RU2233673C1 - Mixed vaccine for immunoprophylaxis of whooping cough, diphtheria, tetanus and viral hepatitis b and d - Google Patents

Abstract

FIELD: biotechnology, medicine, vaccines.

SUBSTANCE: invention relates to a new mixed vaccine. 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 taken in the amount 5-10 mcg; whooping cough microorganisms, 6.0-10 billion; diphtheria anatoxin, 7.5-15 Lf; tetanus anatoxin, 5-10 EC; aluminum hydroxide (Al³⁺) gel, 0.3-0.5 mg; merthiolate, 35-50 mcg, and 0.9% NaCl, up to 0.5 ml. Hepatitis B virus antigens (HBsAg/ayw) and (HBsAg/adw) are taken in the ratio = 1:1. The new mixed vaccine is high-immune, nontoxic preparation with broad spectrum of effect that provides expanding assortment of agents designated for immunoprophylaxis against pathogens of some diseases of viral and bacterial etiology.

EFFECT: valuable medicinal properties of mixed vaccine.

2 cl, 3 tbl, 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 patient in need.

With the simultaneous administration of various vaccines, the problems of reducing the number of injections and reducing the cumulative toxic effects on the body of various vaccine ingredients (sorbent, preservative, etc.) are not solved; the issues of transportation and storage of a large number of thermolabile preparations, as well as the availability of all vaccines indicated for a single use, remain relevant . Therefore, the program for creating associated drugs remains a priority in the development of the modern immunobiological industry.

Known combination vaccine against diphtheria - tetanus - pertussis - hepatitis B.

One 0.5 ml dose contains as active ingredients:

D-toxoid - 7.5 Lf; C - toxoid - 3.25 Lf; Kc - antigen - 15; HBsAg - 10 μg of 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 (the patent refers 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, an antiseptic is used in the vaccine, for example, merthiolate 1: 20,000 to 1: 10,000 or 2-phenoxyethanol from 3 to 6 mg / cell.

The composition of the vaccines includes isotonic saline (RU, patent No. 2160120, A 61 K 39/295, 39/29, publ. 2000).

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

The vaccine has the following composition:

Hepatitis B virus surface antigen (HBsAg) obtained by culturing a recombinant strain of S. cerevisiae VKPM Y2203 5-10 μg HBsAg

Diphtheria purified toxoid concentrated toxoid 10-20 Lf

The tetanus toxoid purified concentrated 5-10 EC

Pertussis microbes killed by formalin 10-15 OE

Mertiolate 0.01 ± 0.002%

Gel of aluminum hydroxide 0.5-1.2 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. 2130778, 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, diphtheria and pertussis.

The problem was solved by constructing a new highly immunogenic combination vaccine in which, as an antigen of hepatitis B virus (HBsAg), contains a new antigen of hepatitis B virus (HBsAg) of serotype adw, obtained by culturing the yeast strain Hansenula Polymorpha, VKPM - Y-2412, hepatitis B virus antigen ayw serotype 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 antigen or mixture of hepatitis virus antigens In the combination vaccine is 5-10 μg HBsAg in 0.5 ml of a solution, which is a vaccination dose of the drug.

In addition, the vaccine contains:

Pertussis microbes killed by formaldehyde 6.0-10 billion

Diphtheria toxoid 7.5-15 Lf

Tetanus toxoid 5-10 EU

Aluminum hydroxide gel (Al ³⁺ ) 0.3-0.5 mg

Merthiolate (preservative) 35-50 mcg

0.9% NaCl Up to 0.5 ml

In contrast to the known vaccine according to the invention, a new hepatitis B virus antigen of ayw and / or adw serotypes is introduced into the designed vaccine and the ratio of active ingredients is changed due to the potentiating effect due to the effect of the new antigen on the immunological activity of other active components of the obtained vaccine. In addition, 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 (expanded spectrum of exposure) with high immunogenic activity and non-toxicity, low reactogenicity.

The vaccine obtained according to the invention, containing hepatitis B virus antigen of two different serotypes, is a directed vaccine against those pathogens that are most widespread in Russia and in neighboring countries.

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 the 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 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 serotypes ay and ad, are new.

Isolated antigens are first used as part of a combination vaccine.

A method of obtaining new strains - producers 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 companies producing enzymes and in vitro DNA manipulation kits. For PCs, 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 lu 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 s, at 50 ° C 30 s, at 72 ° C 90 s. 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 min, 1 μl of the obtained supernatant is added to 25 μl of a mixture containing moxU5 and moxL3 oligonucleotides 1 μM each, 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. The reaction mixture is incubated for 30 cycles of temperature change. Each cycle includes incubation at 95 ° C for 30 s, at 50 ° C 30 s, at 72 ° C 90 s. 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).

2-3 μg of cells of H. polymorpha BKM 4-2559 strain (Pichia angusta) 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 min, 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 s, at 50 ° C 30 s, at 72 ° C 120 s, at 50 ° C 30 s, at 72 ° C 120 s. 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 s, at 50 ° C for 30 s, 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 RKN SC of the Ministry of Health of the Russian Federation) is grown for 15-20 h in YNB-D medium containing tryptophan 20 mg / l at 37 ° C in conditions of intensive aeration. 0.5 ml of culture was added to 30 ml of the same medium and incubated under the same conditions for 4 hours. Cells were collected 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 an incubation of 30 minutes at 30 ° C, the cells are pelleted again 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 h 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.Skryabin. Certificate of deposit of the strain is attached.

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 the culture were collected by centrifugation, suspended in 0.2 ml of distilled water, add 0.2 ml of 0.2 M NaOH and incubate 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 lanes corresponding to strains that synthesize HBsAg / ayw, a band at 24-27 kDa is detected, 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 min, 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 activity of Vent polymerase. The reaction mixture is incubated for 30 cycles of temperature change. Each cycle includes incubation at 95 ° C for 30 s, at 50 ° C for 30 s, 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 1 L 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 KDL mill 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 kDa, 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 solution of HBsAg (ayw) is 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. A certificate of the deposit of the strain is attached), a producer of the hepatitis B virus surface antigen (HBsAg / adw), is carried out similarly to examples 1-5 with the only difference being that they use the following oligonucleotides:

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 the culture were collected by centrifugation, suspended in 0.2 ml of distilled water, add 0.2 ml of 0.2 M NaOH and incubate 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 min, 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 activity of Vent polymerase. The reaction mixture is incubated for 30 cycles of temperature change. Each cycle includes incubation at 95 ° C for 30 s, at 50 ° C for 30 s, 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 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.

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

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

All other active components of the vaccine are known domestic antigens.

The new combination vaccine is a combination of hepatitis B virus surface antigen associated with aluminum hydroxide gel and a mixture of pertussis microbes killed by formaldehyde phase 1 microbes and purified from ballast proteins of diphtheria and tetanus toxoids.

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 the components of the pertussis-diphtheria-tetanus vaccine (DTP) on aluminum gel, taken in about half of its quantity, separately carry out the sorption of hepatitis B virus antigen (HBsAg) of one serotype or a mixture of antigens of 2 serotypes on another part of the aluminum gel, after wherein the sorbed antigens are mixed and a preservative and a pharmaceutically acceptable solvent are added.

2. DTP is sorbed on an aluminum gel, taking all its amount indicated in the formula, after which hepatitis B antigen or a mixture of antigens of 2 serotypes is added to the sorbed DTP, and then as in the 1st embodiment.

3. Mix DTP with HBsAg of the same serotype or with a mixture of ayw and adw antigens and then sorb them on aluminum gel, as in the 1st embodiment.

4. HBsAg or a mixture of ayw and adw antigens are sorbed on an aluminum hydroxide gel, then AKDS and other components are added as in the first embodiment.

The completeness of sorption of diphtheria toxoid is checked by the flocculation reaction, the tetanus component by the antitoxin binding reaction, ELISA for the hepatitis component.

Further, in studies to determine immunogenic activity, the following specific formulations were used:

Specific examples of combination vaccines obtained according to the invention:

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

Virus surface antigen

hepatitis B (HBsAg / ayw),

cultured

Pichia angusta yeast strain

VKM Y-2924D 5 mcg

Formaldehyde-killed pertussis microbes 6.0 billion

Diphtheria toxoid 7.5 Lf

EU tetanus toxoid 5

Gel aluminum hydroxide (A1 ³⁺ ) 0.5 mg

Merthiolate (preservative) 50 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, whooping cough, diphtheria and tetanus contains:

Hepatitis B virus surface antigen (HBsAg / adw) obtained by culturing a yeast strain of N. Polymorpha VKPM Y-2412 5 μg

Pertussis microbes killed by formaldehyde 10 billion

Diphtheria toxoid 15 Lf

EU tetanus toxoid 10

Gel aluminum hydroxide (Al ³⁺ ) 0.5 mg

Merthiolate (preservative) 40 mcg

0.9% NaCl Up to 0.5 ml

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

Hepatitis B virus surface antigen (HBsAg / ayw) obtained by culturing a 2.5 μg strain of Pichia angusta VKM Y-2924D yeast

Hepatitis B virus surface antigen (HBsAg / adw) obtained by cultivating the yeast strain N. Polymorpha VKPM Y-2412 2.5 μg

Pertussis microbes killed by formaldehyde 8 billion

Diphtheria toxoid 10 Lf

EU tetanus toxoid 8

Aluminum hydroxide gel (A1 ³⁺ ) 0.3 mg

Merthiolate (preservative) 35 mcg

0.9% NaCl Up to 0.5 ml

The immunogenic activity of the new combination vaccine was studied on a children's contingent, and a comparative assessment was made of the immunogenic activity of the new drug, DTP vaccine and hepatitis B vaccine when they were simultaneously administered to different parts of the body in children of the first year of life.

The study included 93 children under the age of 1 year.

Group 1 was immunized with the new vaccine three times at 3, 4 and 5 months;

Group 2 received simultaneous vaccination in different parts of the body with DTP - vaccine and hepatitis B vaccine, three times at 3, 4 and 5 months;

Group 3 was immunized with DTP vaccine three times at 3, 4 and 5 months.

Subject to immunization were healthy children who did not have contraindications for vaccination with DTP and hepatitis B vaccines, in the absence of hepatitis cases in the family and in the absence of a history of vaccinations against hepatitis B.

As reference preparations, we used the DTP vaccine manufactured by the State Unitary Enterprise Immunopreparat (Ufa), series No. 254-4 and 251-1 and the hepatitis B vaccine, recombinant yeast liquid according to FS 42-3438-97, series 110 and 113 (10 μg , children's dose).

Immunization was carried out in accordance with the provisions of the Instructions for the use of drugs: vaccines in a dose of 0.5 ml were administered intramuscularly in the upper outer quadrant of the buttock or in the anterior-outer thigh.

To determine the immunogenicity of the vaccines used, 30-45 days after the completion of the vaccination course in children, blood was taken from a vein in an amount of 2.0-5.0 cm ³ .

The obtained serum was frozen and stored until the study at a temperature of minus 30 ° C.

The level of antibodies to HBV virus (HBsAg), to diphtheria and tetanus toxoids was determined using ELISA using IFTS to detect diphtheria and tetanus antibodies produced by NPO Biomed (Perm); anti-HBs antibodies - IFTS "Monolisa" manufactured by Biorat (France). The levels of antibodies to the pertussis component were determined in an agglutination reaction with a standard pertussis diagnosticum produced by Biomed CJSC (Moscow).

Statistical analysis of the results of the study was carried out using descriptive statistics methods. The significance of differences between the groups was evaluated using parametric criteria. In addition, dispersion analysis methods were used. We used Excel spreadsheets for Windows (Microsoft), as well as the DIASTA statistical software package (Moscow State University, Russia), which is a version of the STADIA package.

Evaluation of the immune response showed that the new vaccine induced immunity to all four components of the vaccine. One month after completion of the vaccination course, 100% of the children in all the studied groups had antibody titers against diphtheria and tetanus, significantly exceeding the protective titers (0.03 IU / ml for diphtheria and 0.01 IU / ml for tetanus).

As shown by the results of statistical processing of enzyme-linked immunosorbent assay data with a confidence interval of measurement (95%), the differences in the geometric mean titer (GHT) of diphtheria and tetanus antibodies between the I new vaccine and III (DTP), II (DTP + HBV) and III groups are not significant (p> 0.05). While between groups I and II, the differences in the values of CHT turned out to be significant (p <0.05). In group I, the GHS of diphtheria and tetanus antibodies is 1.65 and 1.6 times, respectively, more than in group II (Table 1).

The results of the evaluation of the immune response to the pertussis component of combination vaccines according to the agglutination reaction are given in table. 2. In the observation group I, only 4 children out of 36 (11.11%) had antibody titers lower (<1:80). While in group II, antibody titers below 1:80 had 9 out of 32 people (28.13%), and in group III - 5 out of 25 (20%). Differences in the value of GHG between I and II, I and III, II and III groups are not significant (p> 0.05) (Table 2).

The structure of the immune response to the hepatitis component is given in table. 3. As can be seen, one month after the completion of the vaccination cycle, 100% of the children in the first group had an anti-HBs level significantly higher than the protective level (> 10 IU / L). Moreover, 50% of children had anti-HBs titers in the range of 100-1000 IU / L. In the second group, 5 children (15.62%) did not give an immune response and the percentage of seroconversion in the group was 84.38%. Meanwhile, the differences in the value of the CGT of anti-HBs between groups I and II are not significant (p> 0.05). It should be noted that the total dose of HBsAg administered as part of the new vaccine was 15 mcg, and with the simultaneous administration of DTP vaccine and hepatitis B vaccine, 30 mcg.

Thus, we can state the following:

1. The new combination vaccine stimulates the immune response to all four of its constituent components. After completing the initial course of vaccination, 100% of children had protective titers of antibodies against diphtheria, tetanus and hepatitis B.

2. Anti-pertussis antibody titers equal to or greater than protective (1: 160) had over 70% of vaccinated children. Moreover, a similar indicator in the comparison groups was not more than 60%.

Claims (2)

1. A combined vaccine for the prevention of pertussis, diphtheria, tetanus and viral hepatitis B and D, containing pertussis, microbes, diphtheria antigens, tetanus, viral hepatitis B (HBsAg), aluminum hydroxide gel, merthiolate and a solvent, characterized in that as a virus antigen The hepatitis B vaccine contains HBsAg / 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 0.9% sodium chloride solution with following quantities components: Virus surface antigen hepatitis B (HBsAg / ayw) obtained culturing a yeast strain Pichia angusta VKM Y-2924D, and / or virus surface antigen hepatitis B (HBsAg / adw) obtained culturing a yeast strain H. Polymorpha VKPM Y-2412 5-10 μg HBsAg Killed with formaldehyde pertussis microbes 6.0-10 billion Diphtheria toxoid 7.5-15 Lf Tetanus toxoid 5-10 EU Gel of aluminum hydroxide (Al ³⁺ ) 0.3-0.5 mg Merthiolate (preservative) 35-50 mcg 0.9% Sodium Chloride Solution Up to 0.5 ml 2. The combined vaccine for the prevention of pertussis, diphtheria, tetanus and viral hepatitis B according to claim 1, characterized in that the surface antigens of hepatitis B virus (HbsAg / ayw) and (HBsAg / adw) are contained in a ratio of 1: 1.