RU2283139C1 - Method for preparing antigens for vaccine against influenza virus - Google Patents

Abstract

FIELD: biotechnology, immunology, medicine.

SUBSTANCE: antigens for vaccine against influenza virus are prepared by preparing the crude viral concentrate and purification of viral concentrate is carried out by gel-filtration method on carrier "DIOL-500". Then viral concentrate is cleaved with detergent β-octylglucoside and vaccine antigens are isolated by gel-filtration method on carrier Sephadex G-50. Advantage of invention involves enhancing prophylactic properties of antigens of influenza viruses. Invention can be used in producing anti-influenza vaccine used in prophylaxis of influenza in population for aim reducing risk and complications of the disease.

EFFECT: improved preparing method.

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Description

The method relates to biotechnology, immunology and medicine and can be used to produce influenza vaccine, which can be used to prevent influenza in the population in order to reduce the risk of disease and complications.

A known method of obtaining surface antigens of influenza virus according to US patent No. 4140762, based on the method of ultracentrifugation. The disadvantage of this method is the lack of internal antigens in the vaccine, which reduces its effectiveness, as well as the use of energy-consuming and capital-intensive ultracentrifugation in a sucrose density gradient.

A known method of producing split antigens according to US patent No. 4,237,182, based on the method of separation of antigens using membrane filters with different pore diameters. The disadvantage of this method is the insufficient purification of the isolated vaccine antigens from impurities of viral lipids and detergent, although the method has high efficiency.

At present, the Grippol vaccine, which is a sterile complex of protective surface proteins of the hemagglutinin and neuraminidase of influenza viruses A ₁ (H1N1), A ₂ (H3N2) and B associated with synthetic high molecular weight, is used for mass influenza prophylaxis in public health practice. immunomodulator polyoxidonium. The method of producing antigens for this vaccine according to the patent of the Russian Federation No. 2164148 and taken as a prototype.

The method is carried out in the following sequence:

1. Obtaining crude viral concentrate.

2. Purification of viral concentrate by ultracentrifugation in a sucrose density gradient.

3. Cleavage of the viral concentrate with the detergent tetradecyltrimethyl ammonium bromide (TTAB).

4. Isolation of vaccine antigens by ultracentrifugation in a sucrose density gradient.

5. Obtaining a sterile prefabricated vaccine.

The disadvantages of the method:

1. The composition of the vaccine obtained by the prototype method, presents only surface antigens of the influenza virus, which reduces the protective capabilities of the vaccine, namely, reduce protection against cross epidemic strains.

2. For the breakdown of whole virions, the detergent TTAB is used, which has a sufficiently high toxicity.

4. The method is quite long in time. So, to obtain purified subunits by ultracentrifugation, 72 hours are needed.

5. The method requires significant energy consumption, because the use of ultracentrifuges leads to significant energy costs.

6. The method requires significant capital costs for equipment in connection with the use of expensive ultracentrifuges.

The technical result is an increase in the prophylactic properties of the vaccine, a decrease in capital and energy costs, and an acceleration of the technological process.

According to the invention, the technical result is achieved due to the fact that, in contrast to the known method for producing antigens for an influenza virus vaccine, which includes obtaining a crude virus concentrate, purifying a virus concentrate, cleaving a viral concentrate with a detergent, and isolating vaccine antigens, in this method the virus concentrate is purified by the method gel filtration on a Diol-500 carrier, then the viral concentrate is cleaved with β-octyl glucoside detergent, and the release of vaccine antigens driven by gel filtration on a Sephadex G-50 carrier.

The advantages of the proposed method are to increase the prophylactic properties of the vaccine by increasing the protective capabilities of the vaccine and increasing protection against cross epidemic strains, using a non-toxic and highly effective detergent β-octylglucoside. In addition, the process of obtaining the vaccine was accelerated and energy and capital costs were reduced.

Causal relationship between the new set of features and the technical result.

For the first time in world production practice, the β-octyl glucoside detergent (certificate of state registration series BT No. 001297), which is a chemical substance with the chemical formula C ₁₄ H ₂₈ O ₆ , is used to break down influenza virus virions.

This detergent makes it possible to more fully split the influenza virus, which allows you to simultaneously isolate both surface and internal antigens of the influenza virus. In addition, β-octylglucoside practically does not form a micel in aqueous solutions and, therefore, is easily separated from influenza antigens after splitting the virions. This makes it possible to use the gel filtration method to isolate vaccine antigens; β-octylglucoside has 10 times less toxicity compared to the TTAB detergent used in the production of the influenza virus vaccine, which allows to reduce the reactogenicity of the vaccine.

The duration of the vaccine's protective effect has increased due to increased protection against cross epidemic strains, since it was possible to simultaneously include not only the surface antigens of influenza viruses, but also internal antigens, namely the membrane protein and nucleocapsid protein, while avoiding the denaturation of these proteins.

The method of industrial gel filtration is used to isolate vaccine antigens, which is much more economical than ultracentrifugation in the sucrose density gradient due to a sharp decrease in energy and capital costs. Due to this, the time from the cleavage of the virus to the preparation of a sterile semi-finished product is also reduced to 12 hours, while it takes 72 hours to obtain purified subunits by ultracentrifugation.

Detailed description of the method.

1. Preparation of crude viral concentrate.

To prepare the inoculum of viral material in the bottle with a 0.1% solution of peptone enter the mother material. The embryo shell in the prebox is treated with a 2% alcohol solution of iodine and burned with a torch dipped in 96% alcohol. Using a syringe, 0.2 ml of inoculated viral material is introduced into each embryo through an opening at the blunt end of the egg. After waxing, the infected embryos enter the thermal rooms, where they are incubated in the technological mode indicated in the passport for the corresponding strain.

At the end of the incubation period, the embryos are ovoscopic, mobile blood vessels with a well-developed network of blood vessels are selected and placed in a refrigerator for cooling (t - 2 ° C, duration 18 hours).

Folders with chilled embryos are delivered to the prebox, where they are disinfected with a 2% alcohol solution of iodine and burned with a torch dipped in 96% alcohol.

In boxing, sterile forceps remove part of the shell above the air sac and the adjacent portion of the chorionallantoic membrane. IMPORTANT from infected embryos is collected using sterile pipettes into bottles, which are then closed with rubber stoppers and placed in the refrigerator.

IMPORTANT Vials are poured into 10-liter bottles through funnels with gauze filters up to an 8-liter mark. Formalized red blood cells are added to each bottle in a volume determined by hemagglutinin titers (hereinafter GA) IMPORTANT. With a titer of IMPORTANT 1: 512 in 0.2 ml, red blood cells are added in an amount of 4% of the volume of IMPORTANT. After thorough mixing, the bottles are placed in a refrigerator for 16 hours. At the end of the specified period, the supernatant is decanted, provided that the flu virus contains no more than 1: 8 titers in it.

In bottles with a sediment of erythrocytes, a saline solution cooled to the appearance of ice crystals is added up to an 8-liter mark. After thorough mixing, the bottles are again placed in the refrigerator for 18 hours. If the titer of HA in the supernatant does not exceed 1: 8, it is again decanted.

Saline solution, heated to 37 ° C in a volume of 300 ml, is added to the bottles with the erythrocyte sediment. After thorough mixing, the red blood cells are poured into 350 ml vials, which are placed in a water bath (t - 38 ° C) for 3 hours. Every 10 minutes, the contents of the vials are shaken vigorously. Warm vials are transferred to a centrifuge and centrifuged at 2.0 thousand rpm. The supernatant of the eluate is poured into vials and recesses are taken for control studies.

Concentrated eluate is carried out on a Sartikon-mini unit with two cartridge modules with a pore size of 100 nm in a tangential flow. Immediately before work, the installation and piping system is sterilized with a 3% formalin solution, washed with sterile distilled water and the pH is restored using the Tris buffer to 7.2.

The sterile eluate is poured into a 5-liter bottle, which is connected through the pump to the input of the system, the return of the ultrafiltration system with cassette modules is connected to the same bottle.

2. Purification of the viral concentrate.

The purification is carried out by gel filtration on a Diol-500 chromatographic medium (BioChemMak company, Russia.).

A column with a Diol-500 carrier is washed from a 3% formalin solution with distilled water. The pH level is restored with a Tris buffer solution to 7.2. A vial of eluate concentrate is attached to the column "entrance" through a siphon. The filtration rate of 0.12 l / m ² per minute. In the process of passing through a column of a chromatographic buffer at the "exit", a sample is taken. Protein peaks are monitored using the Uvikord instrument (LKB company, Sweden).

3. Cleavage of the viral concentrate.

The purified viral concentrate is treated with β-octyl glucoside detergent as follows. Pre-determine the amount of protein in the concentrate. Then, depending on the amount of protein, a detergent is weighed at a ratio of 1:10 to the viral protein.

The contact time is 25 minutes with constant stirring, the incubation temperature is room temperature.

4. Isolation of vaccine antigens.

The selection of vaccine antigens is carried out by gel filtration on a Sephadex G-50 chromatographic medium (Pharmacea Sweden) as follows. A Sephadex column is washed from a 3% formalin solution with distilled water. The pH level is restored phosphate-buffered saline to a pH of 7.2. A vial with a solution of the destroyed virus concentrate is attached to the column "entrance" through a siphon. The filtration rate of 0.12 l / m ² per minute. In the process of passing through a column of a chromatographic buffer at the "exit", a sample is taken. The control of protein peaks is carried out on the device "Uvikord" (company "LKB", Sweden).

5. Obtaining a sterile prefabricated vaccine.

Isolated vaccine antigens for sterile prefabrication are sterilized through a Sartobran filter from Vladisart (Russia). To do this, a bottle with a solution of antigens is connected to the "input" of the installation. Pressure is supplied to the bottle by an aquarium pump. Sampling is carried out from the "exit" of the installation in bottles under the cap.

The time for sterile filtration is 2 hours. The time from the splitting of the virus to the receipt of the semi-finished product is not more than 12 hours.

Information confirming the possibility of carrying out the invention

Example. Preparation of semi-finished products (antigens) for conducting State clinical trials on volunteers.

Obtaining a semi-finished product And ₁

7200 pieces of 10-day-old chicken embryos were infected with the uterine material of strain A ₁ . After incubation in a thermal room for 48 hours at a temperature of 37 ° C and cooling for 18 hours in a refrigerator at a temperature of 4 ° C, a virus-containing allantoic fluid (IML) was obtained in an amount of 48 liters with a hemagglutinating activity (GA) titer of 1: 512.

This volume of IMPORTANT is poured into 10 liter bottles of 8 liters each and red blood cells are added, in the amount of 4% of the volume of IMPORTANT. After decantation of the supernatant after 18 hours and washing with saline with ice, 300 ml of warm 37 ° C saline solution was added to the erythrocyte sediment, poured into 350 ml vials. Vials are placed in a water bath at a temperature of 37 ° C for 3 hours. Warmed vials were centrifuged at 2.0 thousand rpm for 30 minutes. After centrifugation, the supernatant was drained into a bottle and the HA was determined in it, which amounted to 1: 8000. The volume of the eluate was 2.7 liters.

The eluate was concentrated on a Sartikon-mini unit with cassette modules with pore sizes of 100 nm to 300 ml. Purification from impurity proteins was carried out on a chromatographic column with a carrier Diol-500 with a volume of 2.0 liters. The volume of purified concentrate was 400 ml. The protein content in it was 200 μg / ml, the hemagglutinin concentration was 100 μg / ml according to the ARID, there was no ovalbumin.

To destroy the virus, 200 ml of glycerol concentrate was taken and 400 mg of β-octyl glucoside was added. The selection of vaccine antigens (split) was carried out on a column with Sephadex with a volume of 1 liter, the volume of the selected split was 290 ml. To carry out sterilizing filtration, Sartobran filter capsules were used. The total protein content in the semi-finished product was 180 μg / ml, hemagglutinin in the ARID was 88 μg / ml.

The time from the destruction of the virus to the receipt of semi-finished products is 11 hours.

Preparation of Prefabricated A ₂

7200 pieces of 10-day-old chicken embryos were infected with uterine material of strain A ₂ . After incubation in a thermal room for 48 hours at a temperature of 37 ° C and cooling for 18 hours in a refrigerator at a temperature of 4 ° C, a virus-containing allantoic fluid (IML) was obtained in an amount of 48 liters with a hemagglutinating activity (GA) titer of 1: 256.

This volume of IMPORTANT is poured into 10 liter bottles of 8 liters in each and red blood cells are added, in a volume of 2% of the volume of IMPORTANT. After decantation of the supernatant after 18 hours and washing with saline with ice, 300 ml of warm 37 ° C saline solution was added to the erythrocyte sediment, poured into 350 ml vials. Vials are placed in a water bath at a temperature of 37 ° C for 3 hours. Warmed vials were centrifuged at 2.0 thousand rpm for 30 minutes. After centrifugation, the supernatant was drained into a bottle and GA was determined in it, which amounted to 1: 4000. The volume of the eluate was 3.4 liters.

The eluate was concentrated on a Sartikon-mini unit with cassette modules with pore sizes of 100 nm to 300 ml. Purification of impurity proteins was carried out on a chromatographic column with a Diol-500 carrier with a volume of 2 liters. The volume of purified concentrate was 400 ml. The protein content in it was 250 μg / ml, hemagglutinin was on ORID 151 μg / ml, ovalbumin was absent.

To destroy the virus, 200 ml of glycerol concentrate was taken and 500 mg of β-octyl glucoside was added. The selection of vaccine antigens was carried out on a Sephadex G-50 chromatographic column with a volume of 1.0 liter, the volume of the selected split was 300 ml. To carry out sterilizing filtration, Sartobran filter capsules were used. The protein content in the semifinished product was 270 μg / ml, Hemaglutinin by ARID was 130 μg / m.

The time from the destruction of the virus to the receipt of semi-finished products is 11 hours.

Preparation of Prefabricated B

8260 pieces of 10-day-old chicken embryos were infected with the uterine material of strain A ₂ . After incubation in a thermal room for 72 hours at a temperature of 33 ° C and cooling for 18 hours in a refrigerator at a temperature of 4 ° C, a 40-gallon-containing allantoic fluid was obtained with an hemagglutinating activity (HA) titer of 1: 256.

This volume of IMPORTANT is poured into 10 liter bottles of 8 liters in each and red blood cells are added, in a volume of 2% of the volume of IMPORTANT. After decantation of the supernatant after 18 hours and washing with saline with ice, 300 ml of warm 37 ° C saline solution was added to the erythrocyte sediment, poured into 350 ml vials. Vials are placed in a water bath at a temperature of 37 ° C for 3 hours. Warmed vials were centrifuged at 2.0 thousand rpm for 30 minutes. After centrifugation, the supernatant was drained into a bottle and GA was determined in it, which amounted to 1: 4000. The volume of the eluate was 2.0 liters.

The eluate was concentrated on a Sartikon-mini unit with cassette modules with pore sizes of 100 nm to 300 ml. Purification of impurity proteins was carried out on a chromatographic column with a Diol-500 carrier with a volume of 2 liters. The volume of purified concentrate was 400 ml. The protein content in it was 290 μg / ml, hemagglutin in the ARID was 150 μg / ml, ovalbumin was absent.

To destroy the virus, 200 ml of glycerol concentrate was taken and 580 mg of β-octylglucoside was added. The selection of antigens was carried out on a column with Sephadex volume of 1.0 liter. The split volume was 310 ml.

To carry out sterilizing filtration, Sartobran filter capsules were used. The protein content in the semifinished product was 400 μg / ml, Hemaglutinin by ARID was 200 μg / ml.

The time from the destruction of the virus to the receipt of semi-finished products is 12 hours.

State clinical trials on volunteers obtained semi-finished products (antigens) as part of an experimental vaccine

Volunteers were randomly divided into groups II, individuals of one group (133 people) were vaccinated with an experimental vaccine consisting of three obtained semi-finished products. One vaccine dose of this vaccine contained 10 μg for semi-finished product hemagglutinin A ₁ , 10 μg for semi-finished product hemagglutinin A ₂ and 15 μg for semi-finished product hemagglutinin B. Persons of the second control group (132 people) were vaccinated with a placebo drug (control group). Before the test, the vaccine and placebo were encrypted by a GISK employee. L.A. Tarasevich. Volunteers selected for clinical trials were vaccinated once intramuscularly with an encrypted preparation in a volume of 0.5 ml (the volume of one vaccinating dose) in the upper third of the shoulder with a syringe.

Reactogenicity of the obtained semi-finished products as part of an experimental vaccine

All volunteers prior to the introduction of the experimental vaccine and placebo were interviewed and examined by physicians. Each had an individual observation map, which included the results of daily medical examinations, surveys, measurements of body temperature, general and local reactions. Particular attention was paid to the symptoms corresponding to the clinical manifestations of influenza, allergic reactions. Local reactions were assessed by the diameter of the hyperemic skin area at the injection site, soreness, swelling of the skin, the presence of infiltrates, lymphadenitis, abscess.

Vaccinations were monitored daily for 5 days from the time of vaccination. Those who gave deviations were monitored until these reactions completely disappeared. Along with this, for 6 months, the vaccine and the control group were vaccinated with monthly registration of somatic, infectious and allergic morbidity among the observed population.

The results are shown in tables 1, 2, 3.

When collecting an anamnesis prior to vaccination, it was revealed that none of the volunteers complained, the skin and mucous membranes were clean. Body temperature and blood pressure were normal in all subjects. Immediately after the administration of the vaccine and placebo, no local reactions were noted.

During daily thermometry and examination by a general practitioner, placebo vaccines noted: malaise, headache, runny nose, pharyngeal hyperemia, nausea, abdominal pain, which in absolute numbers was 6 people. Of the local reactions, 1 person showed pain at the injection site (Table 1).

The vaccine vaccinated had complaints of headache, runny nose, nasal congestion, only 3 people (one complaint). Local reactions: pain at the injection site was noted by 3 people and pain by pressure of 9 (Table 2).

An increase in temperature from 37.1 to 37.4 ° C was observed in 2 vaccinated in the placebo group, and in 5 people vaccinated with the vaccine, the temperature was from 37 to 37.3 ° C. In 4 people, a temperature increase was noted on the second day, on the third day, a temperature above 37 ° C was not detected (Table 3).

From the data presented in the tables, it follows that the experimental vaccine has a weak reactogenicity.

Antigenic activity of the obtained semi-finished products as part of an experimental vaccine

Antigenic activity was assessed in the hemagglutination inhibition reaction (RTHA) according to the generally accepted method based on the results of blood tests collected before vaccination and after vaccination after 28 days. As antigens for the production of serological reactions used commercial diagnosticums, created on the basis of strains of influenza viruses A ₁ (H ₁ N ₁ ); A ₂ (H ₃ N ₂ ) and B, which were recommended by GISK to them. L.A. Tarasevich.

Antigenic activity of vaccines was characterized by the following indicators:

- four-fold seroconversion compared to background serum;

- the level of antigenic response before and after 28 days after vaccination, while the geometric mean antibody titers (CHTA), the rate of titer increase compared to background serum were determined;

- the level of serological protection by determining the percentage of individuals with a protective titer of antibodies before and 28 days after vaccination.

All collected quantitative indicators were processed by methods of variation statistics. The significance of differences was determined by the Student-Fisher criterion, the differences were considered significantly significant at P <0.05.

The results obtained in the study of paired blood serum vaccinated with a “placebo” and an experimental vaccine using commercial diagnostics are presented in Tables 4, 5, 6. The tables show that in the sera vaccinated with an experimental vaccine, a statistically significant (p <0.05) increase in antibody titers expressed in individuals with a low baseline level (<20). The multiplicity of increase in antibody titers to the strain of the virus H ₁ N ₁ is - 27.9, to H ₃ H ₂ - 7.5, to the strain of virus B - 6.5.

In the group with a high initial level of antibodies (> 40), the indicators are lower: to the strain of the virus H ₁ N ₁ - 3.7, to the strain of the virus H ₃ N ₂ - 3.0 and to the strain of virus B - 2.1.

The frequency of a fourfold increase in antibodies 28 days after vaccination was 93.7% for the H ₁ N ₁ virus strain, 90.0% for the H ₃ N ₂ virus strain and 77.3% for strain B. In the placebo group, the level of antibodies in blood serum taken before vaccination and after 28 days remained virtually unchanged.

The percentage of individuals with a protective titer of antibodies (> 40) (Table 7) was 47.1% for vaccinated vaccines for the H ₁ N ₁ virus strain before vaccination, 95% after vaccination; 66.4% for the H ₃ N ₂ virus strain before vaccination, 97.5% after the vaccination, 26.1% for the B virus strain before vaccination, 88.2% after vaccination.

In the placebo group, the protective titer of antibodies to the H ₁ N ₁ virus strain before vaccination was 38.9%, after vaccination 41.6%; 79.6% for the strain of the H ₃ N ₂ virus before vaccination, 82.3% after the vaccination, and 32.7% for the virus B strain before vaccination, 35.4% after vaccination. The percentage of individuals with a titer> 40 for all strains became slightly higher than before vaccination, but the difference between these indicators is not statistically significant.

Thus, the results show that the experimental vaccine, consisting of the obtained semi-finished products A ₁ , A ₂ and B, has high antigenic activity for strains of viruses A ₁ , A ₂ and B.

Conclusion on the results of state clinical trials on volunteers obtained semi-finished products as part of an experimental vaccine

As a result of field trials under conditions of strictly controlled epidemiological experience, it was found that an experimental vaccine consisting of the obtained semi-finished products A ₁ , A ₂ and B has weak reactogenicity and high antigenic activity against influenza viruses A ₁ , A ₂ and B. The results obtained allow us to recommend this experimental vaccine for the prevention of influenza in humans.

Table 1
Frequency of general and local disturbances in vaccinated placebo Vaccinated condition Absolute amount % ± m I. The nature of violations of the general condition: malaise one 0.75% ± 0.75 headache 3 2.25% ± 1.29 runny nose one 0.75% ± 0.75 nasal congestion 2 1.5% ± 1.05 pharyngeal hyperemia 2 1.5% ± 1.05 nausea one 0.75% ± 0.75 stomach ache one 0.75% ± 0.75 Total eleven 8.3% ± 2.4 II. Local reaction Skin soreness at injection site one 0.75% ± 0.75 (with pressure or physical activity) - - Total one 0.75% ± 0.75 Total were monitored 132

table 2
The incidence of general and local disturbances in vaccinated experimental vaccines Vaccinated status Absolute amount % ± m I. The nature of violations of the general condition: malaise - - headache 2 1.50% ± 1.05 runny nose 2 1.50% ± 1.05 nasal congestion 2 1.50% ± 1.05 pharyngeal hyperemia one 0.75% ± 0.75 nausea - - stomach ache - - Total 7 5.26% ± 1.94 II. Local reaction Skin soreness four 3.00% ± 1.48 at the injection site (with pressure or physical activity) 10 7.52% ± 2.60 Total fourteen 10.53 ± 2.66 Total were monitored 133

Table 4
The results of the study of sera inoculated with an experimental vaccine before and after vaccination using a diagnosticum for a strain of influenza virus A ₁ (H ₁ N ₁ ). Group A drug Baseline antibody Number of persons with paired
serums Of these, ^4-fold conversions
in the second portion (in%) CGTA The increase in the titers
antibodies Comparison by criterion
Student's The number of individuals with a protective titer of antibodies (> 40) during
second portion (in%) Before vaccination After vaccination one vaccine <20 63 93.7 ± 3.1 1: 7 1: 195 27.9 p <0.05 95.2 ± 2.7 placebo <20 69 2.9 ± 2.0 1:10 1:12 1,2 p> 0.05 4.3 ± 2.4 eleven vaccine > 40 56 58.9 ± 6.6 1:91 1: 339 3,7 p <0.05 one hundred placebo > 40 44 2.3 ± 2.3 1:72 1:85 1,2 p> 0.05 one hundred Note: an indicator is emphasized that is statistically significantly different from the corresponding indicator of the group (p <0.05)

Table 5
The results of the study of sera vaccinated with an experimental vaccine before and after vaccination using a diagnosticum for the strain of influenza virus A ₂ (H ₃ N ₂ ). Group A drug Baseline antibody Number of persons with paired
serums Of these, ^4-fold conversions
in the second portion (in%) CGTA The increase in the titers
antibodies Comparison by criterion
Student's The number of individuals with a protective titer of antibodies (> 40) during
second portion (in%) Before vaccination After vaccination one vaccine <20 40 90.0 ± 4.7 1:15 1: 112 7.5 p <0.05 92.5 ± 4.2 placebo <20 23 13.0 ± 7.0 1:16 1:22 1.4 p> 0.05 13.0 ± 7.0 eleven vaccine > 40 79 46.8 ± 7.9 1:76 1: 229 3.0 p <0.05 one hundred placebo > 40 90 0 1:98 1: 100 1,0 p> 0.05 one hundred Note: an indicator is emphasized that is statistically significantly different from the corresponding indicator of the group (p <0.05)

Table 6
The results of the study of sera vaccinated with an experimental vaccine before and after vaccination using a diagnosticum for a strain of influenza B. Group A drug Baseline antibody The number of persons with paired sera Of these, ^4-fold conversions
in the second portion (in%) CGTA The increase in the titers
antibodies Comparison by criterion
Student's The number of individuals with a protective titer of antibodies (> 40) during
second portion (in%) Before vaccination After vaccination one vaccine <20 88 77.3 ± 4.5 1:10 1:65 6.5 p <0.05 84.1 ± 3.9 placebo <20 76 2.6 ± 1.8 1:13 1:15 1,2 p> 0.05 3.9 ± 2.2 eleven vaccine > 40 31 19.4 ± 7.1 1:48 1: 100 2.1 p <0.05 one hundred placebo > 40 37 0 1:43 1:45 1,0 p> 0.05 one hundred Note: an indicator is emphasized that is statistically significantly different from the corresponding indicator of the group (p <0.05).

Claims (1)

A method for producing antigens for a vaccine against influenza virus, including obtaining a crude virus concentrate, purifying a viral concentrate, cleaving it with a detergent, isolating vaccine antigens, characterized in that the viral concentrate is purified by gel filtration on a Diol-500 carrier, the viral concentrate is cleaved with detergent β -octylglucoside, and the selection of vaccine antigens is carried out by gel filtration on a Sephadex G-50 carrier.