GB2248241A

GB2248241A - Cultivation method for preparing an oral live typhoid vaccine

Info

Publication number: GB2248241A
Application number: GB9117918A
Authority: GB
Inventors: Bong Wha Park
Original assignee: BORYUNG BIOPHARMA CO Ltd
Current assignee: BORYUNG BIOPHARMA CO Ltd
Priority date: 1990-09-05
Filing date: 1991-08-19
Publication date: 1992-04-01
Anticipated expiration: 2011-08-19
Also published as: TW209244B; KR920006496A; JPH05336950A; GB2248241B; IL99138A0; KR930001382B1; ES2037599B1; CN1056876C; IL99138A; CH683187A5; CN1060110A; JPH0771471B2; GB9117918D0; ES2037599A1

Abstract

A cultivation method for obtaining oral live typhoid vaccine comprises the steps of cultivating seed bacteria obtained from typhoid strain Ty21a in BHI medium, suspending the cultivated bacteria in protective medium and lyophilizing the suspended bacteria in an ampoule, reviving the lyophilized bacteria and inoculating the revived bacteria in production medium, cultivating the bacteria in a fermentor, and lyophilizing the bacteria and formulating the bacteria for oral use. The preferred protective medium is composed of 8% lactose, 1% carboxymethyl cellulose (or 1% gelatin), 5% skimmed milk, 0.2% MgSO4.7H2O and 1% monosodium glutamate. The preferred growth medium is composed of 37g BHI, 5g L-lysine, 30g sorbitol, 1.5g K2HPO4, 0.5g MgSO4 and 1 litre distilled water pH 7.0. The cultivating temperature in the fermentor is controlled so that it is 30 DEG C for the first six hours, 25 DEG C for the next two hours and 20 DEG C for the final fourteen hours.

Description

Cultivation Method For Preparing An Oral Live Typhoid Vaccine This

invention relates to a cultivation method for preparing an oral live typhoid vaccine, more specifically, a cultivation method using novel growth medium, protective medium and controlled cultivating temperatures in order to obtain the increased harvest of the bacteZial cells after cultivation and the high survival of immuno-active attenuated live bacteria after lyophilization.

1, Typhoid fever, the germ of which is known as Salmonella typhi, is generally s-Pread by contact with contaminated water or foods, and versonal contacts in crowded areas.

For the prevention of typhoid fever, non-oral killed vaccine or oral live vaccine has been normally used.

Nowadays, the use of oral live vaccine is gradually increasing because the killed vaccine does not show the desired effective immunogenic activity in spite of its high safety.

The preparation method of live vaccine is different from that of killed vaccine. The live vaccine is prepared by the following known process:

i) cultivation of an attenuated mutant of typhoid bacteria in liquid or solid medium, ii) centrifugation and lyophilization, and iii) formulation, whereas the killed vaccine has been prepared by the following process: i)cultivation of typhoid bacteria in liquid or solid medium, ii) sterilization using acetone, phenol or formalin, and iii) formulation.

For producing live vaccine, the attenuated mutant Salmonella typhi Ty21a which is isolated by double step mutation treatments from Salmonella typhi Ty2 by R. Geimanier and E. Furei is normally used. Such mutant is characterized by the lack of enzyme activity of uiidine-diphosphogalactose -4- epimerase which is a necessary intermediate in the formation of lipopolysaccharides in the cell walls of the bacteria. Therefore, such mutant has been practically free of virulence since only incomplete cell walls are formed.

However, such mutant shows outstanding immunization effects by synthesizing the lipopolysaccharides in the presence of galactose. And the excess supply of galactose causes the accumulation in the form of galactose -1- phosphate and UDP-galactose which bring about bacteriolysis. R. Germaniei et al. obtained the US Patent No. 3,856,935 issued on December 24, 1972 regarding the mutation, cultivation and formulation of Ty21a strain. Following is a process for preparing the typhoid vaccine disclosed in this patent.

Selected mutant Ty21a was cultivated in brainheart infusion on a shaking machine at 371C for 6 hours, By centrifuging at 6,000g, the bacteria were harvested and suspended in protective aqueous medium containing 8% saccharose, 1.5% gelatine and 5% skimmed milk powder. After lyophilizing the suspension in vials, the bacteria were prepared by opening one of the vials and were inoculated on a nutrient agar slant which was then incubated at 371C. The bacteria were suspended in physiological saline solution and the suspension was used as an inoculum for 600m1 of a nutrient solution. The nutrient solution was prepared by dissolving 28g of casein hydrolyzate, 10g of yeast extract and 2g of glucose in one liter of distilled water with pH adjustment to 7.2. And the culture was then transferred to a 25 liter batch of the same nutrient solution which was maintained at 371C for 12 hours after inoculation while being aerated at a rate of 5 liteis of air per minute. After cultivation, the bacterial cells were harvested by centrifuging at 6,000g and suspended in the aforementioned aqueous protective medium, and lyophilized in vials.

But the bacteria cultivated according to abovementioned method has a low survival rateafter lyophilization because Ty21a strain is easily killed during the lyophilization due to the weak resistant property against the change of environment.

The present - invention may provide an improved cultivation method using novel growth medium, protective medium and controlled cultivating temperatures in order to obtain an increased harvest of the bacterial cells after cultivation and the high survival rate of immuno-active attenuated live bacteria after lyophilization.

The growth medium prepared in this invention is designed so that the bacteria grow up effectively and easily adapt to the envioinmental changes by means of controlling the cultivating temperatures.

The protective medium prepared in this invention is designed so that bacteria can endure the highly rapid freezing during the lyophilization so that. 1yophilized bacteria can be preserved for a long time without change of their properties.

The ' oral typhoid vaccine obtained according to the cultivation method using new growth medium and protective medium shows the high survival rate of the attenuated bacteria and the high immunoactivity against typhoid fever.

The detailed cultivation method according to this invention can be explained as follows. The typhoid strain for preparing the oral typhoid vaccine is the attenuated mutant strain Ty21a of Salmonella typhi which has been deposited in American Type Culture Collection (11ATCC11) under deposit No. ATCC-33459 and which has been also deposited in Korea Institute of Science and Technology (11KIST11) under deposit No. KCTC2425. The above deposited typhoid strain can be also obtained by reviving the commercially available product Wivotif Bernall [marketed by Swiss Serum & Vaccine Institute Berne] as a single colony in the revival medium composed by Biain-Heart Infusion agar.

f., A The seed bacteria isolated from the single colony of the above typhoid strain were inoculated in 100m1 of Blain-Heart Infusion (11BHP) medium and the inoculated strain was cultivated for 18 hours at 301C on a shaking machine. By centrifuging the culture at 6,000g for 10 minutes, the bacteria were harvested and suspended in protective medium. The protective medium contained 8% lactose, 1% caiboxymethyl cellulose, 5% skin-med milk, 0.2% MgSO47H20, and 1% monosodium glutamate.

The above suspension was lyophilized in the ampoule. The lycphilized cawm:>oule was stored at 4-8t before producing the live vaccine.

The bacteria were prepared by opening the ampoule and were inoculated on BHI medium. For the seed cultivation, the inoculated bacteria were cultivated for 18 hours at 301C on a shaking machine. The seed bacteria so obtained were inoculated to the productive growth medium which was composed of 37g of BHI, 5g of L-lysine, 309 of soibitol, 1.5g of K2HP04, 0.5g Of MgS04.7H20, and 1 liter of distilled water with pH adjustment to 7.0 for main cultivation.

In case of mass production, the culture obtained from the production medium can inoculate to 250 liter feimentoi. During the main cultivation, the temperature for cultivation,which is one of the main characteristics of this invention,should be changed in order to fully endure the rapid temperature change during the lyophilization.

The cultivating temperature during the main cultivation was i) at 301C for first 6 hours, ii) at 2512 for second 2 hours, and iii) at 201C for last 14 hours.

The aeration for the feimentor was maintained at 0.5 Wm(Volume/ Volume/minute).

At the completion of the cultivation, the bacteiial cells were harvested by refrigerated centrifuge at 6,000g and suspended in aforementioned protective medium for 2 hours at 0-41C.

And the suspension was lyophilized in a freeze dryer. Finally the lyophilized bacteria were mixed with lactose in order to contain 2.5 x 10" live bacteria per one gram, and then the prepared vaccine was f filled in the capsules in an amount of 0.2g per each capsule and the capsules were made by enteric coating for oral use.

The present invention can be explained in more detail in the following examples.. Thes.e examples are only illustration and are not to be regarded as limitation for the scope of this invention.

Example 1

The attenuated mutant strain Salmonella typhi Ty2la from ATCC-33459, KCTC-2425 or Vivotif Berna was inoculated in 100ml BHI medium and cultivated for 18 hours at 30r, on a shaking machine, and then the bacteria harvested by centrifuging were lyophilized in the ampoule The lyophilized bacteria as the seed were inoculated in main growth medium composed of 37g of BHI, 5g of L-lysine, 30g of sorbitol, 1.5g of Kz HP04, 0-59 Of MgS04.7HzO and 1 liter of distilled water with pH adjustment to 7.0.

For carrying out the main cultivation, 5 liters of the main production medium was transferred to a 10 liter fermentor.

aeration for fermentor was at a rate of 0.5VVm with stirring at 300 rpm. The temperature during the main cultivation was i) at 301C for first 6 hours ii) at 251C for second 2 hours, and iii) at 201C for last 14 hours.

Total cultivation time required 22 hours. The properties of cultivated solution finally obtained showed that the turbidity of the cultivated solution was 0.516 X 20 on OD 660 and the number of live bacteria was 4.6 X 1010 per ml.

Example 2

The cultivation was carried out in the same manner aj Example 1 except that the temperature of main cultivation was i) at 301C for first 6-8 hours ii) at 251C for second 2-4 hours, and iii) at 20-25C for last 12-14 hours.

The property of cultivated solution finally obtained showed that the turbidity was 0.452 x 20 - 0.516 x 20 on OD 660 and that the number of live bacteria was 3.2 x 101c per ml.

Examr)1e 3 The cultivated solution obtained in Example 1 was centrifuged at 6,000g and was suspended in 2.5 liters of protective medium composed of 8% of lactose, 1% of carboxymethyl cellulose, 5% Of skimmed milk, 0.2% C)J- M9S04.7H20, 1% of monosodium glutamate. The suspension in protective medium was allowed to stand at 0-4r- for 2 hours in order to increase the resistance to low temperature. In the freeze dryer, the resistance increased suspension was frozen at -70t for 2 hours and dried for 12 hours in vaccum state. The number of live bacteria, after the lyophilization, was 6.8 x 1TO per gram.

Example 4

The lyophilization was carried out in the same manner as Example 3 using 1% of gelatin instead of 1% of carboxymethyl cellulose. The number of live bacteria obtained after the lyophilization was 4.1 x 10to per cram.

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Claims

1. A cultivation method for obtaining oral live typhoid vaccine comprising the steps of:

i) cultivating seed bacteria obtained from typhoid strain Ty21a in BHI medium, suspending the cultivated bacteria in protective medium and lyophilizing the suspended bacteria in theamilDoule iii) reviving the lyophilized bacteria and inoculating the revived bacteria in production medium, cultivating the revited, bacteria in a fermentor, and lyophilizing the bacteria Dremared in step -(iv) and formulating said bacteria for oral use.

A cultivation method according to claim 1, wherein the protective medium is composed of 8% lactose, 1% carboxymethyl cellulose, 5% ski milk, 0. 2 % M9S04.7H20 and 1% monosodium glutamate.

A cultivation method according to claim 1, wherein the growth medium is composed of 37g BHI, 5g L-lysine, 30g sorbitol, 1.5g K2HP04, 0.5g MgSO4 and 1 liter of distilled water with pH adjustment to 7.0.

A cultivation method according to one of claims 1 to 3, wherein the cultivating temperature in fermentor is i) at 301C for first 6 hours, ii) at 2513 for second 2 hours, and iii) at 201C for last 14 hours.

A cultivation method according to claim 2, wherein the 1% caiboxymethyl cellulose in protective medium is replaced by 1% gelatin.

A cultivation method according to claim 1 wherein the bacteria are lyophilized, after the suspension is laid at 0-4t for 2hours, under the freezing at -701C for 2hours and the drying for 12hours under vaccum.

ii) IV) v) 7. A live typhoid vaccine obtained according to the cultivation method described in any preceeding claim.

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