UA35488C2 - Method for preparing antiviral vaccines - Google Patents

Abstract

The method for preparing the antiviral vaccines has been proposed. The aim of the invention consists in improving the efficacy of the vaccination and preventing the side reactions. This aim is attained by immobilizing the live or inactivated vaccine onto the adsorbent and by peroral administration of the vaccine in the volume of excipient causing the swallowing reflex. Enterosgel (the hydrogel of methyl silicic acid) used as an adsorbent is added to virus-containing solution at a dose of 100 mg per 1 ml of solution. The mixture is shaken for 10 minutes and incubated for 24 hours at the temperature of 40С. The method proposed does not require the huge expenditures and is not time-consuming. The method of administering the vaccine is quite safe.

Description

The invention belongs to the production of medical and biological preparations and can be used for live and inactivated vaccines.

There is a known method of obtaining an inactivated influenza vaccine (b), which involves stepwise purification and concentration of the influenza virus by ultrafiltration and ultracentrifugation in a sucrose density gradient; as well as the polymer subunit vaccine "grippol" (2), which is a sterile conjugate of surface proteins, to increase the immunizing activity - as an adjuvant - the synthetic immunostimulant polyoxidonium is added (4, 5).

The most serious problem with the use of DNA vaccines for human immunization is the theoretical possibility that foreign DNA introduced parenterally can integrate into the chromosomes of human cells (1).

The means of obtaining a glycoprotein herpes vaccine (3) involves the isolation and purification of surface glycoproteins followed by lyophilization.

All mentioned vaccines are used parenterally, but the means of obtaining them require special equipment, reagents, significant material costs, and a long time.

The invention is based on the task of increasing the effectiveness of vaccination, preventing adverse reactions during the administration of vaccines.

The goal is achieved by the fact that the live or killed vaccine is immobilized on an adsorbent (silicic acid methyl hydrogel) and administered orally.

The proposed method does not involve large costs, is fast, and the method of vaccine administration is safe.

The tool is carried out as follows: the virus-containing liquid is combined with enterosgel (methyl silicic acid hydrogel) and is administered orally in the volume of the filler, which causes a swallowing reflex.

Examples of specific execution:

Example 1.

Receiving herpes inactivated vaccine.

The herpes vaccine is produced in accordance with the Production Regulations for the inactivated, liquid and dry herpes culture vaccine (7).

A monolayer of primary trypsinized chicken embryo cells is infected with one of two strains of the herpes simplex virus (HSV): US (HSV-1) or VN (HSV-2) at a multiplicity of infection of 1-0.01 plaque-forming units (PSU) of the virus per cell. Cultivation is carried out at 36"C on roller installations for the development of cytopathogenic effect (CPE) at 90-10095. The virus-containing liquid (VVR) of one strain is combined by draining into one container.

Inactivation with formalin is carried out for 10 days (1122000 formalin by volume). Formolvaccine is a transparent reddish liquid, pH 7.5 ± 0.2, protein content 1500-2000 μg/ml. The active basis is the antigens of the herpes simplex virus, which stimulate the cellular mechanisms of antiviral resistance of the human body in the interrecurrence period.

Enterosgel manufactured by MP "Kreoma", M.Kyiv, at the rate of 100 mg/ml, is added to the liquid herpes vaccine. After shaking for 10 minutes on a magnetic stirrer, the mixture is left for 24 hours at 4"С.

The immunogenicity of the obtained preparation was studied during the immunization of white rats in parallel with the non-adsorbed commercial vaccine, which was administered according to the production regulations three times in 0.1 ml with an interval of

From days, alternating intraperitoneal and subcutaneous injection of material. The adsorbed vaccine was administered orally, three times, in the same volume (0.1 ml) and with the same interval (3 days). Blood was taken 5 days after the last immunization. The sera of immunized animals after heating at 567"C were used to determine the neutralization reaction. For this, 10-fold dilutions of HSV strain US up to 1:500,000 were made, strain

VN up to 1:50 thousand. Equal amounts (0.3 ml each) of each dilution were mixed with undiluted immune serum and with the serum of non-immune animals. The mixture was incubated for 1.5 hours, then 0.1 ml was added to vials with a monolayer of chicken embryo cells. After incubation at 367C for 30 minutes, 0.1 ml of the medium with antibiotics was added, the calculation after 96-144 hours by CPE of the monolayer.

The neutralization index is the logarithm of the virus titer in TKID5O (tissue infectious dose) in the control minus the logarithm of the virus titer in the presence of immune serum.

Thus, in both experiments, when the adsorbed vaccine was administered orally, the neutralization index was slightly higher (in one case - equal) than when the commercial vaccine was administered parenterally.

Example 2.

Obtaining influenza live adsorbed vaccine.

The work used allantoic virus-containing fluid obtained during the cultivation of influenza viruses on 10-day-old chicken embryos. Enterosgel was added at the rate of 100 mg/ml. The same method was used as for the preparation of the herpes vaccine: intensive shaking for 10 minutes and incubation for 24 hours at 426. The immunogenicity of the obtained preparation was studied during oral immunization of white mice with 0.1 ml (25 animals in each group). Native virus-containing fluid with the same hemagglutinating titer as in the experimental group was used as a control. Sera/obtained 21 days after immunization were tested in the hemagglutination inhibition reaction (HAI).

4 strains of influenza virus of 2 serosubtypes were studied. Immunization with adsorbed vaccine from the specified strains leads to the production of antibodies in higher titers than in the control group.

It should be noted that enterosgel adsorbent has a general detoxifying effect, has a number of other beneficial properties for the body, is quickly excreted from the body, does not penetrate into the epithelial cells of the mucous membrane, does not have a damaging effect on the mucous membrane of the stomach and intestines.

In addition, the oral method of administration does not cause post-vaccination reactions.

List of references. 1. Hendon Yu.3. Popynucleotidne (DNA) viral vaccine! and problems of their safety. ZhMZY, 1997, Mo1, 78-81. 2. Elshina G.N., Gorbunova M.A. et al. Evaluation of the effectiveness of influenza trivalent polymer-subunit vaccine "Grippol". ZhMZY, 1998, Me3Z, p. 40-43.

Z. Novitsky V.A., Klyushnyk S.Yu., Lviv N.D., Barinsky I.F., Zaides V.M. etc. The method of obtaining a glycoprotein herpes vaccine. Author's certificate Mo 1580618, Ab1K39/245. 02.08.88. chipboard 4. Petrov R.V., Khaitov R.M. Vaccines of the new generation based on synthetic polyones. History of creation, mechanism! actions, implementation in practice. Allergology. Asthma. Clinical immunology. 1998, Mo 7, 6. 1-11. 5. Petrov R.V., Hayitov R.M., Nekrasov A.V. and others. The method of obtaining vaccines! against the influenza virus. Author's certificate Me 1580617, A/61K39/145. 05/13/88. chipboard 6. Pushkarev M.A., Petukhov A.8., Eremeeva L.Y. etc. Method of obtaining inactivated vaccines! against the flu. Patent No. 1522494, Russia, MKY5 Ab1K39/145. The bulletin was invented on May 21, 1994. 7. The regulation of vaccine production! herpetic cultural inactivated liquid and dry; 1990.

Table 1

Index of neutralization in sera of rats after immunization

Strain Oral immunization Parenteral immunization of the virus with an adsorbed vaccine with a commercial vaccine 7 UsE 30717 from 17711125 |. 225

Table 2

Arithmetic average titers of antihemaplutins in sera of immunized mice

Native virus

A/NZM2I .A/Odesa/1/197 127.61-14.24 106.004-16.22

ALoganesburg/33/94 93.33-4-8.87 87.00411.72

A/Н1ТМИ1/

A/Dnipropetrovsk/ 310/95 89.28-4-12.90 61.434-7.42 d/Khabarovsk/74/77 79.28.-9.05 45.66-4.88

Claims (1)

The method of obtaining antiviral vaccines by immobilizing a live or inactivated virus on an adsorbent, which differs in that enterosgel is used in a ratio of 100 mg/ml of virus-containing liquid with subsequent shaking for 10 minutes, incubation for 24 hours at 4"C and oral administration into the body at with a filler that causes a swallowing reflex.