JPH05336950A - Cultivation for preparing oral live typhoid vaccine - Google Patents

Abstract

PURPOSE: To provide a cultivation method for preparing oral live typhoid vaccine.

CONSTITUTION: This 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 a protective medium, lyophilizing the suspended bacteria in an ampule, reviving the lyophilized bacteria, seeding the revived bacteria in a production medium, cultivating the bacteria in a fermentor, lyophilizing the obtained bacteria and formulating the lyophilized bacteria for oral use.

COPYRIGHT: (C)1993,JPO

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a culturing method for the preparation of a live oral typhoid vaccine, more specifically, to increase the yield of bacterial cells after culturing, which has immunoreactivity after lyophilization and is attenuated. , A novel growth medium, a protective medium and a culturing method using a controlled culturing temperature in order to increase the viability of living bacteria.

[0002]

2. Description of the Related Art Salmonella typhi
Typhoid fever, a pathogen known as phi), is usually infected by contact with contaminated water or food, or people in crowded areas. For the prevention of typhoid fever, parenteral killed vaccine or oral live vaccine is usually used. Today, the use of live vaccines is gradually increasing as killed vaccines do not show the desired effective immunoreactivity despite their high safety. The method for preparing a live vaccine is different from the method for preparing a killed vaccine. Killed vaccines are prepared by the following steps: i) culturing S. typhi in a liquid or solid medium, ii) sterilizing using acetone, phenol or formalin, and iii) formulating The vaccine is prepared by the following known steps: i) culturing the attenuated mutant strain of S. typhi in liquid or solid medium, ii) centrifugation and lyophilization, and iii) formulation.

For the production of live vaccines, R. Ger
manier and E.I. 2 from Salmonella typhi Ty2 strain by Furer
The attenuated typhoid fever mutant Ty21a isolated by stepwise mutagenesis is commonly used. Such mutants are characterized by uridine-galactose-2, an intermediate required in the formation of lipopolysaccharides in the bacterial cell membrane.
It lacks the enzymatic activity of phosphate-4-epimerase. Therefore, such mutants are virtually non-toxic because only incomplete cell membranes are formed.

[0004] However, such a mutant strain exhibits a remarkable immune effect by synthesizing lipopolysaccharide in the presence of galactose. When galactose is supplied in excess, galactose-1-phosphate and UD
It accumulates in the form of P-galactose, causing bacterial lysis. R. Germanier et al. Obtained US Pat. No. 3,856,935 issued Dec. 24, 1972, regarding mutation, culture and formulation of Ty21a strain. The following is a method for preparing the typhoid vaccine disclosed in this patent specification.

The selected mutant strain Ty21a was incubated in brain-heart exudates for 6 hours at 37 ° C. on a shaker. Bacteria were recovered by centrifugation at 6,000g
Suspended in liquid protective medium containing% sucrose, 1.5% gelatin and 5% skim milk powder. After the suspension was lyophilized in vials, the bacteria were prepared by opening one of the vials and culturing at 37 ° C by inoculating a gradient culture of nutrient agar. Bacteria were suspended in saline and the suspension was used as an inoculum into 600 ml of nutrient medium. This nutrient medium was prepared by dissolving 28 g of casein hydrolyzate, 10 g of yeast extract and 2 g of glucose in 1 liter of distilled water to obtain a pH of 7.2.
It was prepared by adjusting.

The culture was then transferred into 25 liters of the same nutrient medium and placed at 37 ° C. for 12 hours with aeration at a rate of 5 liters per minute after inoculation. After culturing, bacterial cells were harvested by centrifugation at 6,000 g and suspended in the liquid protection medium described above and freeze-dried in vials. However, since the Ty21a strain is easily killed during freeze-drying due to its weak resistance to environmental changes, the bacteria cultured according to the above-mentioned method survived only slightly after freeze-drying.

[0007]

The object of the present invention is to increase the yield of bacterial cells after culturing and to increase the viability of immunologically active, live attenuated bacteria after lyophilization. To provide an improved culture method using a novel growth medium, a protective medium and a controlled culture temperature. The growth medium prepared in the present invention is intended for effective growth of bacteria by controlling the culture temperature and easy adaptation to changes in the environment. And the protective medium prepared in the present invention is intended such that the bacteria can withstand rapid freezing during freeze-drying and that the freeze-dried bacteria can be stored for a long time without changing their properties. The oral typhoid vaccine obtained by the culture method using the new growth medium and the protective medium has a high survival rate of attenuated bacteria and a high immunological activity against typhoid fever.

[0008]

The detailed culture method according to the present invention will be described below. A typhoid strain for the preparation of an oral typhoid vaccine is available under the deposit number ATCC-33459, American Type Culture Col.
depository (ATCC), and under the deposit number KCTC-2425 by Korea Institute.
ute ofScience and Technology
It is an attenuated mutant strain of typhoid fever Ty21a deposited at ology (KIST). The deposited typhoid strain is a commercial product "Vivotif Berna" using a resuscitation medium composed of brain-heart exudate agar.
[Swiss Serum & Vaccine In
marketed by site Bene] can be revived as a single colony. Bacteria for inoculation isolated from a single colony of the typhoid strain were treated with 100 ml of brain-heart exudate (BH
I) Inoculated into medium and inoculated strain 1 on shaker
Incubated at 30 ° C. for 8 hours. 6,000g of culture solution
Bacteria were harvested by centrifugation at 10 min and suspended in protective medium. Protective medium is 8% lactose, 1% carboxymethylcellulose, 5% skim milk, 0.2
% MgSO ₄ .7H ₂ O, and 1% glutamic acid 1
Contains sodium. The suspension was freeze dried in ampoules. Lyophilized ampoules were stored at 4-8 ° C prior to production of live vaccines.

Bacteria were prepared by opening ampoules and inoculated into BHI medium. For inoculation culture,
The inoculated bacteria were cultivated on a shaker at 30 ° C. for 18 hours. The inoculated bacteria thus obtained were treated with 37 g of BHI, 5
g L-lysine, 30 g sorbitol, 1.5 g K
₂ HPO ₄ , 0.5 g of MgSO ₄ .7H ₂ O, and 1 liter of distilled water were inoculated into the production growth medium for the main culture adjusted to pH 7.0. For mass production, the culture broth obtained from the production medium can be inoculated into a 250 liter fermentor. During the main culture, it is necessary to change the culture temperature so that one of the main features of the present invention is to completely withstand the abrupt temperature change during freeze-drying. The culture temperature during the main culture is i) 30 ° C. for the first 6 hours, ii) 25 ° C. for the next 2 hours, and iii) 2 hours for the last 14 hours.
It was 0 ° C. Aeration in the fermentor was maintained at 0.5 VVm (volume / volume / minute). After completion of culture,
It was collected by chilling centrifugation at 000 g and 0-
Suspended at 4 ° C for 2 hours. Then, the suspension was freeze-dried in a cooling dryer. Finally, the lyophilized bacteria were mixed with lactose in order to obtain a viable count of 2.5 × 10 ¹⁰ per gram, and the prepared vaccine was made to an amount of 0.2 g per capsule. Capsules were prepared by filling the capsules with an enteric coating for oral administration.

The invention is explained in more detail in the following examples. However, these examples are merely examples, and do not limit the scope of the present invention.

[0011]

【Example】

Example 1 ATCC-33459, KCTC-2425 or V
The typhoid attenuating mutant strain Ty21a from Ivotif Berna was inoculated into 100 ml of BHI medium and incubated on a shaker at 30 ° C. for 18 hours, and the bacteria recovered by centrifugation were lyophilized in ampoules. Lyophilized bacteria for inoculation 37g BHI, 5g L
- lysine, sorbitol of 30g, 1.5g of K ₂ HP
A growth medium for main culture adjusted to pH 7.0 with O ₄ , 0.5 g of MgSO ₄ .7H ₂ O, and 1 liter of distilled water was inoculated.

To carry out the main culture, 5 liters of the main production medium were added to a 10 liter fermenter. Aeration of the fermentor was performed at a speed of 0.5 VVm with stirring at 300 rpm. The temperatures during the main culture were i) 30 ° C. for the first 6 hours, ii) 25 ° C. for the next 2 hours, and iii) 20 ° C. for the last 14 hours. The total culture time required 22 hours. The characteristics of the finally obtained culture solution are OD66.
The turbidity of the culture solution at 0 was 0.516 × 20, and the viable cell count was 4.6 × 10 ¹⁰ per 1 ml.

Example 2 The temperature during the main culture was i) the first 6-8 hours at 30 ° C., i
i) the next 2-4 hours at 25 ° C, and iii) the last 1
Example 1 except for 2-14 hours at 20 ° C-25 ° C.
The culture was performed in the same manner as in. The characteristics of the culture broth finally obtained are that the turbidity of the culture broth at OD660 is 0.452.
× a 20-0.516 × 20, the number of viable cells was 3.2 × 10 ¹⁰ per 1 ml.

[0014] The culture solution obtained in Example 3 Example 1 was centrifuged at 6,000 g, and 8% lactose, 1% carboxymethylcellulose, 5% skim milk, 0.2% MgSO ₄ · 7
It was suspended in 2.5 liters of protective medium consisting of H ₂ O and 1% monosodium glutamate. To increase resistance to low temperatures, suspend the suspension in protective medium at 0-4 ° C.
And allowed to stand for 2 hours. Freeze the suspension with increased resistance in a chill dryer for 2 hours at -70 ° C,
Then, it was dried in a vacuum state for 12 hours. The viable cell count after lyophilization was 6.8 × 10 ¹⁰ per gram.

Example 4 Freeze-drying was carried out in the same way as in Example 3, substituting 1% gelatin for 1% carboxymethylcellulose. The viable cell count obtained after freeze-drying was 4.1 × 10 ¹⁰ per gram.

Claims (7)

[Claims] 1. i) S. typhi in a BHI medium
Culturing a seed culture obtained from strain 1a, ii) suspending the cultivated bacteria in a protective medium, and freeze-drying the suspended bacteria in ampoules, iii) resuscitating the freeze-dried bacteria, and resuscitating Typhoid fever, which comprises inoculating the bacterium in a production medium, iv) culturing the bacterium in a fermenter, and v) lyophilizing the bacterium and formulating the bacterium for oral administration. Culture method for obtaining live vaccine. 2. A protective medium is 8% lactose, 1% carboxymethylcellulose, 5% skim milk, 0.2% M.
The culture method according to claim 1, which comprises gSO ₄ .7H ₂ O and 1% monosodium glutamate. 3. The growth medium is 37 g BHI, 5 g L-lysine, 30 g sorbitol, 1.5 g K ₂ HPO ₄ ,
0.5 g MgSO ₄ and pH adjusted to 7.0 1
The culture method according to claim 1, comprising liters of distilled water. 4. The culture temperature in the fermenter is i) 30 ° C. for the first 6 hours, ii) 25 ° C. for the following 2 hours, and ii.
i) The method of culturing according to any one of claims 1 to 3, which consists of bringing the last 14 hours to 20 ° C. 5. The culture method according to claim 2, which comprises replacing 1% carboxymethylcellulose in the protective medium with 1% gelatin. 6. After the suspension has been placed at 0-4 ° C. for 2 hours,
The culture method according to claim 1, which comprises freeze-drying the bacteria for 2 hours at 70 ° C. and freeze-drying under vacuum for 12 hours. 7. A live typhoid vaccine obtained by the culture method according to any one of claims 1 to 6.