RU2225223C1 - Lyophilized antiherpetic vaccine - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: the present innovation deals with obtaining vaccine against viruses of herpes simplex (VHS) types I and II. The suggested vaccine contains suspension of VHS I and VHS II grown upon standardized cell line Vero B. The present vaccine is useful to be applied in research institutions and clinical practice in the field of health care. EFFECT: higher efficiency of vaccine. 3 ex

Description

 The alleged invention relates to medicine, namely to obtain a vaccine against herpes simplex viruses of types 1 and 2.

 Chronic herpetic infection is one of the most common immunodeficiencies in the world with a lifelong persistence of the virus in the nerve ganglia and periodic exacerbation of the disease with localization, usually “lokus minoris” on the skin, on the mucous membrane of the genitals, in the eyes, etc.

 The total number of patients with chronic forms of herpes infection in Russia is more than 20 million people. According to the Moscow Antiherpetic Center, the infection rate in the population of Moscow and the Moscow Region is about 90%. According to the WHO, mortality from herpetic encephalitis and disseminated forms of the disease is 15.8%, ranking second after influenza (35.8%) as the cause of death from viral infections.

 In connection with the above, the urgency of the problem of obtaining highly effective drugs against herpes simplex viruses 1 (HSV1) and 2 types (HSV2) becomes obvious.

 An antiherpetic vaccine is known, including HSV1 and HSV2 grown on fibroblasts of chicken embryos, and residual formalin (1).

Also known is an antiherpetic vaccine, which is a dosage form in the form of suppositories, containing a suspension of the vaccine (20 ml of suspension with 10 ⁸ PFU), 1.5-2 g of L-ascorbic acid, 1-2 g of hyaluronic acid, 1-2 g of L- tocopherol, (3-5) 10 ⁶ international units of L ₂ -interferon. Each candle weighing 1.5-2 g, containing <500 μg / g protein, has a residual moisture content of <2.5% and a physiological pH value of 7.5 + 0.2 (2). It is prepared as follows: HSV1 and HSV2 viruses were grown on a transplantable Vero cell line (green monkey kidney), purification and concentration were performed by filtration through porous membranes, and viruses in the filtrate were inactivated by gamma radiation (2).

 The objective of our research was to obtain a lyophilized antiherpetic vaccine that ensures the creation and maintenance of a long-lasting intense immunity in the human body against herpes virus types 1 and 2, which preserves the stability of its properties over its shelf life and does not cause allergic reactions.

The proposed vaccine is, vol.%:
A suspension of strains of HSV1 and HSV2 grown on a standardized Vero B cell line (green monkey kidney) in 179-190 passages with an activity of 10 ^8.0 -10 ^8.5 lg TCD _{50 / ml} and 40% offical formalin solution to a final concentration 0.014% - 91.5 - 92
70-75% Sucrose solution - 7.0 - 7.5
10% Official gelatose solution - 1.0
A method of manufacturing a vaccine is as follows:
in a support medium (Eagle’s medium without adding serum of a cow embryo and donor human albumin) to a monolayer of transplantable Vero B cell lines with a seed cell concentration (0.5-1.0) • 10 ⁵ cells / ml we cultivate herpes simplex viruses HSV1 and HSV2; viruses were released from cells by freezing and thawing, and then processed in an ultrasonic disintegrator; virus inactivation was carried out with formalin followed by deformalization; purification from destroyed cells (detritus) was carried out by centrifugation at 2000-2500 rpm for 30-60 minutes, then the vaccine was lyophilized.

Specific examples of implementation are as follows:
Example 1
To prepare 1 L of the vaccine, a monolayer culture of Vero B cells with a concentration of 0.7 × 10 ^{5 was} taken, grown monolayer at 37 ^° C for 48 h until a dense cell monolayer was formed. Then, virus-containing material was added at a dose of 0.03 TCD ₅₀ / ml, viruses were adsorbed for 30 minutes and incubated for 24 hours (HSV1) and 48 hours (HSV2) until the monolayer was completely destroyed, after which the mattresses with the contents were subjected to a single freeze at temperature minus 40-45 ^o C and thawing at room temperature, followed by treatment in an ultrasonic disintegrator until the virus is completely released from cells with a virus concentration of 10 ^8.0 TCD ₅₀ / ml. Formalin (1: 1800) was added to the virus-containing liquid and kept on a magnetic stirrer for 3 days at a temperature of 37 ^° C. Deformalization was carried out for 10 days at a temperature of + 4 ^{° C.} to a final formalin content of 170 μg / ml. By centrifugation at 2000 rpm for 60 min, Vero B cells and their residues were separated. The resulting suspension was lyophilized. The proposed vaccine was immunized with 10 male white rats weighing (110 ± 10) g three times (intraperitoneally, subcutaneously, intraperitoneally) in 1.0 ml of the vaccine; intervals between injections 3 days. The vaccine induced the synthesis of virus-neutralizing antibodies with a neutralization index for HSV-2,7 IgTCD ₅₀ / ml; for HSV2 - 2.5 lgTCD ₅₀ / ml.

Example 2
To prepare 1 l of the vaccine, a monolayer culture of Vero B cells with a concentration of 0.8 • 10 ⁵ cells / ml was taken, and the cultivation, adsorption of viruses, destruction of the monolayer was carried out analogously to example 1, the virus titer was 10 ^8.0 lgTCD ₅₀ / ml. Formalin was introduced at a dilution of 1: 1900 and deformalization was carried out to a final formalin content of 160 μg / ml. By centrifugation at 2500 rpm for 30 min, Vero B cells and their residues were separated.

 The resulting suspension was lyophilized.

In the experiment, 10 male white rats weighing (110 ± 10) g were examined, immunized three times (intraperitoneally, subcutaneously, intraperitoneally) with 1.0 ml of the vaccine; intervals between injections 3 days. The vaccine induces the synthesis of virus-neutralizing antibodies with a neutralization index for HSV1-3.0 lgTCD, ₅₀ / ml; for HSV 2-2.7 lgTCD ₅₀ / ml.

Example 3
To prepare 1 l of the vaccine, a monolayer culture of Vero B cells with a concentration of 0.1 • 10 ⁵ cells / ml was taken, and the cultivation, adsorption of viruses, destruction of the monolayer was carried out analogously to Example 1, the virus titer was 10 ^8.5 lgTCD ₅₀ / ml. Formalin was introduced at a dilution of 1: 1800 and deformalization to a final formalin content of 150 μg / ml. By centrifugation at 2000 rpm for 60 min, Vero B cells and their residues were separated. The resulting suspension was lyophilized.

10 male white rats weighing (110 ± 10) g were taken into the experiment; they were immunized three times (intraperitoneally, subcutaneously, intraperitoneally) with 1.0 ml of the vaccine; intervals between injections 3 days. The vaccine induced the synthesis of virus-neutralizing antibodies with a neutralization index for HSV1 1-3.0 lgTCD ₅₀ / ml; for HSV2-3.0 lgTCD ₅₀ / ml.

 The proposed Vitagerpavak vaccine can also be used in the form of suppositories (suppositories). At the same time, filler is additionally introduced into it - confectionery fat in the amount of 97-98%.

In the proposed vaccine due to:
- the exclusion of cow embryo and donor human albumin from the serum support medium allowed to reduce the content of foreign proteins to 100-200 μg / ml (causing allergic reactions when administered to humans), while maintaining the volume of virus-containing biomass and the activity of viruses;
- the use of methods of freezing and thawing Vero B cells with subsequent processing in an ultrasonic disintegrator, allowed to increase the titer of viruses to 10 ^8.0 - 10 ^8.5 lgTCD ₅₀ / ml;
- Subsequent treatment with formalin of the virus-containing fluid made it possible to completely inactivate the herpes virus, and also contributes to the complete isomerization of the residual DNA of the transplanted Vero B cell line and the denaturation of cell proteins, which eliminates the risk of tumorigenicity of this vaccine.

The proposed vaccine was tested in full in the framework of state preclinical trials under the guidance of the State Institute of Standardization and Control of Medical and Biological Preparations named after L.A. Tarasevich of the Ministry of Health of the Russian Federation in 2001-2002 with a positive result:
- Vitagerpavak vaccine has high immunogenicity, provides the body with the creation and maintenance of long-term intense immunity;
- non toxic;
- does not cause allergic reactions;
- not tumorigenic.

 - in a lyophilized form preserves the antigenicity and stability of the drug over the entire shelf life.

 The Vitagerpavak vaccine will find application in research institutions and in the clinical practice of healthcare in the Russian Federation.

 We ask you to keep the term “Vitagerpavak” in the name of the vaccine, according to the documentation for it approved by the Ministry of Health of the Russian Federation.

 Sources of information.

 1. RF patent 2014084, cl. A 61 K 39/245, 1994

 2. RF patent 2142816, cl. A 61 K 39/12, 1997

Claims (1)

A lyophilized antiherpetic vaccine containing a suspension of the herpes simplex virus strains HSV1 and HSV2 obtained by culturing these strains on a standardized Vero B cell line followed by treatment of the culture medium with 40% formalin solution and lyophilization with the addition of a sucrose-gelatinous protective medium.