SU1318149A3 - Method of producing vaccine against russian tick-borne ence phalitis - Google Patents

Abstract

Meningo-encephalitis virus (TBE-virus) vaccines are produced by culturing the virus in tissued cultures or in suspensions of avian embryo cells, separating the cells and cell debris by centrifugation, inactivating and concentrating the virus, purifying the suspension containing the virus by subjecting it to a continuous flow density gradient ultracentrifugation in which the gradient content is divided into fractions and the fractions exhibiting an increased absorbance at 260 nm and containing particles with a sedimentation constant in the range of 200 S are collected, and processing the purified suspension into vaccines.

Description

The invention of oTrUici-rrcyi to the melio-il bic icypogia hk; 1s; 1: rc: ;; iijK-ir Ho ;; i - i iin 15 ists1pp ny ;: eiir; ):

Pel in the product - no ibniic-ii:.: Purity of the target product; i ;: sch-g Ni iju: - (l), HH NreTo; j, a cleaning and.

The method is improved cjTCv iyKii Hir-i on - time.

Embryo5. chicken chickens (mustache: ne: Ng: i- in the cell culture medium (VOL 199) (Tissue C-lture Medium S99), i f.hr 1 virus is infected with the virus and kept in AI-s visa for aerobic conditions at , within 23-38 ° C for 5 days, Then the cells and cell debris are separated by 1:; e grifying. The resulting suspension of the virus,, to giviru from the cbopi4.u-: Hu; i rhpropiolactone is then concentrated by ultrafiltration. Std - tiviip and concentrating are also carried out in reverse EEO sequence – telg-o (tee. Suspension, along with TBIE, the virus contains various impurities that It is miteed partially faster than HJ and slower than TBLE-3: 1). Preemptive-: - it is easily separated faster than se: n-sl; tt py:: parts by sedimentation with rii:) Moir, b (j Protamine sludge, , prey, let us swindle off the coagulation of soybean nehrenra (n-, gdisssOzOtsentrifugirovaniya with razpre ;: cjii: niy gradients of density of density} ;.

In the implementation of the proposal of the ac -; - th method in the rotor of an ultracentrifuge; filled with bu solution (|; erra "corrupted;) horn-1 (|; (1fat} gag buffer at 3000 oG /; i poured buffer solution displaced with / with 50% m solution of sucrose and r6i;: j is due to Ztoku 1 for 1 h; t silt :: - Hoci H 0-50% sucrose.

After this suspension, g; od (: rzhl 1; s- ;; narus, / vnoT, drain out of the ro / ng -fn i g ;.- growth r-n 3500L rotor with () prp lh, mipz tu speed is 1 n - :: - respectfully 3 l / h. When selected -: G: o v; - large Bnpyciibix chaglu goes to G radieity of density. Like TOJibKO all the material is noshe / t atgchar, /, centrifuged another i h, hp i iei; one Bremenpo is allowed to flow rasp; head diaTHor o buffer. Then carefully rotate the rotor to the dokgg state and the cor ci rotor section lt tta:) tds. fractions. After that, in each tail coat ;;: an extinct is determined,) at 2.60 N. W;:;.: .- C:; C; I;) ;;;;:: sucrose in fraction. Loka I1: 1a1; -gl vk- ovca in goadiergt density vsk can ;:

 : h rts ode; w egg. -k.: ri:; iiu H npri 260 nm. T Irus with i: i - ii ia (T (- (and about: gus;. With i: :( viep, - 1; n {and s.: X a; M: I 3 s measured: 0% and obus - dorlinet with g etli: | C) e ut unit; ke extti:; n: j 26C nm.

In about. Costi OKO iC 40% sucrose fraction 10; 11 and 12 years old, and Shkdi 11 -, - ax amal extinction. In this context, they are found in OCIUOBHOM-virus particles with a constant of 1x tsni and 200 S without sed. Impregnating more quickly or slow-forming 5 than a virus. The peak corresponds to a concentration of sugar of 39%. Another peak is in the fraction} homer 25, which corresponds to the sucrose concentration: less than 10%, 1:;

About 6paCF:: aiOTi: .4. ,

Frakp1-; : 1 has the highest protective effect;

The collected virus containing fracn, 1-; and dilution, g: are boiled, containing human albumin, in order to be able to. improve stub pipost nirus - an-chigena a s; 1, in the usual way, is not processed further: in vaccines.

ioluchenta; e vaccines have more than-;: the eye of a doctor and are tolerated ,; ts garaty, made known - with a crest; what is the difference about;: - ablits,

In the case of a situation with a number of vacancies, d: n, tulles according to the invention,; c, no cases were observed: g;: yelits;): stocks: neither: go; and:; s, nor systems ;) 1e,

Sravgn-gs; 1n1: those results of lerenosity: 0sti preiarato ;; ,, obtained information- .i; iiT4 and H oil; lie ;;pn--: in ways

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Vaccines produced according to the method proposed are distinguished by a low protein content, not taking into account the added human albumin. This protein content, with an equal antigome action, is many times, at least 10 times less, than in the case of the use of well-known drugs.

Primer p. Suspension of chicken embryonic cells in TCM 199 (G / SSVF - culture - medium) is infected with TLE-virus. After incubation for 4 days at 33 ° C, the suspension containing the virus is collected and centrifuged at 3000 g 5 for 15 min, the cells are separated,

Formalin is added to the resulting suspension of the virus in such a quantity that the final dilution is -20 to 1: 2000, and then the suspension of vagaries is kept for 30 hours at 37 ° C and 48 hours at room temperature.

Then the inactivated suspension of the virus is mixed with 0.5 mg / l of protamine sulfate and the mixture is withdrawn overnight at 4 ° C. The formed precipitate is separated by centrifuging at 10,000 g for 30 minutes.

at 4 ° C. The suspension pretreated by the inventive method is a starting material for continuous ultracentrifugation in a density gradient. , 35

A virus suspension with a flow rate of 3 l / h is passed by means of a pump through a centrifuge, in which E.Kopcha is the quality editor.

Compiled G.Smirnova TehredN.Glushchenko Proofreader A.Zimokosov

Order 348.3 Circulation 595 Subscription

VNIIPI USSR State Committee

for inventions and discoveries 113035, Moscow, Zh-35, Raushsk nab,. 4/5

. Production and Printing Prefecture, Company, Uzhgorod, st. Project, 4

five

0

five

494

ve sucrose density gradient contains 0-50% sucrose in phosphate buffered saline at p 7.6,

The volume is 4.5 liters, the centrifugation time is 1.5 hours. After the centrifuge has stopped, the contents of the rotor are pumped out and divided into 16 fractions of 100 ml each. For each individual fraction, the extinction is measured at 260 nm on a spectrophotometer and the specific gravity on a refractometer. Peak fractions 10, II, and 12 with a sugar concentration of about 40% are combined and diluted to 1:10 with a phosphate buffered saline solution at a pH of 7.6, which contains 0.1% human albumin.

The protective activity of the preparation obtained by the proposed method is determined by direct protective experiment on Mbmiax.

The study proposed was diluted (1: 3, 1: 9, 1:27, 1:81, 1: 243) and mixed with 0.2% alumina pvdroxia, used as a means of enhancing the effect of the drug. When applying in each case 0.2 ml of the indicated diluted solutions, 2 times with a duck in 1 week subcutaneously immunized with .. in 10 mice (weighing approximately 10 g each). After the next two weeks, mice are infected with the SEA - 1000 ZDjg TVLE virus and the spread-; the protective dose was read by the method of Reed and -Munha after observation for 3 weeks.

The protective dose of ZDgg is determined at a dilution of 1:30.

Claims (3)

1. A METHOD FOR OBTAINING A VACCINE AGAINST TAIGA SPRING SUMMER ENCEPHALITIS by cultivating the virus in tissue culture of bird embryonic cells, separating the virus-containing fluid by centrifugation, followed by inactivation, clarification, concentration and purification of the virus by zonal ultracentrifugation in a gradient of density gradient. selection and dilution of the obtained fractions, characterized in that, in order to increase the purity of the target product, cultivation7 is carried out in suspensions of bird embryonic cells, after clarification before zonal ultracentrifugation, the virus-containing liquid is treated with protamine sulfate, fractions with increased extinction at 260 nm and containing particles with a constant are selected sedimentation in the region of 200 S, and the dilution of the fraction is carried out with phosphate buffer pH 7.6 2. The method according to claim 1, characterized in that the zone ultracentrifugation is carried out with continuous centrifugation with a sucrose density gradient of 507 ,. 3. The method according to claim 1, with the fact that, in order to stabilize the target product, the fractions are diluted with a buffer solution containing 0.1% human albumin. SU <„> 1318149 Joint venture IC! / ABOUT