RU2095083C1 - Rotaviral inactivated vaccine for animal immunization - Google Patents

Abstract

FIELD: virology, immunology, veterinary science. SUBSTANCE: vaccine has strain of simian rotavirus SA-11 and platinum (II) derivative with DNA polyanion at concentration (5.3-81) x 10^-3 (as measured for platinum). EFFECT: high immunogenicity of vaccine, safety and stability. 3 tbl

Description

 The invention relates to the field of virology, in particular to the production of inactivated vaccines for immunization of animals, and can be used for specific prophylaxis of rotavirus gastroenteritis.

Known inactivated rotavirus vaccine for passive immunization of calves against rotavirus gastroenteritis (Gastrucei G. Fregeri F. Ferrari M. // Comp. Immunol. Microbiol. And Infect. Disiases. 1989, vol. 12, N 3, p. 71-76) [ 1] The vaccine consists of cattle rotavirus with a biological activity of 3 • 10 ⁸ CPD 50 / ml and contains formalin as an inactivator in a final concentration of 0.05 to 0.10% and sodium bisulfate.

 Before the vaccine is administered to animals (cows), an adjuvant is added to it - a water-in-oil emulsion, which increases its immunogenicity.

 As follows from the technical essence, the known technical vaccine is toxic, because it is practically impossible to achieve complete inactivation of formalin, which is toxic to microorganisms (Methods of laboratory studies on rabies. Part V. Methods for determining the harmlessness and immunogenicity of vaccines / Edited by Martin M Kaplan, H. Lary Koprorosky. WHO. M. Medicine, 1975, 358 pp.) [2] has low immunogenicity due to the need to introduce an adjuvant into it, the action of which is aimed at increasing immunogenicity (Reference ju of bacterial and viral preparations / Under the editorship of Dzagurov SG Rezepov F.F.M. Medicine, 1975, 223 p.) [3] and is characterized by instability during storage, because it is known (Petrov R.V. Khaitov R.M. Artificial antigens. M. Medicine, 1988, 288 pp.) [4] that the stability of rotavirus vaccines does not exceed 24 months.

Closest to the invention in technical essence and the achieved effect is a rotavirus vaccine containing monkey rotavirus SA-11, inactivated by formalin (WO 94/01134, class A 61 K 39/42, 1994) [5]
For the vaccine [5] the same disadvantages are inherent as for the vaccine [1] toxicity, low immunogenicity, storage instability. Confirmation of these properties of the vaccine [5] are the data obtained in the study of rotavirus vaccine using viral material obtained by us from a monkey strain of rotavirus SA-11 with antigenic activity of 1,024 in RNGA (indirect hemagglutination reaction) GAE / 0.25 ml and biological activity 10 ^5.5 CPD 50 / ml. The vaccine is prepared by adding formalin to the viral material to a final concentration of 0.1% and subsequent inactivation of the virus for 72 hours at 37 ^° C. The obtained formalized vaccine is tested for immunogenicity in rabbits and the stability of rotavirus antigens during storage. The results are shown in table 1, which reflects the main properties of the vaccine.

 As follows from the data in Table 1, a rotavirus vaccine obtained from a virus with high antigenic activity has low immunogenicity against rotavirus gastroenteritis, expressed as a titer of antirotavirus antibodies in RTNGA (inhibition of indirect hemagglutination), equal to 1,640; characterized by low stability of only 24 months and is toxic.

 The disadvantages of obtaining a known vaccine should include a rather long duration of its receipt, due to the slow progress of inactivation of the virus with formalin.

 The basis of the invention is the task to develop the composition of a rotavirus inactivated vaccine for immunization of animals, consisting of a monkey strain of rotavirus SA-11 and a platinum derivative (II) with a polyanion of deoxyribonucleic acid (DNA), which allows to obtain a non-toxic vaccine characterized by high immunogenicity against rotavirus gastroenteritis and stability at the minimum cost of time for its receipt.

To solve this problem, a rotavirus vaccine inactivated for immunization of animals is proposed, containing a monkey rotavirus vaccine strain SA-11 and platinum (II) derivatives with a polyanion of deoxyribonucleic acid (DNA) of the general formula [1] [PtCl (NH ₃ )] ₆ (OH) _x (H ₂ O) _y } _n -DNA ^-m ,
where DNA ^-m = - (C ₃₉ H _55-r O ₃₂ N ₁₅ P ₄ ) - _n ^-m ;
n = 2000 ± 200;
at x = 0, y = 12, m = 6n, r = 6;
x = 2, y = 10, m = 4n, r = 4;
x = 0, y = 6, m = 6n, r = 6;
in the following ratio of components, in wt.

Derivatives of platinum (II) with a DNA polyanion of the general formula [1] (in terms of platinum) (5.3 81) • 10 ^-3
Vaccine strain of monkey rotavirus SA-11 with antigenic activity (1-64) - (1-1024) in RNGA GAE / 0.25 ml and biological activity of 1 • 10 ^5.5 -1 • 10 ^7.5 CPD 50 / ml Else
The difference of the proposed vaccine is the presence in its composition of platinum (II) derivatives with a polyanion of DNA as an inactivating agent.

 We have shown that a system consisting of a vaccine strain of monkey rotavirus SA-11 and the claimed platinum (II) derivatives with DNA polyanion provides the formation of an immunostimulating complex "rotavirus protective antigens of platinum (II) derivatives with DNA polyanion". We believe that the complex provides effective inactivation of the virus, enhances immunogenicity and increases the stability of the vaccine while significantly reducing the time it takes to receive a non-toxic inactivated vaccine.

Derivatives of platinum (II) with a polyanion of DNA are known (Patent EP N 0374267, class A 61 K 35/28, 31/555, 1990) [6] These compounds are synthesized for use as an active substance of an antitumor drug. Platinum compounds do not have an immunosuppressive effect and are not toxic in the concentrations used to prepare the vaccine (a half-lethal dose of LD ₅₀ is 102 mg / kg body) [6] while a maximum of 144 mg is consumed per animal immunization.

 Thus, on the basis of the properties inherent in these compounds of platinum, it does not follow explicitly that their introduction into the composition of the vaccine will give it high immunogenicity and stability.

 According to the invention, the vaccine obtained is non-toxic, has high immunogenicity, characterized by titers of antibodies in RTNGA at the level of 1 16400, increased stability, characterized by the constancy of titers of antirotavirus antibodies in RTNGA for at least 40 months under standard storage conditions.

The vaccine is prepared as follows. A cell culture, for example Vero, is infected with rotavirus of group A monkeys with antigenic activity (1 64) (1 1024) RNA GAE / 0.25 ml and biological activity 1 • 10 ^5.5 -1 • 10 ^7.5 CPD 50 / ml, activated trypsin. The infected cells are incubated at 37 ^° C. for 48 hours, and then the infected cells are destroyed by freezing and thawing three times to increase the yield of the virus. Then the viral material is clarified by low-speed centrifugation and introduced into interaction with a platinum derivative (II) with a DNA polyanion, thermostated at 37 ^° C for at least 90 minutes to inactivate the virus.

As derivatives of platinum (II) with a DNA polyanion of the general formula [1] [PtCl (NH ₃ )] ₆ (OH) _x (H ₂ O) _y } _n -DNA ^-m ,
where DNA ^-m = - (C ₃₉ H _55-r O ₃₂ N ₁₅ P ₄ ) - _n ^-m ;
n = 2000 ± 200
use the following compounds:
compound I at x = 0, y = 12, m = 6n, r = 6;
poly {hexakis [chlorammindiaquaplatine (II)] - μ-deoxyribonucleate of the formula
[PtCl (NH ₃ )] ₆ (H ₂ O) ₁₂ } _n -DNA ^-m ,
where n = 1900 ± 90; m is 6n;
DNA- ^m = - (C ₃₉ H ₄₉ O ₃₂ N ₁₅ P ₄ ) - _n ^-m ;
compound II at x = 2, y = 10, m = 4n, r = 4;
poly {bis [gidroksohloroamminakvaplatina (II)] - tetrakis [hloroammindiakvaplatina (II)] - m-deoxyribonucleat formula [PtCl (NH _{3)] 6} (OH) ₂ (H ₂ O) _{10} n} -DNA ^-m,
where where n = 2000 ± 100; m is 4n;
DNA- ^m = - (C ₃₉ H ₅₁ O ₃₂ N ₁₅ P ₄ ) - _n ^-m ;
compound III at x = 0, y = 6, m = 6n, r = 6;
poly {hexakis [chloroamine-quinaplatine (II)] - m-deoxyribonucleate of the formula [PtCl (NH ₃ )] ₆ (H ₂ O) ₆ } _n -DNA ^-m ,
where n = 2100 ± 100; m is 6n;
DNA ^-m = - (C ₃₉ H ₄₉ O ₃₂ N ₁₅ P ₄ ) - _n ^-m .

 Compounds I, II, III are fine crystalline yellow powders, odorless, stable in air, capable of being stored for one year without changing properties.

 The resulting final product is an inactivated rotavirus vaccine for immunizing farm animals against rotavirus gastroenteritis and has the following ingredient composition, in wt.

Derivatives of platinum (II) with a DNA polyanion of the general formula [1] (in terms of platinum) (5.3 81) • 10 ^-3
Monkey rotavirus vaccine strain SA-11 with antigenic activity (1 64) - (1 1024) in RNGA GAE / 0.25 ml and biological activity 1 • 10 ^5.5 -1 • 10 ^7.5 CPD 50 / ml Else
A series of the resulting rotavirus vaccine is tested for immunogenicity, toxicity, sterility, safety and stability.

Immunogenicity of the vaccine specific activity is investigated in laboratory tests with the erythrocyte rotavirus diagnosticum of the company "Rostepidkompleks" in the inhibition of indirect hemagglutination and expressed by antitavirus antibody titers in RTNGA (Zarubinsky V.Ya. Kolpakov S.A. Virology, 1989, N 2, pp. 250-253) [7]
The stability of the vaccine is characterized by the time during which the titer of antirotavirus antibodies in RTNGA remains constant.

The toxicity of the vaccine is determined by the standard method and is evaluated by cytological doses of CTD 50 / ml [2]
The safety of the vaccine is characterized by the absence of infectious activity in the cell culture and is determined by the CTD 50 / ml [2]
Sterility of the vaccine is characterized by the absence of m / v growth when plated on sugar broth, MPA, Saburo medium (General requirements for the sterility of biological preparations // Requirements for biological preparations. N6 Wld.Hlth Org. Techn. Rep. Ser. 1972, N 486 ) [8]
An example implementation according to the invention. A suspension of Vero cell culture in a growth medium consisting of Eagle’s basic medium with 10% fetal calf serum, inoculated at a concentration of 300 thousand cells per ml, is introduced into the culture vessels and grown under stationary conditions until monolayers form in them. Then, the culture medium from the vessels is removed and introduced onto a monolayer of inoculated rotavirus of monkeys of strain SA-11, activated by trypsin, in a final concentration of 10 μg per 1 ml of viral suspension, with antigenic activity 1024 in RNGA GAE / 0.25 ml and biological (infectious) activity 10 ^5.5 CPD 50 / ml. The adsorption of the virus is carried out for 2 hours at a temperature of 37 ^o C. Then the non-adsorbed virus is removed, and monolayers of cells are washed twice with any balanced saline solution, for example, Hanks solution. After that, the virus propagation medium consisting of the standard basic medium Eagle and trypsin (final trypsin concentration in the medium 1 μg / ml) is added to the vessels. The infected cultures are incubated at 37 ^° C for 48 hours. After the incubation is over, the remains of the cell monolayer together with the virus-containing liquid are subjected to three freezing and thawing, virus-containing material is collected and it is freed from cell debris by centrifugation at 1000 g for 20 minutes.

Then, to a 100 ml clarified viral suspension, add 10 ml of a solution of poly {hexakis [chloroamminacquaplatine (II)] - m-deoxyribonucleate (compound III) at a final concentration of 0.2136 mg / l (in terms of platinum) and incubated at 37 ^o C in within 90 minutes

 The resulting product is an inactivated rotavirus vaccine in the following ratio of components, in wt.

Derived platinum (II) with a polyanion of DNA (in terms of platinum) (compound III) 21 • 10 ^-3
Vaccine strain of monkey rotavirus SA-11 with antigenic activity of 1024 in RNGA GAE / 0.25 ml and biological activity of 1 • 10 ^5.5 CPD 50 / ml - Else
The vaccine is non-toxic, safe, sterile, has an immunogenicity of 1 16400 RTNGA in rabbits, stable for 40 months (table 2, example 5).

 Table 2 shows the vaccine compositions obtained using monkey SA-11 rotavirus, which has different antigenic activity, and containing platinum (II) derivatives with DNA polyanion, the amount of which is both in the claimed range and beyond.

 It was found that the quantitative and qualitative composition of the vaccine is selected from conditions that ensure complete inactivation of SA-11 monkey rotavirus, immunogenicity in rabbits, and vaccine stability with a minimum virus inactivation time (Table 2, examples 1-19).

The optimal from the point of view of achieving a technical result is the content in the vaccine of platinum (II) derivatives with a polyanion of DNA in the range (5.3 81) • 10 ^-3 wt. (in terms of platinum).

 The content of platinum (II) derivatives in the vaccine with a DNA polyanion below the claimed limit, even when using SA-11 monkey rotavirus with high antigenic activity, does not provide the vaccine with sterility, safety and high immunogenicity against rotavirus gastroenteritis: the titer of antirotavirus antibodies in RTNGA is only 1,640 that is at the level achieved by the known vaccine (table 2, example 21).

 The upper limit of the content of platinum (II) derivatives with DNA polyanion in the vaccine is limited in that a further increase does not contribute to improving the quality of the vaccine and is impractical (Table 2, Example 22).

 The optimal value of the antigenic activity of rotavirus SA-11, which provides high vaccine efficacy in the claimed range of platinum (II) derivatives with a DNA polyanion, is (1 64) (1 1024) in RNGA GAE / 0.25 ml.

 The use of rotavirus with antigenic activity below the claimed limit at the maximum content of compound III (platinum) results in a vaccine with low immunogenicity: the antiviral antibody titer is 1 1280, which practically corresponds to the immunogenicity of the known vaccine (Table 2, Example 20).

 The upper limit of the antigenic activity of rotavirus monkeys SA-11 is limited by the biological properties of the virus-cell system. It was experimentally established that within 1.5 hours complete inactivation of monkey rotavirus SA-11 is achieved in the presence of a platinum derivative (II) with a DNA polyanion in the claimed range of its content, which ensures the production of an inactivated vaccine.

 Reducing the time of inactivation of rotavirus leads to a vaccine, which is dangerous, because contains non-inactivated viral material (table 2, example 24).

 Reducing the time of inactivation of rotavirus does not affect the improvement of the quality of the vaccine and is impractical (table 2, example 23).

 The data in table 3 confirm the high immunogenicity of the proposed rotavirus vaccine when vaccinating various species of animals.

 The advantages of the proposed rotavirus inactivated vaccine for immunization of animals compared with the known rotavirus vaccine are confirmed by the data presented in tables 1 and 2.

As follows from a comparative analysis of the quality and effectiveness of vaccines, the proposed vaccine is superior to the known in the following indicators:
by immunogenicity against rotavirus gastroenteritis by 6 25 times, which is expressed in an increase in titers of antirotavirus antibodies by 6 25 times, for example, in rabbits;
stability approximately 2 times, which is expressed in an increase in the constancy time of the titers of antirotavirus antibodies approximately 2 times;
is non-toxic.

 The proposed vaccine, as well as the known one, is sterile and safe.

 In addition, to obtain the proposed vaccine requires 48 times less time due to faster and more effective inactivation of the virus.

 It should be emphasized that the developed inactivated rotavirus vaccine corresponds to the main directions of creating modern vaccine preparations, which consists in improving their compositions by using immunostimulating complexes (ISKOMs).

Claims (1)

A rotavirus vaccine inactivated for immunization of animals, comprising a vaccine strain of monkey rotavirus Sa II and an inactivating agent, characterized in that platinum (II) derivatives with a polyanion of deoxyribonucleic acid (DNA) of the general formula I [PtCl (NH ₃ )] ₆ are used as an inactivating agent. (OH) _x (H ₂ O) _y } _n - DNA ^{- m} ,
where DNA ^{is m} (C _{3 9} H _{5 5} - rO _{3 2} N _{1 5} P ₄ ) _n ^{- m} ;
n 2000 ± 200,
at x 0, y 12, m 6n, r 6;
x 2, y 10, m 4n, r 4;
x 0, y 6, m 6n, r 6,
in the following ratio of components, wt. Derivatives of platinum (II) with a DNA polyanion of general formula I (in terms of platinum) (5.3 81) • 10 ^{- 3}
The vaccine strain of rotavirus monkeys SA II with antigenic activity (1 - 64) (1 1024) in RNGA GAE / 0.25 ml and biological activity 1 • 10 ^{5 , 5} 1 • 10 ^{7 , 5} CPD 50 / ml - Remains

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