RU2053297C1 - Method of measles virus cultivation - Google Patents

Abstract

FIELD: virology. SUBSTANCE: strain L-16B is cultured at infecting dose 0.5-0.05 TCD₅₀/kl on the supporting Eagle medium or medium 199 in the presence of 3-9 vol.% directively added amino acids from the group of essential amino acids or aminopeptide. The yield of virus on the 1-l flat bottle is 560 ml at titer value 6.5 Ig TCD₅₀/0,5 ml. EFFECT: improved method of cultivation. 5 cl, 4 tbl

Description

 The invention relates to biotechnology and can be used to obtain measles vaccine.

 Known methods for producing measles vaccine from producer strains: Schwartz strain, Edmonston-Zagreb, CAM-70, AIK-C (S. J. Clements, et al. Research into alternative measles vaccines in the 1990's World Health Organization, EPI / GEN / 88.11).

 The disadvantage of the methods of cultivation of these strains is their prolonged passage on human tissue cultures, which in turn reduces reproductive activity and, as a result, causes a decrease in the immunogenic activity of the vaccine obtained from it.

 The main characteristics of the process of obtaining measles vaccine are determined by the technology of cultivation of strains of certain species.

 In this regard, it is known that Schwartz, AIK-C, and CAM-70 strains are grown in chicken embryo fibroblast culture, and Edmonston-Zagreb in human diploid culture (C. J. Clements et. Al. Specified work). No other technological information was found by the authors.

 Two domestic strains are also known: Leningrad-16 and Leningrad-16V (L-16 in accordance with the regulation N 196-74 of the measles culture live dry vaccine, change N 175/4, 1988 and its variety L-16V in accordance with the State Standard N 2277).

 So strain L-16 is cultured by a known method in a support medium 199 with one of the following additives: 0.4% albumin; 5% sorbitol and antibiotics; 5% aminopeptide. (prototype method).

Strain A-16B was obtained by passage of the prototype strain L-16 taken after 22 year of storage in the collection of production strains in tissue culture of quail embryos fibroblasts (FEP) at a low temperature, namely 30 ° ^C. Selection was performed by the authors on the criterion of absence of neurovirulence, which was tested on monkeys, because the original strain L-16 contains neurovirulent viral particles.

 However, known methods of culturing these producers give a low biomass yield, which also reduces the immunogenic activity of the resulting vaccine.

 The aim of the invention is to increase the yield of viral biomass, i.e., increase its reproductive activity.

This goal is achieved by the fact that according to the method of cultivating measles virus in a support medium containing amino acids, the measles virus L-16B is cultivated in medium 199 or Needle medium with the additional introduction of amino acids with their total content of 3.0.9.0 vol. while the specific infectious dose of the virus is set equal to 0.5.0.05 TCD ₅₀ / cell.

 In addition, amino acids are administered as an aminopeptide. Amino acids from the group of essentials are also introduced: valine, trionin and tryptophan.

In this case, the group of essential amino acids is contained in the following concentrations, g / l; Valine 0.5.1.0 Trionin 0.5.1.0 Tryptophan 0.5.0.8 Isoleucine 1.0.1.5 Leucine 1.0.1.5 Lysine 1.0.2.0 Methionine 0.5.0.8 Phenylalanine 0.7.1.4
In addition, the infectious dose is administered on a porous carrier.

 According to our data, the Needle medium was not used for the cultivation of measles virus. The proposed specific infectious dose exceeds the known one by an order of magnitude, which, in our opinion, is not obvious in relation to the achievement of the above goal. The amino acid content (3.0.9.0 vol.) In this formulation also has novelty. The sharing of support medium 199 with additional directed administration of amino acids has not been previously practiced either.

 Introduction to the supporting medium of the missing amino acids: valine, trionin and tryptophan creates the conditions for the full development of microorganisms and, consequently, a significant increase in the total yield of useful viral biomass.

 The amino acid content in the support medium in very small concentrations up to 100 mg / l creates a qualitative barrier between the conditions for the survival of microorganisms and the conditions for their reproduction. Compliance with the latter is necessary in terms of their industrial cultivation. The introduction of basic amino acids from the group of indispensable, i.e., non-reproducible cells, in the above concentrations allows you to create just such optimal conditions.

In addition, to increase the yield of the virus, an aminopeptide is used as an additive to the supporting medium, which is produced at meat processing plants by treating cattle tissues with aminopeptidase enzymes contained in the stomachs of cows. As a result, an unpredictable composition of short fragments of protein molecules is obtained: short peptide chains, amino acids, the COOH carboxyl group of one amino acid is connected to the amino group NH _{2 of} another amino acid. All these short chains do not possess antigenicity and the ability to stimulate the formation of antibodies in the body. But the aminopeptide additive supplies the nutrient medium (supporting medium + amino acids in any form) with all 20 amino acids necessary for the growth and functioning of cells in the synthesis of the virus in them, for the construction of its protein coat.

 The introduction of an infectious dose on a porous carrier, together with a significant increase in the infectious dose (by an order of magnitude), can significantly increase the yield of cultured viral biomass due to the more developed surface used to form a producing cell monolayer.

 Compared with the prototype, the invention has a new set of essential features, that is, meets the criterion of "novelty."

 The set of distinguishing features of the invention among the known solutions was not found. This allows us to argue that the invention meets the criterion of "materiality of differences."

 The combination of general and private essential features of the invention ensures the achievement of the purpose of the invention.

 PRI me R 1.

In one-liter mattresses with tissue culture of solar cells in the growth medium 199 with the addition of 10 vol. bovine serum making A-16B strain measles virus infecting doses of 0.04 to 0.6 TCD ₅₀ / c (for each infection dose on the mattress 5) and incubated in roller at ³⁵ ° C and about 0.3 speed / min before the formation of a monolayer of cells, which occurs no later than 3 days of incubation. Then the growth medium is removed, and the monolayer is washed 6 times from the serum with Hanks pH 7.4.

Then, 160 ml of maintenance medium Needle with 6 vol. Are added to mattresses with an infected cell monolayer. aminopeptida or amino acids from the group of essential and produce virus in roller culture at ³⁵ ° C and 0.3 rev / min during the term of preservation of tissue culture (in this period various mattresses ranges from 18 to 22 days). With increasing cytopathic changes in the cell monolayer, typical of measles virus (observed under a microscope), virus-containing liquid is decanted into vials and stored under a rubber stopper at -20 ^{° C} for later use, and in mattresses added to 160 ml of fresh maintenance Eagle medium with the above amount of aminopeptide or amino acids and cultured under the same conditions. This procedure is repeated many times. Moreover, the medium replacement interval is 1-2 days. Under these conditions, the number of useful removals (cycles in which the virus content is not less than 4 lgTCD ₅₀ / 0.5 ml) is not less than 5. All operations of the method are performed under sterile conditions.

 The cultivation results are given in table. 1.

As can be seen from table 1, an increase in the infecting dose of 10-30 times in comparison with the prototype allows to obtain viral biomass with a titer of 6.2-6.5 lg TCD ₅₀ / 0.5 ml on this medium, while at a lower infectious dose ( 0.04 TCD ₅₀ / cell) this titer is only 5.2 lg TCD ₅₀ / 0.5 ml. At the same time, the maximum yield by volume (700-760 ml / mattress) is achieved in the range of infectious doses from 0.1 to 0.3 TCD ₅₀ / cell due to an increase in the number of useful virus removals (possible cultivation cycles).

 PRI me R 2.

In 25 1-liter mattresses with tissue culture FEP in the growth medium 199 with the addition of 10 vol. serum of cattle contribute measles virus of strain L-16B at an infectious dose of 0.2 TCD ₅₀ / cell and incubated as in example 1. Next, 160 ml of maintenance medium Needle with 2.3 are added to mattresses with an infected cell monolayer; 5.8 and 13 vol. aminopeptide or the specified group of amino acids (Fibonacci parametric series, used, as is known, to increase the accuracy of a one-factor experiment), using 5 mattresses for each value of this parameter.

 The cultivation of the virus is carried out as in example 1.

 The cultivation results are given in table. 2.

From the table. Figure 2 shows that when the aminopeptide content in the supporting medium is 5–8 vol. the maximum virus yield is 500 ml / mattress with a titer of 5.6-6.3 lg TCD ₅₀ / 0.5 ml. Outside this range of aminopeptide concentration, the virus yield is much smaller both in volume and in titer.

 PRI me R 3.

 In 25 1-liter mattresses contribute 50 million cells of solar cells in the growth medium 199 with the addition of 10 vol. cattle serum. After the formation of the monolayer of cells, the growth medium is removed, the cell monolayer is washed from the serum as in example 1 and infected with the measles virus of strain L-16B in the same doses as in example 2. The remaining operations are carried out as in example 2.

 The cultivation results are given in table. 3.

From table 3 it is seen that when the content of aminopeptide or amino acids in the supporting medium, the Needle 5-8 vol. the maximum virus yield is 550 ml / mattress with a titer of 5.5-6.2 lg TCD ₅₀ / 0.5 ml. Outside this range of aminopeptide or amino acid concentration, the yield of the virus decreases both in volume and in titer.

 PRI me R 4.

 A cell culture of FEP in 10 mattresses is infected with the L-16B virus as in Example 1.

 In 5 mattresses, the Needle medium with the addition of 5 vol. aminopeptide or amino acids, and in the remaining 5 mattresses, the virus is cultured using medium 199 with the same amount of aminopeptide or amino acids.

 Other cultivation conditions set in example 1.

 The cultivation results are given in table. 4.

 As can be seen from the table. 4, with equal infectious doses, the yield of the virus on the Eagle medium is greater than on the 199 medium both in volume and concentration.

 PRI me R 5.

 Comparative cultivation of strains L-16 and L-16B.

5 1-liter FEP cell culture mattresses are infected with measles L-16 virus (prototype strain) at a dose of 0.01 TCD ₅₀ / cell. The other 5 mattresses are infected with measles L-16B virus at a dose of 0.1 TCD ₅₀ / cell.

 For the cultivation of strain L-16 using support medium 199 with 5 vol. aminopeptide or amino acids, and for strain L-16B, Needle medium with the same amount of nutritional supplement.

 Conditions for removal of virus-containing liquid and cultivation as in example 1.

The average yield of L-16 virus from one mattress is 360 ml with a titer of 5.2 lg TCD ₅₀ / 0.5 ml, and of L-16B virus 560 ml with a titer of 6.5 lg TCD ₅₀ / 0.5 ml.

The collected virus-containing materials are combined by strain, add a stabilizer (gelatin with sucrose at a final concentration of 1 vol.), Diluted with Eagle medium to a concentration of 4.5 lg TCD ₅₀ / 0.5 ml, poured into ampoules and freeze-dried.

The result is 36,000 doses of the vaccine strain L-16 and 526,000 doses of the vaccine strain L-16B in a single dose of 2000 TCD ₅₀ / 0.5 ml).

 In this example, the volume of the vaccine from strain L-16B is 14 times greater than from the prototype.

 All the above examples were tested on porous and conventional carriers. Micro- and macroporous glass were used as porous structures, as well as fermenters with latex or cytodex particles. In all cases, the use of porous carriers was achieved up to 15-40% increase in useful biomass.

 PRI me R 6.

Strain L-16B is cultivated as in example 1 at an infectious dose of 0.2 TCD ₅₀ / CL. The grown virus was controlled by immunization of 83 seronegative children at the age of 1 year and older. Seroconversion (the formation of specific antibodies) was detected in 100% vaccinated. According to RTGA, the geometric mean antibody titer in these children is 6.44 log ₂ .

 Reactogenicity of the vaccine virus does not exceed the norm regulated by scientific and technical documentation. A specific reaction to the introduction of the vaccine (short-term catarrhal phenomena and hyperemia of the pharynx) was observed in 7 children.

When compared with the immunogenicity of the prototype vaccine from strain L-16, seroconversion was observed in 97.9% of 46 vaccinated seronegative children. The geometric mean antibody titer was 6.05 log ₂ , which is lower than the immunogenicity of the vaccine from strain L-16B.

 As explained by the above examples, the proposed new method for the cultivation of measles vaccine provides, in comparison with the prototype, an increase in the virus yield by titer and metabolism, which allows a 20-fold increase in the vaccine yield. Moreover, the resulting target product has a higher immunogenicity and, therefore, meets the criterion of "positive effect".

Claims (4)

1. METHOD FOR CULTIVATION OF KORI VIRUS in a support medium containing amino acids, characterized in that the strain of measles virus L-16 or L-16B is cultured in medium 199 or in Needle medium with an additional introduction of amino acids with their total content of 3.0-9.0 vol .%, the specific virus infecting the dose is set to 0.5 - 0.05 CDT _{5 0 / k l.}  2. The method according to claim 1, characterized in that the amino acids are administered as an aminopeptide.  3. The method according to claim 1, characterized in that the amino acids from the group of irreplaceable are introduced: valine, trionin and tryptophan. 4. The method according to claim 3, characterized in that the group of essential amino acids is contained in the following concentrations, g / l:
Valine - 0.5 - 1.0
Trionin - 0.5 - 1.0
Tryptophan - 0.5 - 0.8
Isoleucine - 1.0 - 1.5
Leucine - 1.0 - 1.5
Lysine - 1.0 - 2.0
Methionine - 0.5 - 0.8
Phenylalanine - 0.7 - 1.4
5. The method according to claim 1, characterized in that the infectious dose is administered on a porous carrier.

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