CS199547B2 - Process for production mks-virs - Google Patents

Abstract

1375154 Foot and mouth disease vaccine BAYER AG 12 Feb 1973 [12 Feb 1972] 6801/73 Heading A5B [Also in Division C6] A vaccine for foot and mouth disease consists of the virus cultured in a suspension of surviving cells of cattle tongue epithelium and ruminant stomach. Preferred epithelium cells are from the periglottis and stomach cells from the rumen pillars and floor. The fresh tissues may be irradiated with U.V. light prior to use.

Description

The invention relates to a method for the production of MKS-viruses in bulk. .

The following methods of multiplication of MKS-viruses are already known from the literature:

1) Growing of the virus on the mucosa of the live cattle tongue (O. Waldmann, G. Pyl, K. O. Hobohm and H. Mohlmann Zbl. Bakt. 1. 'Orig. 148, 1, (1941).

2) Growing surviving isolated epithelial cells of cattle tongue mucosa in vitro (H. S. Frenkel, Bull. Off. Int. Epiz. 28, 155, (1947) and 39, 91, (1953).

3) Virus multiplication · surviving enzymatically degraded tissues · isolated stomach of ruminants in vitro. (K.O. Hobohm, H.U. Rehneberg, H.S. Fiege, Germany DAS Interpretation Document No. 1,937,760.

4) Virus multiplication in monolayer primary cell cultures of calf kidneys (B. Ubertini, B. L. Nardelli, A. Dal Prato, G. Panin and G. Santero, Zbl. Ver. Med. 1963, 93-101).

5) Virus multiplication · on monolayer culture or in suspension · BHK (Baby humster kidney) (Macpherson and Stoker, Virology 16, · 147 (1962)).

The virus thus obtained is suitable for the production of a vaccine against MkS (foot-and-mouth disease) of ungulates, such as cattle, pigs, sheep and goats.

The above processes have a number of disadvantages:

Cultivation of viruses · on the mucosa · of the tongue of the live cattle (Waldmann procedure) is · banned in most countries in the world for the production of the vaccine against limpid because of the possibility that the disease may spread undesirably.

The cultivation of the MKS-virus by the Frenkel process encounters a number of difficulties due to the need to continuously prepare a sufficient amount of isolated tongue mucosa from freshly slaughtered cattle. In addition, the method is using tissue cultures, especially single-layer tissue cultures. in the case of producing large quantities of viruses very expensive and time consuming.

MKS-vaccines from viruses that have been grown on heterologous cell lines, such as the hamster kidney, carry the disadvantage that allergic diseases may occur in vaccinated animals at repeated vaccination and sometimes at first vaccination.

If the virus is grown on surviving enzymatically degraded stomachs of ruminant stomachs in vitro, virus synthesis proceeds for 1595 to 40 hours and the virus yield is at 10β to 107. infectious doses in ml very small.

It has been found that the disadvantages of the above methods can be overcome by growing the virus using both isolated epithelium of cattle tongue and using surviving tissues from the stomach of ruminants.

Accordingly, the present invention provides a process for the production of large-scale MKS-viruses, characterized in that rumen and epithelium-free bovine tongue epithelium is suspended in isotonic antibiotic sodium chloride solution and homogenized, the homogenate thus obtained is washed and shaken. with an isotonic sodium chloride solution containing antibiotics. The antibiotic is removed and the cell slurry is infected with a suspension of MKS-vi.ru, after which the slurry is grown in a fermenter filled with nutrient medium at pH 7.5 at 37 ° C, maintaining the pH by adding glycol buffer. , after 22 hours, the cultivation is discontinued and the fermentation medium is cooled to 4 ° C, parts of the tissue are crushed, and the thus obtained viral cell slurry and the culture medium are extracted at 4 ° C for one hour, then centrifuged and recovered the clear virus suspension is mixed with a preservative, preferably Thimerosal or chloroform. ‘

The process according to the invention is suitable for the production of MKS-virus and for the production of MKS-vaccine in bulk. The method is very simple, is cheaper than pure Frenkel cultivation, and also has more favorable conditions than cultivation on surviving, enzymatically distributed tissues of ruminant stomach, especially in terms of virus yields. The combination of tissues reduces the difficulties that arise during. the need to extract large amounts of fresh epithelium from the cattle tongue. The MKS-virus is grown on homologous tissue in accordance with the method of the invention, so that the use of the vaccine thus produced does not cause allergic reactions. The vaccine is effective, durable and well-tolerated. Thus, the method of the invention represents a major advance in the field of veterinary medicine.

The method according to the invention is based on cattle tongue tissue and stomach tissue of ruminants. Preferably, the bovine stomach is used, namely papilla tissue and rumen mucosa, as well as tongue mucosa containing epithelial cells.

The bovine tongues are processed according to the Frenkel method (H. S. S. Frenkel, Bull. Off. Int. Epiz. 28, 155, (1947) and 39, 91, (1953), the mucosa of the tongue is isolated and Immediately after the stomach is taken from the slaughtered animals, the cocci and the smooth part of the rumen mucosa are separated, washed with lukewarm water and free of fat. irradiate from both sides with ultraviolet light from a distance of 30 cm and then store in an ice-cooled isotonic salt solution containing antibiotics.

The bovine tongue epithelium obtained by the Frenkel method and the isolated rumen sections described as described are ground in a meat grinder having a hole size of 12-20 mm and then washed three times with isotonic cold salt solution. Then the ground rumen and tongue epithelial tissues are shaken for 1 hour in isotonic antibiotic salt solution.

The isotonic saline solution containing antibiotics is preferably a physiological saline solution of the following composition: NaCl - 8 grams, KCl - 0.4 g, NaHCO3 - 0.35 g, penicillin - 0.5 g, chloramphenicol · - · 0 25 g, Nystatin - 0.075 g, distilled water to 1000 ml.

After treatment with an isotonic antibiotic salt solution, the supernatant is decanted and the tissue slurry is infected with a MKS virus suspension of 0.1 of the total virus slurry volume.

Any known virus type, for example European types O, A and C, South American types O, A and C, African types sAt 1, 2 and 3 as well as Asian types, such as Asia 1 and all subtypes of these basic virus types.

After an aborning time of 20 minutes, the infected rumen and tongue epithelial tissue are placed under a sterile fermentor under virus conditions.

The fermenter already contains the amount of culture medium required to grow the virus. This nutrient medium is used at a pH of 7.4 to 7.6 and preferably consists of a physiological nutrient solution of the following composition:

NaCl 720 G KC1 18 G CaCh. 6H2O 18 G MgCl2. 6H2O 9 G NaHzPO. H2O • 11.1 G NaHCCb 200 G Na2HPO4.2H2O 48.06 G Glucose 100 ALIGN! G peptone 240 - G casein hydrolyzate »300 G 0.02% hemin solution 3.6 · 1 ml DL-Methionine 12.8 G DL-Tryptophan 8.0 G DL-Threonine 2.5 G 5.2 G L-Thyroxine 0.0009 G D-Ca Pantothenate 0,032 G Niacin 0.16 G Insulin 80 IU Chloramphenicol 10 G Penicillin 32 G Neomycin sulfate 10 G Polymixin B 0.5 G Nystatin 0.75 G Distilled water to 100 1

A viral tissue slurry is then added to the fermentor and the total amount of nutrient solution is adjusted to pH 7.4-7.6 with glycol buffer after addition of the components, and the fermentor content is grown at 37 ° C and 25 rpm. minute.

Preferably, a glycol buffer is used. of the following composition: Glycokol - 158. g, NaCl - 1.4 g, NaOH - 51 g, distilled water to 1000 ml.

At the same time 2 liters per minute are fed into the fermenter. sterile filtered pure oxygen. . The pH is constantly controlled and. if necessary, adjust the glycol buffer back to pH 7.4 to 7.6.

After 18 to 24 hours, depending on the type of vortex, the cultivation is stopped and the suspension is cooled to 4 ° C. Then the tissues they contain. the virus, ie rumen tissue and tongue epithelium, is homogenized in a colloid mill to release the virus contained in the cells. The tissue slurry thus treated is extracted together with the culture medium for 1 hour at 4 ° C with slow stirring at 25 rpm. Mertoga (Thimerosal) is added to the tissue suspension at a concentration of 1: 40,000 or 0.2 to 1% chloroform. ·

After extraction, the virus suspension is centrifuged in a Scherples or Westfalia flow centrifuge. The clear MKS-virus extract thus obtained is used as an antigen for the production of the MKS-vaccine after quantitative determination of the antigenic MKS-virus content.

The determination of the virus content and hence the content of the infectious antigen and the antigen which binds complete is carried out quantitatively.

The content of infectious antigen in mice is determined as follows:

10 mice, 4 to 7 days old, receive 0.10 ml of virus at 10 to ⁴ , 10 to ⁵ , 10 to ⁶ , 10 ⁷ , 10 ⁸ and 10 to ⁹ under ether anesthesia by intraperitoneal injection. Each experiment lasts 7 days. Deaths within · 48 hours after infection are not counted because the incubation period is at least 48 hours. For each dilution, 50% endpoints were determined according to the method of B. Behrens, Naunyn, Schmiedeberg's Arch, exp. Path. Pharmak. 140, 237, (1920) and G. Karber idem. 162,. 489, (1931). The content of infectious MKS-antigen (virus titer) is expressed in MsLD50 / ml. '

Complement-binding antigen content is determined quantitatively by response to complement binding (KBR) by the so-called "50% method" (Traub, E. and Mohlmann H. Zbl. Bakt. I. Orig. 150, 289 (143 a)) Orig. 300 (1943b), Camargo, NF Eichhorn, BA, De vine JM, and Telloz Giron, A., Proc., 87th, Ann., Meet Amer., Med., Assoc., 1950, 207-211) spectrophotometrically.

P Rikli dl _r kg net ultraviolet infectious germs partially stripped tissue 15 · rumen. kg of purified, ultraviolet radiation partially, germ-depleted infectious germs are introduced into an isotonic salt solution containing antibiotics and ground in a meat grinder.

The tissue slurry thus obtained is washed with physiological saline solution and then shaken for 1 hour with an isotonic antibiotic salt solution. Then isotonic saline containing antibiotics and tissue removed by decantation · slurry were infected with a suspension of 30 kg · MKS South American virus type C, the filter is MsLDso 10 · ⁷⁷⁵ · ml.

After an absorption time of 20 minutes, the infected tissue slurry is introduced under sterile conditions into a fermenter containing 240 kg of culture medium. The culture medium is pre-heated to 37 ° C. The contents of the fermenter were adjusted to pH 7.5 with 250 ml of glycol buffer and stirred at 25 rpm at 37 ° C.

During cultivation, sterile filtered pure oxygen is fed into the fermenter at a rate of 2 liters per minute. The pH is constantly controlled · and · 5 hours and 8 hours after infection of the contents · is adjusted by adding 300 ml, 14 · hours after infection by adding 400 ml of glycol buffer back to pH 7.4.

Samples are taken at 12 hour intervals after infection of the contents until 22 hours after infection of the contents for quantitative determination of the content of the infectious and complement-binding antigen. Cultivation was discontinued after 22 hours and the mixture was cooled to 4.degree. The parts are crushed in a colloid mill to extract the virus. It processes tissue mash. se. extracts into the culture medium,. also containing virus, for 1 hour at 4 ° C with slow stirring at 25 rpm.

After extraction, the virus suspension is centrifuged in a flow centrifuge (Westfalia type). To the clear virus suspension thus obtained is added (Thiomersal) (Mertogan) at a concentration of 1: 40,000.

The virus titer and the complement titer antigen titre develop over 22 hours of cultivation as follows:

Time in hours MsLD 50% inhibition of haemolysis after antigen dilution infection

1:10 1:20

12 10-Sat 15 Dec 0 0 14 10-6. « 22nd 0 0 16 10-7.25 41 0 0 18 10-7.5 100 ALIGN! 38 0 20 May 10-8.0 100 ALIGN! 85 15 Dec 22nd 10-8,5 100 ALIGN! 100 ALIGN! 68

Example 2

By the method of Example 1, a suspension is prepared from the epithelium of the bovine tongue and from the rumen tissue which is infected with the South American

MKS-type O virus, Oi-Campos subtype (MsLD 50 virus titer = 10 , ^57.5 / ml).

The virus culture is discontinued after 20 hours. The complement and titre virus and antigen titres develop as follows:

Time in hours after infection MsLDso 1: 5 o / o inhibition of hemolysis at 1:10 antigen dilution 1: 20 12 10-5.5 0 0 0 14 10-625 21 0 0 16 7.5 38 0 0 18 10-8.0 100 ALIGN! 72 10 20 May 10-9.’5 100 ALIGN! 100 ALIGN! 82 Example 3 MKS-type A virus, subtype A25 (titre vi- rtl MsLDso = 10 ^{7 '6} / ml). By the method of Example 1, tissue is After 24 hours the cultivation is discontinued and disease and epithelium of cattle tongue prepare tissue rus is isolated. Evolution of virus titer and anti- the suspension which is formed vaccinates south- the complement binding gene, is this: Time in hours MsLD.50 o / o inhibition of haemolysis at after infection antigen dilution 1: 5 1: 10 1: 20 12 Ю-45 0 0 0 14 5.0-5.0 10 0 0 16 6.25 10 0 0 18 7.5 63 5 0 20 May 80- ⁸⁰ 100 ALIGN! 10 0 22nd 10-925 100 ALIGN! 75 10 24 8.7-8.75 100 ALIGN! 100 ALIGN! 87

The virus suspension obtained by the method of Examples 1, 2 and 3 is used as MKS-virus and antigen in the trivalent MKS-vaccine.

This vaccine protects cattle against expert mental infection by three types of virus, namely Oi-Campos, A26 and C, which have been used to produce it.

Claims (1)

the subject of the invention Method for the large-scale production of MKS-viruses, characterized in that the germ-free rumen and epithelial epithelium of the bovine tongue are suspended in an isotonic sodium chloride solution and homogenized, the homogenate thus obtained is washed and shaken with an isotonic sodium chloride solution containing antibiotic, then the sodium chloride solution containing the antibiotics is removed and the cell slurry is infected with a suspension of MKS-virus, after which the slurry is grown in a fermenter filled with nutrient medium at pH 7.5 at 37 ° ^C while maintaining the pH at that value by addition glykokolového buffer after 22 hours cultivation was stopped and the fermentation broth is cooled to 4 ° C, part of the tissue is crushed and the resulting cell slurry containing virus and the culture medium is extracted at 4 DEG C. for 1 hour, centrifuged and the clear virus suspension obtained is mixed with a preservative, preferably Thlmerosal or chloroform.