) and (HBsAg/adw) are taken in vaccine in the ratio = 1:1. The novel mixed vaccine is high-immune, nontoxic preparation providing carry out immunization against pathogens of some diseases of viral and bacterial etiology. The application of invention provides expanding assortment of agents designated for simultaneous prophylaxis of viral hepatitis B and D, tetanus and diphtheria.

EFFECT: valuable medicinal properties of mixed vaccine.

2 cl, 10 ex

Description

The invention relates to biotechnology, namely to the development of new associated vaccines containing several antigens in a single preparation, which makes it possible to simultaneously immunize against several pathogens.

The development of vaccinology and vaccination has led to a significant increase in the number of vaccinations received by each individual.

Immediate vaccination, which is not accompanied by a suppression of the immune response to any of the antigens, or an increase in reactogenicity, allows not only to more successfully implement vaccinations on maternal dates, but also reduce the workload of medical workers. However, with simultaneous vaccination, unresolved issues such as the possibility of using three or more drugs, excessive inoculation of the sorbent and preservative if they are contained in the drug, and, finally, the inevitability of two injections. All these circumstances determine the need to develop and introduce new combination vaccines.

Known combination vaccine against diphtheria, tetanus and hepatitis B.

At a dose of 0.5 ml, the vaccine contains as active components:

D - toxoid 25 Lf (u);

T - toxoid 10 Lf (u)

HbsAg 10 mcg protein

Hepatitis B virus surface antigen is any known hepatitis B virus surface antigen antigens or fragments thereof exhibiting antigenicity of hepatitis B surface antigen (references are given in the patent description to known antigens).

All antigens, except hepatitis B surface antigen, are adsorbed onto aluminum hydroxide. Hepatitis B surface antigen is adsorbed onto aluminum phosphate. If necessary, use an antiseptic in the vaccine, for example, merthiolate, from 1: 20,000 to 1: 10,000 or 2-phenoxyethanol from 3 to 6 mg / cell.

The vaccine contains isotonic saline. RU, Patent No. 2160120, A 61 K 39/295, 39/29, publ. 2000 year

A combination vaccine is also known for the immunoprophylaxis of viral hepatitis B and D, tetanus and diphtheria.

The vaccine has the following composition:

Hepatitis virus antigen

B (HBsAg) obtained

culturing transformed

cell strain

S. cerevisiae VKPM Y-2203 5-10 μg HBsAg

Diphtheria toxoid

refined concentrated 5-30 Lf

Tetanus toxoid

purified concentrated 5-10 EC

Mertiolate 0.01 ± 0.002%

Aluminum gel

hydroxide 0.6-1.2 mg

in terms of aluminum

Phosphate buffered saline

50 mM Na-phosphate buffer,

pH 6.8-0.13 M NaCl) Up to 1 ml

RU, Patent No. 2130779, 1998, A 61 K 39/29.

The objective of the invention is to expand the arsenal of funds intended for the immunoprophylaxis of viral hepatitis B and D, tetanus and diphtheria.

The problem was solved by constructing a new highly immunogenic combination vaccine in which the hepatitis B virus antigen (HBsAg) contains a new hepatitis B virus antigen (HBsAg / adw) obtained by culturing the Hansenula Polymorpha VKPM Y-2412 yeast strain or hepatitis B virus antigen ( HBsAg / ayw) obtained by culturing a strain of the yeast Pichia angusta (Hansenula Polymorpha) VKM Y-2924D, or a mixture of these antigens in equal amounts (1: 1 ratio).

In connection with the introduction in 2003 of a new classification, Hansenula Polymorpha is now designated as Pichia angusta.

The amount of hepatitis B virus antigen or mixture of antigens in the combination vaccine is 5-10 μg HBsAg in 0.5 ml of the solution, which is the vaccine dose.

In addition, the vaccine contains:

Diphtheria toxoid 5-10 Lf

Tetanus toxoid 5-7.5 EU

Gel of aluminum hydroxide (Al ³⁺ ) 0.35-0.55 mg

Mertiolate (preservative) 15-35 mcg

0.9% NaCl Up to 0.5 ml

Unlike the known vaccine according to the invention, a new hepatitis B virus antigen of serotypes adw and / or ayw is introduced into the designed vaccine, the ratio of active ingredients is changed due to the revealed potentiating effect, and the amount of solvent is significantly reduced.

When developing a new vaccine, it was required to combine several antigens (one of them is new) in one injection drug so that a new combination of components taken in certain quantities does not reduce the safety and immunogenicity of the newly created immunobiological preparation, as well as its epidemiological effectiveness.

Studies have shown that the created drug has wide potential for use with high immunogenic activity, non-toxicity and the absence of any adverse reactions.

The new combined preparation is a surface antigen of hepatitis B virus associated with an aluminum hydroxide gel and a mixture of diphtheria and tetanus toxoids purified from ballast proteins, and it has a wider spectrum of action than previously known drugs.

It should be noted that the new combination drug is also effective in the immunoprophylaxis of viral hepatitis D, since the hepatitis D virus cannot participate in the development of hepatitis B infection without the simultaneous replication of hepatitis B virus, and it is possible to protect against delta infection by vaccination against viral hepatitis B as a result the formation of antibodies to HBsAg (anti HBs).

New yeast producers are used to isolate new hepatitis B virus antigens contained in the vaccine.

Yeast Pichia angusta (Hansenula 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 (Hansenula Polymorpha), which are producers of the hepatitis B virus surface antigen of the adw and ayw serotypes, are new and are not described in the patent or in the scientific literature.

A method of obtaining new producer strains and the selection of hepatitis B virus antigens of serotypes ayw and adw:

all routine genetic engineering operations are carried out in accordance with standard procedures and instructions of manufacturers of enzymes and in vitro DNA manipulation kits. 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:

Example 1. Obtaining a DNA fragment encoding HBsAg / ayw (fragment A). The reaction mixture containing 1 μM oligonucleotides 1u and 8L, oligonucleotides 2u to 8u; 1L to 7L, 10 pM, ThermoPol buffer (10 mM KCl, 20 mM tris-HCl 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 activity of Vent polymerase 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 containing the MOX gene promoter (fragment B).

Suspend 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, in 0.1 ml of 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 KCl, 20 mM tris-HCl 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 Pichia angusta genomic DNA fragment containing a portion of the TRP 3 gene (fragment B).

Suspend 2-3 μg of cells of the strain Pichia angusta (Hansenula Polymorpha) BKM 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 containing 1 μM trpU5 and trpL3 oligonucleotides, ThermoPol buffer (10 mM KCl, 20 mM tris-HCl 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 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 KCl, 20 mM tris-HCl 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 Pichia angusta genome.

The strain Pichia angusta (Hansenula Polymorpha) DLT (Agaphonov et al., 2002; a collection of strains of the laboratory of molecular genetics of the 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 3000g for 5 min and suspended in 15 ml of T buffer (10 mM tris-HCl 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 cannot grow on this medium are selected and tested for their ability to synthesize HBsAg / ayw. The resulting strain of Pichia angusta yeast 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. Scriabin.

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 10000 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 KCl, 20 mM tris-HCl 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 cells. After fermentation of the parent strain of Pichia angusta yeast, HBsAg is isolated according to the conventional 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, the solution is concentrated, which is essential for the next stage of absorption on 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 N. polymorpha VKPM Y - 2412 (deposited at the State Research Institute of Genetics - producer of the hepatitis B virus surface antigen (HBsAg / adw) is carried out analogously 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 containing 1 μM MOX and TRP oligonucleotides, ThermoPol buffer (10 mM KCl, 20 tM tris-HCl 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 specific names: Hansenula polymorpha and Pichia angusta. The name 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 for 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.

Method, conditions and composition of 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 combination vaccines as active components.

The vaccines obtained according to the invention can be presented in several variants:

1. The vaccine contains the ayw serotype antigen obtained by culturing the Pichia angusta VKM Y-2924D yeast strain as a hepatitis B virus antigen.

2. The vaccine contains the hepatitis B virus antigen as the adw serotype antigen obtained by culturing the Hansenula Polymorpha VKPM Y-2412 yeast strain.

3. The composition of the vaccine as a hepatitis B virus antigen contains a mixture of antigens of ayw and adw serotypes, taken in equal amounts (1: 1 ratio).

All active components of the combination vaccine, except for the hepatitis B virus antigen, are known domestic antigens.

A method of obtaining a combination vaccine consists in the usual mixing of the components of the composition, while equally good results can be obtained by implementing the method in various ways:

1. Sorb diphtheria-tetanus toxoid (ADS) on an aluminum hydroxide gel, taken in about half its amount, separately carry out the sorption of hepatitis B virus antigen (HBsAg) of one serotype or ayw and adw mixture on another part of the aluminum hydroxide gel, after which the sorbed antigens a preservative and a pharmaceutically acceptable solvent are mixed and added.

2. ADS is sorbed on an aluminum hydroxide gel, taking all its amount indicated in the formula, after which hepatitis B antigen of one serotype or a mixture of antigens of 2 serotypes and further, as in option 1, are added to the adsorbed ADS.

3. ADS is mixed with HBsAg of the same serotype or with a mixture of ayw and adw antigens and then sorbed on an aluminum hydroxide gel, as in the first embodiment.

4. HBsAg of one serotype or a mixture of antigens of 2 serotypes are sorbed on an aluminum hydroxide gel, then ADS and other components are added, as in the first embodiment.

The completeness of sorption is checked by the coagglutination reaction for diphtheria and tetanus components, ELISA for hepatitis component.

Specific examples of vaccines prepared according to the invention.

1. The combined vaccine for the immunoprophylaxis of viral hepatitis B and D, tetanus and diphtheria contains:

Surface antigen

hepatitis B virus

(HBsAg / ayw) obtained

cultivation

yeast strain

Pichia angusta VKM Y-2924D 5 mcg

Diphtheria toxoid 7.5 Lf

EU tetanus toxoid 5

Gel aluminum hydroxide (A1 ³⁺ ) 0.5 mg

Mertiolate 25 mcg

0.9% NaCl Up to 0.5 ml

0.5 ml of the drug is one vaccination dose.

2. The combined vaccine for the immunoprophylaxis of viral hepatitis B and D, tetanus and diphtheria contains:

Virus surface antigen

hepatitis B (HBsAg / adw),

cultured

yeast strain

N. Polymorpha VKPM Y-2412 10 mcg

Diphtheria toxoid 10 Lf

EU tetanus toxoid 7.5

Aluminum hydroxide gel (Al ³⁺ ) 0.35 mg

Mertiolate 15 mcg

0.9% NaCl Up to 0.5 ml

3. The combined vaccine for the immunoprophylaxis of viral hepatitis B and D, tetanus and diphtheria contains:

Surface antigen

hepatitis B virus

(HBsAg / ayw) obtained

cultivation

yeast strain

Pichia angusta VKM Y-2924D 4 mcg

Surface antigen

hepatitis B virus

(HBsAg / adw) obtained

cultivation

H. polymorphaVKPMY-2412 yeast strain 4 μg

Diphtheria toxoid 10 Lf

EU tetanus toxoid 5

Gel aluminum hydroxide (A1 ³⁺ ) 0.5 mg

Merthiolate 35 mcg

0.9% NaCl Up to 0.5 ml

In accordance with the current procedure for registering vaccines and the research program, State clinical trials of the reactogenicity and immunogenicity of the obtained vaccine in adults and children were conducted.

The reference drugs were ADS-M toxoid and hepatitis B vaccine recombinant yeast produced by NPK Kombiotekh.

The studies were conducted under the conditions of controlled experience among organized adult contingents aged 16 years and older and organized children aged 6 years.

The observation and comparison groups were made up of healthy individuals who did not have hepatitis B (HB) and diphtheria, had no contraindications to vaccination, had not previously been vaccinated against HB, and were subject to age-related revaccination with ADS-M toxoid, provided that the previous revaccination against diphtheria was carried out at least 4 years ago.

Vaccination was carried out after obtaining written informed consent.

Separately, work was carried out to study the reactogenicity and immunogenicity of a new vaccine when vaccinating weakened children with various somatic pathologies (14 people).

The reactogenicity of the new vaccine and the immunogenicity of diphtheria and tetanus components with the composition of the vaccine was evaluated after a single injection.

To assess the immunological activity of the HBsAg component in the composition of the new vaccine, those who were single vaccinated with the new vaccine were additionally vaccinated twice (after 1 and 6 months) with a single drug of a hepatitis B vaccine in children or adults according to age. In addition, a group of adults was identified that, after vaccination with a new vaccine, was twice inoculated with a pediatric dose of a single drug of the hepatitis B vaccine. Reactogenicity of the vaccine was assessed by the total frequency and intensity of local and general reactions (26 symptoms) to each drug administration after 30 minutes and then for 1 -5 days after immunization by interviewing and examining the injection site.

The level of antibodies to diphtheria and tetanus toxoids was determined using enzyme-linked immunosorbent assays to detect diphtheria and tetanus antibodies produced by Biomed Scientific-Production Association (Perm); the level of anti-HBs using enzyme immunoassay systems of the company “Sanofi” (France) and “Abbott” (USA).

All serums obtained before their study in ELISA were encrypted and formed into blocks. Statistical processing of the material was carried out using methods of variation statistics. Significance of differences was determined by Student's t-test at p <0.05.

In the course of the studies, unusual and strong post-vaccination reactions and complications were not found among both adults and children.

A comparative assessment of the reactogenicity of the new vaccine, ADS-M, and the hepatitis B vaccine in adults revealed the following: 80% of those who received the new vaccine showed no symptoms. Among the simultaneously vaccinated with ADS-M and hepatitis B vaccine, the number of people with the absence of post-vaccination reactions was only 52.3%. Among the noted side effects in both groups, the most frequent were local reactions in the form of hyperemia, edema, and mild soreness. General reactions were observed much less frequently. So, 2 general reactions (temperature 37.0 ° С and 37.7 ° С) were registered to the new vaccine 24 hours after the first injection of the vaccine.

Evaluation of the reactogenicity of the vaccine during immunization of both healthy and weakened children confirmed that the new combination vaccine is less reactogenic. The number of post-vaccination reactions in the group vaccinated with a new vaccine (25.9%) did not statistically significantly differ from the number of reactions in the group of children who received ADS-M toxoid and hepatitis B vaccine when they were combined in different parts of the body (26.7%) ( p> 0.05).

A study of the immunological activity of the new combination vaccine revealed the following: with almost the same background level of diphtheria and tetanus antibodies (p> 0.05), after the completion of the primary vaccination, the immune response to both the tetanus and diphtheria components of the vaccine in adults was higher than when using ADS-M (p <0.05). It should also be noted that while immunization with a new vaccine showed a 100% increase in antibody titers for diphtheria and tetanus components, with the separate administration of ADS-M toxoid and hepatitis B vaccine, titers increased only in 95% of those vaccinated. Before vaccination, anti-HBs antibodies in adults were absent in the 2 observation groups and the comparison group. After completion of the vaccination cycle, seroconversion in the first comparison group was 100%. In the second (additional) observation group, the proportion of individuals with a protective level of antibodies was 92%.

A more pronounced immune response occurred in the first observation group (geometric mean antibody titer was 658.6 mIU / ml). The total dose of the introduced HBs antigen was 50 μg. There were no statistically significant differences in the geometric mean titer of antibodies in the second observation group (222.4 mIU / ml) and the comparison group (273.3 mIU / ml), although the total doses of HBsAg administered were 30 μg and 60 μg, respectively. These data not only confirm the high immunological activity of the new vaccine, but also justify the possibility of switching to vaccination of adults with a single drug for hepatitis B vaccine in a children's dosage.

Evaluation of the immunological activity of the vaccine during immunization of children showed that after 1 month after completion of the initial course of vaccination, all children in the test group and in the comparison group showed a significant increase in the level of anti-diphtheria and tetanus antibodies (p <0.05). The titer of tetanus antibodies in the test group was statistically significantly (p <0.05) higher than the corresponding indicator in the comparison group. At the same time, the content of anti-diphtheria antibodies after vaccination in the comparison group was slightly higher than in the first (p <0.05), although the differences in titer increase (by 33.7 and 39.9 times, respectively) were not significant.

An immune response to the HBs component of the new vaccine was observed 1 month after completion of the vaccination cycle in 100% of children. Moreover, in the group vaccinated with a new vaccine, the level of antibodies was significantly higher than the protective level (CHT antibodies 13721 mIU / ml). With the combined administration of drugs, the level of seroconversion was 95%, and GGT - 2441 mIU / ml.

Studies have shown that the immunological activity of tetanus, diphtheria and hepatitis components in the combined preparation is higher than each of them individually, which confirms the presence of a potentiating effect.

Thus, a combination vaccine for the prevention of diphtheria, tetanus and hepatitis D and B is characterized by weak reactogenicity, safety and high immunogenicity.

Claims (2)

1. A combined vaccine for the immunoprophylaxis of viral hepatitis B and D, tetanus and diphtheria containing hepatitis B antigens (HBsAg), diphtheria and tetanus, merthiolate, aluminum hydroxide gel and a solvent, characterized in that the vaccine contains HBsAg as an antigen of the hepatitis B virus / ayw obtained by cultivating the yeast strain Pichia angusta VKM Y-2924D and / or HBsAg / adw obtained by cultivating the yeast strain Hansenula Polymorpha VKPM Y-2412, and as a solvent, a 0.9% sodium chloride solution with the following amounts of components: Surface antigen hepatitis B virus (HBsAg / ayw) obtained cultivation yeast strain Pichia angusta VKM Y-2924D, and / or superficial hepatitis virus antigen B (HBsAg / adw) obtained cultivating the strain H. polymorpha yeast VKPMY-2412 5-10 mcg HbsAg Diphtheria toxoid 5-10 Lf Tetanus toxoid 5-7.5 EU Gel of aluminum hydroxide (Al ³⁺ ) 0.35-0.55 mg Mertiolate 15-35 mcg 0.9% Sodium Solution chloride up to 0.5 ml 2. The combined vaccine for the immunoprophylaxis of viral hepatitis B and D, tetanus and diphtheria according to claim 1, characterized in that the surface antigens of hepatitis B virus (HBsAg / ayw) and (HBsAg / adw) are contained in a 1: 1 ratio.