JPH06234659A - Stabilized live vaccine - Google Patents

Abstract

PURPOSE:To obtain a stabilized varicella-zoster virus live vaccine excellent in preservability and heat resistance or a stabilized mixed live vaccine containing varicella-zoster virus as one of virus ingredients an improved stabilizing agent capable of being advantageously used for the stabilization of a live vaccine containing the varicella-zoster virus. CONSTITUTION:Ca<2+> ions and Mg<2+> ions contained in a live vaccine containing varicella-zoster virus or in the ingredient of a stabilizing agent therefor are masked or converted into chelate compounds with a chelating agent to stabilize the live vaccine. And a novel stabilizing agent is characterized by containing gelatin purified by a treatment such as a chelating reagent treatment, a dialysis treatment or a gel filtration treatment, and/or a gelatin derivative such as gelatin hydrolysate.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a live vaccine which has excellent storage stability and heat resistance and is stabilized. More specifically, it consists of a virus component consisting of chickenpox virus and a stabilizer,
It relates to a stabilized varicella vaccine which is substantially free of Ca ²⁺ and Mg ²⁺ ions. The present invention also provides a stabilization comprising at least one selected from Ca ²⁺ ion-free and Mg ²⁺ ion-free gelatin and gelatin derivatives useful for stabilizing a live vaccine comprising varicella virus as a virus component. Regarding agents.

[0002]

2. Description of the Related Art WHO has a global expansion plan 1
Since starting in 974, demand for various vaccines having both long-term storage stability and heat resistance has been increasing more and more. In response to such demand, research and development of various vaccines with further improved stability are being energetically promoted around the world, and for certain vaccines, it can be applied to cold regions and developing regions of the tropics. However, a material that can withstand use anywhere in the world under the outside temperature has already been put into practical use. For example, pertussis toxoid, and a DPT (diphtheria, pertussis, tetanus) three-type mixed vaccine using the toxoid (US Pat. No. 4,
849,358), dried Japanese encephalitis inactivated vaccine (F
fields “Virology”, Second Edition, Volume 1, 78
2-783, Raven Press [New York, USA], issued in 1990) and the like. However, such improved stabilization has not been achieved for all vaccines. For example, the heat resistance of live virus vaccines such as measles, rubella, mumps and chickenpox has been improved by freeze-drying, but it can be said that there is still room for improvement. In particular, chickenpox virus for live vaccines is extremely unstable to heat and usually requires storage at -60 ° C or lower. In general, viruses belonging to the Herpesviridae family and having a tendency to have cell affinity (cell-association), for example, varicella virus, are extremely unstable to heat, so development of stabilizers for such virus vaccines, and Varicella virus and other viruses, for example,
At present, it is very difficult to secure heat resistance of a mixed live vaccine with measles, rubella, mumps, etc.

By the way, examples of main substances commonly used as stabilizers for vaccines include the following. Amino acids such as sodium glutamate, arginine, lysine, cysteine; glucose,
Monosaccharides such as galactose, fructose and mannose;
Disaccharides such as saccharose, maltose, lactose; sugar alcohols such as sorbitol and mannitol; oligosaccharides, polysaccharides such as starch and cellulose and their derivatives; serum albumins such as human and bovine; gelatin; gelatin hydrolyzate, etc. Gelatin derivative; Ascorbic acid, etc. as antioxidant (Nei, "Freeze-drying and protective substance", 1
-176 pages, published by The University of Tokyo Press in 1972; Ota et al., "Vacuum Technology Course (8) Vacuum Drying", pp. 176-182.
It is reviewed in Nikkan Kogyo Shimbun, Inc. 1964). Since these substances usually have insufficient functions as stabilizers when used alone, for example, amino acids,
From each group of saccharides, sugar alcohols, and peptones, each one type that has synergistic or additive effects on the stabilizing effect in the coexistence is selected and adopted. Performed and used as a stabilizer.

[0004]

As described above, the varicella live virus vaccine is extremely unstable to heat, and the amino acids, saccharides, and amino acids conventionally used as stabilizers in other types of live virus vaccines, Stability is not obtained even when sugar alcohols, gelatin, gelatin derivatives, etc. are added, and its infectivity when stored at ambient temperature for a long time, namely,
The titer decreases. That is, improved stability cannot be obtained for a plain varicella vaccine containing live varicella virus as the only virus component, and other live viruses such as measles, rubella, mumps, polio and influenza are added. Even in mixed mixed (multivalent) vaccines, sufficient stability cannot be obtained by adding an effective stabilizer to a live vaccine containing a live virus other than live varicella virus as a virus component. It is extremely difficult to provide various stable combination vaccines containing saccharin.

[0005] In particular, when gelatin, which effectively acts to stabilize live vaccines such as measles, rubella, and mumps, and / or a gelatin derivative such as a gelatin hydrolyzate, is added to a vaccine containing live varicella virus, it is surprising. However, there was the difficulty that the stability of the vaccine would decrease. Also,
Even when only other stabilizers were used without gelatin and / or gelatin derivatives such as gelatin hydrolysates, major problems often occurred with mixed vaccines containing live varicella virus. Specifically, when preparing a live vaccine using a live virus other than varicella virus, such as measles, collected from its culture system, conventional stabilizers other than gelatin and gelatin derivatives are simply added. In this case, the stability of the vaccine is maintained, but the addition of live varicella virus collected from the culture system may reduce the stability of the mixed vaccine to the varicella virus. There is a difficulty that the function as a mixed vaccine containing live virus cannot be satisfactorily exerted.

[0006]

Under these circumstances, the present inventors have solved the above-mentioned difficult problems, and have at least stabilized live virus containing a live attenuated varicella virus as a virus component. Diligent research was conducted to provide a vaccine. As a result, satisfactory stability has not been obtained regardless of the presence or absence of gelatin and / or gelatin derivatives such as gelatin hydrolysates, which have been conventionally effective as stabilizers for other types of live virus vaccines. It was surprisingly found that the addition of ethylenediaminetetraacetic acid (EDTA) to a varicella virus vaccine improves the stability of the vaccine. As a result of further research on the cause,
Surprisingly, the stability of the varicella virus vaccine is C
is determined by the presence or absence of a ² + ions and Mg ² + ions, contains no Ca ² + ions and Mg ² + ions substantially have been found to be an essential requirement to the stability of the varicella virus. This is conventional, for example, stability of the vaccine polio live virus is, Ca ² + ions and Mg
^This is really surprising given the fact that the presence of 2+ ions would rather improve.

[0007] Ca ² + ions and Mg ² + ions are often introduced in the preparation of cultured cells, and gelatin, which has been widely used as a typical stabilizer component in recent years for the stabilization of various vaccines, is not the Ca ² + of about 0.1% just under,
Commercially available gelatin hydrolyzate contains about 0.1% C
It is known to contain a ² + ions and about 0.01% Mg ² + ions.

It has not yet been clarified how such Ca ² + ions and Mg ² + ions inhibit the stability of a live vaccine containing a live varicella virus, but heat-induced varicella virus. It is presumed that the inactivation of the virus particles is promoted to reduce the infectious titer, or the virus particles are aggregated to destabilize the virus particles. Although EDTA is originally considered to be unfavorable as an additive to vaccines from the viewpoint of its stimulating property, the inventors of the present invention have surprisingly discovered that during the course of research, live vaccines containing varicella virus and a stabilizer are included. It was found that the addition of EDTA to the vaccine significantly improves the stability of the vaccine. The reason for this is that EDTA, which the present inventors accidentally added, acts as a chelating agent for Ca ² + ions and / or Mg ² + ions that are considered to be derived from cultured cells and stabilizers, It is considered that these ions are masked. The present invention has been made based on the above new findings.
Accordingly, one object of the present invention is to provide a live vaccine containing an attenuated varicella virus and a stabilizing agent and having high stability. Another object of the present invention is that gelatin is said to be extremely effective for stabilization of live vaccines other than live varicella virus vaccine, but is harmful rather than effective for the stabilization of live varicella virus vaccine. ,
And a stabilized live vaccine containing at least one selected from gelatin derivatives such as gelatin hydrolyzate and exhibiting its excellent stabilizing action, and containing at least a live varicella vaccine as a virus component. .

Still another object of the present invention is to provide a generally available gelatin which is not used as a stabilizer as it is in a live vaccine containing live varicella virus, and gelatin such as a gelatin hydrolyzate. Per derivative,
At least varicella containing at least one selected from a treated gelatin obtained by subjecting the Ca ² + ion and Mg ² + ion contained as the cause to a masking treatment or a removing treatment and a gelatin derivative to be treated. It is intended to provide a stabilizer for a live vaccine containing a virus as a virus component. These and other objects, features and benefits of the present invention will become apparent from the detailed description and claims set forth below. That is, according to the present invention, Ca ² + containing a viral component consisting of at least one attenuated varicella virus selected from the group consisting of live attenuated varicella virus and recombinant varicella virus, and a stabilizer,
Stabilized live vaccine is provided which is substantially free of ions and Mg ² + ions.

The attenuated live chickenpox virus used in the present invention is
It comprises at least one selected from the group consisting of an attenuated live varicella virus and an attenuated recombinant varicella virus produced by a conventionally known method. Also, as a preferred attenuated varicella virus for live varicella vaccine, there is Oka strain (US Pat. No. 3,985,615; virus deposit number ATC).
C VR-795) is well known. A live attenuated recombinant chickenpox virus can be produced by recombining the genomic gene of Oka strain with a foreign gene. Recombination is easily possible by conventional methods. For details of the method for recombining a viral gene with a foreign gene, European Patent Application Publication No. Reference can be made to 0334530A1. This virus strain (Oka strain) can be advantageously used in the present invention, but of course, the chickenpox virus used in the present invention is not limited to this Oka strain.

It is essential that the live vaccine of the present invention contains substantially no Ca ² + ion or Mg ² + ion.
“Substantially free of Ca ² + and Mg ² + ions” means that Ca is determined by a colorimetric titration method using a chelating reagent.
^This means that ² + ions and Mg ² + ions are not detected. When Ca ² + ions and Mg ² + ions derived from cultured cells are mixed in the vaccine, these ions can be masked as chelate compounds by adding and mixing a chelating reagent. As the chelating agent, ethylenediaminetetraacetic acid (hereinafter, abbreviated as “EDTA”) and its salts, polyaminocarboxylic acids, oxycarboxylic acids such as citric acid and its salts, condensed phosphates such as ultraphosphates, and the like, Water-soluble substances that form chelate compounds can be used. Especially, regarding EDTA, EDTA itself and its Ca, Co, K, N
Various salts of a, Li, Ni, Ba, Bi, Mg, Mn, La, ammonium and the like are commercially available. Among these, it is desirable to avoid the use of Ca salt or Mg salt and to use Na salt or K salt.
It is preferable to use EDTA-2Na or EDTA-3Na in consideration of reducing the fluctuation of Also, water-soluble ethylene glycol tetraacetic acid and its salts, which are analogs of EDTA, can be used as the chelating agent according to the present invention.

As the stabilizer used in the present invention, various conventionally known stabilizers are used, for example, saccharose, lactose, sorbitol, sodium glutamate, cysteine, gelatin, gelatin derivatives such as gelatin hydrolysate, and the like. Can be mentioned. The use of at least one selected from gelatin and gelatin derivatives is particularly useful as a stabilizer component. The gelatin and gelatin derivatives used in the present invention can be used alone or in combination. Examples of gelatin include purified gelatin described in the Japanese Pharmacopoeia. Gelatin derivatives include gelatin hydrolysates and their chemical derivatives. "Gelatin hydrolyzate" means high molecular weight gelatin
deg clear by making it tic clear
Hydrolyzate manufactured by addition
d polypeptides or polys of these
It means merized polypeptides. Such gelatin hydrolyzate is water-soluble and has a molecular weight of about 35,000 or less. Specific examples of the gelatin derivative that can be used in the present invention include a commercially available product under the trade name of gerysate (BBL, USA).
Hydrolyzed gelatin manufactured by K.K.), trade name Physiogel, Neoplasma gel (Neo)
plasmagel), Gerifandor (Gelfif)
Undol) and Haemacel (a gelatin hydrolyzate manufactured by Hoechst, Germany, or a chemical derivative thereof).
For more information on these gelatin derivatives, see Develo.
pments in Biological Stan
darzation, Vol. 48, pp. 207-234 (S. Karger 1981). Of these, gelatin hydrolyzate easily available under the trade name of gerisate is preferably used in the present invention. The amount of gelatin and gelatin derivative is
Usually, the concentration in the stabilized live vaccine is about 0.
02 to about 1% (w / v) and about 0.5 to about 10% (w /
v) is preferably used. Regarding gelatin, and gelatin derivatives, especially gelatin hydrolyzate, commercially available products are usually Ca ² + ions and / or Mg ² +.
Since it contains ions, when a commercially available product is used as it is, it is preferable to add and mix the above-mentioned chelating agent such as EDTA or a salt thereof to the live vaccine. The amount of the chelating agent added to the live vaccine of the present invention is not critical and may be an amount necessary for converting existing Ca ² + ions and Mg ² + ions into a chelate compound for masking, but As a concentration in a stabilized live vaccine, about 0.
It can be used at a concentration of 001 to about 0.1% (w / v).

The solution for suspending the viral antigen was 0.005 prepared according to the procedure described in Reference Example 4 below.
~ 0.01 M PBS (-) can be used. Also, Ca
And 199 medium (see Reference Example 10) or MEM medium (Reference Example 1) prepared without adding and mixing the Mg compound in advance.
See also) can be used. The concentration of each component of the stabilizer in the live virus vaccine prepared using such a basic solution is, for example, about 0.01 to about 5.0% sodium glutamate as the concentration in the stabilized live vaccine. (W /
v), cysteine is about 0.02 to about 1.0% (w /
v), about 0.5 to about 10% (w /
v), lactose is about 0.5 to about 10% (w / v), sorbitol is about 0.2 to about 6.0% (w / v), and gelatin is about 0.02 to about 1.0% (w / v). w / v), gelatin derivatives such as gelatin hydrolysates are about 0.5 to about 10%
The chelating agent such as (w / v) and ethylenediaminetetraacetic acid or a salt thereof may be about 0.001 to about 0.1% (w / v). Final pH after adding stabilizer
Is preferably 6.5 to 7.5, and it is preferable that the osmotic pressure of the vaccine is in the vicinity of isotonicity in consideration of pain due to injection and damage to the vaccination site.

The following four examples are listed below as preferable examples of the composition of the stabilizer effective for obtaining the stabilized live vaccine of the present invention. (1) As a concentration in a stabilized live vaccine, about 0.01 to about 5% (w / v) sodium glutamate, about 0.5 to about 10% (w / v) sucrose, about 0.02 to about 1 %
A stabilizer containing (w / v) gelatin, about 0.5 to about 10% (w / v) gelatin hydrolyzate. (2) As a concentration in the stabilized live vaccine, about 0.01 to about 5% (w / v) sodium glutamate, about 0.5 to about 10% (w / v) sucrose, about 0.02 to about 1 %
(W / v) gelatin, about 0.5 to about 10% (w / v) gelatin hydrolyzate, and about 0.001 to about 0.1% (w).
/ V) A stabilizer containing a chelating agent. (3) As a concentration in the stabilized live vaccine, about 0.5 to about 1
0% (w / v) lactose, about 0.2 to about 6.0% (w
/ V) sorbitol, about 0.02 to about 1.0% (w /
v) cysteine, about 0.01 to about 5.0% (w / v) sodium glutamate, about 0.02 to about 1.0% (w /
v) A stabilizer containing gelatin and about 0.5 to about 10% (w / v) gelatin hydrolyzate. (4) As a concentration in the stabilized live vaccine, about 0.5 to about 1
0% (w / v) lactose, about 0.2 to about 6.0% (w
/ V) sorbitol, about 0.02 to about 1.0% (w /
v) cysteine, about 0.01 to about 5.0% (w / v) sodium glutamate, about 0.02 to about 1.0% (w /
v) A stabilizer containing gelatin, about 0.5 to about 10% (w / v) gelatin hydrolyzate and about 0.001 to about 0.1% (w / v) chelating agent.

Next, practical means for carrying out the present invention will be described. (1) Preparation of virus suspension containing no Ca ² + ion and Mg ² + ion: For example, in the case of varicella virus, after culturing the virus using host cells, the culture solution is discarded and the final concentration of 0.05 0.01 M PBS (-) containing% (w / v) EDTA-3Na is added to detach the infected cells from the inner surface of the culture container. Then, the infected cells were collected by low-speed centrifugation, and the above-described stabilizer was added to the solution for varicella virus suspension to resuspend the infected cells, and then the cells were disrupted with a homogenizer or ultrasonic waves. A cell suspension can be removed by centrifugation to prepare a virus suspension. For each virus such as measles, rubella, mumps, a virus culture solution containing both Ca ²⁺ and Mg ²⁺ ions was prepared without adding Ca and Mg compounds in advance during or after the virus culture. Each virus suspension can be prepared by substituting 199 medium or MEM medium. For example, with measles virus, infected cells in a culture vessel are washed with PBS (−) of Reference Example 4 described below to remove Ca ²⁺ and Mg ²⁺ ions, and then suspended in the above-mentioned medium, A virus suspension can be prepared by freeze-thawing and low-speed centrifugation. For both rubella and mumps viruses, for example, after concentrating the virus culture solution with a membrane, infectious virus particles collected by ultracentrifugation can be suspended in the above-mentioned medium to prepare these virus suspensions. . Each component of the stabilizer can be added to and mixed with the virus suspension.

(2) Preparation of a virus suspension in which Ca ² + ions and Mg ² + ions derived from virus-cultured cells are masked or converted into chelate compounds: For example, a chelating reagent is added to and mixed with the virus suspension after virus culture. By doing so, the above metal ions can be masked or converted into a chelate compound.

(3) Preparation of gelatin and gelatin hydrolyzate in which Ca ² + ions and Mg ² + ions are masked or converted into chelate compounds: For example, a chelating agent is added to each solution of gelatin and gelatin hydrolyzate and mixed. By doing so, the above metal ions can be masked or converted into a chelate compound. Such gelatin and gelatin hydrolyzate are very useful as live vaccine stabilizers.

(4) Preparation of purified gelatin and gelatin hydrolyzate containing no Ca ² + ion and Mg ² + ion: can be purified by a conventional method using dialysis, gel filtration, cation exchange resin, chelate resin, etc. . For example, gel filtration of each of the above-mentioned gelatin and gelatin hydrolyzate masked with metal ions, dialysis of each solution of gelatin and gelatin hydrolyzate, and the like, purified gelatin not containing the above metal ions and purified gelatin Gelatin hydrolysates can be obtained respectively. Such purified gelatin and purified gelatin hydrolyzate are extremely effective as a live vaccine stabilizer.

(5) Production of Stabilized Live Vaccine: After culturing varicella virus using human diploid cell culture, the culture is purified, for example, by centrifugation, and the varicella virus fraction is collected. Vaccine stabilizer is added to this mixture, and the virus content per live vaccine dose is 1,000P.
Dilution is prepared so that it is more than FU (plaque forming unit),
Produce a live vaccine. As for the live vaccine, for some samples, the biological standard (1989) specified in Ministry of Health and Welfare Notification No. 195, for example, "dried attenuated live varicella vaccine" is used.
In accordance with the above, various test tests regarding safety, efficacy and homogeneity are performed, and the eligibility as a vaccine is confirmed,
After confirmation, it is ready for use. Dry vaccine is about 3-30
It is lyophilized in a ml vial bottle or ampoule, provided in a hermetically sealed state, and stored at 5 ° C or lower. The use of such a vaccine is as described in the instruction manual attached to it.
Immediately before use, the dry contents are completely redissolved in sterile distilled water, and then 0.5 ml of 1 dose is subcutaneously inoculated.

As described above, each virus such as measles, rubella, and mumps is relatively stable to heat compared to varicella virus, and the stabilizer components of these virus vaccines are similar to each other. It is well known that the combination of known and commonly used sugars, amino acids, sugar alcohols, gelatin and gelatin derivatives is effective. In comparison, the stabilizer of varicella virus according to the present invention is specific, but not only varicella virus vaccine and recombinant varicella virus vaccine, such varicella virus and other viruses, for example, measles, rubella, mumps. ,influenza,
Japanese encephalitis, mixed vaccine with each virus such as hepatitis E,
For example, it is effective for a mixed live vaccine containing varicella virus or recombinant varicella virus as one of active virus components, such as a mixed live vaccine for chickenpox, measles, rubella, and mumps (Japanese Patent Publication 58-5169). As described above, various dried vaccines, dried mixed vaccines, liquid vaccines, liquid mixed vaccines and the like, which have extremely good storage stability and are stable in heat, are provided. In the case of the mixed live vaccine, the live vaccine stock solution is diluted and mixed so that each virus content per dose of the live vaccine is 1,000 or 5,000 PFU or more, or 5,000 TCID ₅₀ or more.

Hereinafter, with reference to Reference Examples and Examples,
The present invention will be described more specifically. However, the present invention is not limited to these reference examples and examples.

[0022]

[Reference example]

Reference Example 1 Culture of human diploid fibroblast MRC-5: Culture at 37 ° C. MEM medium [Gibco (USA)] is used as a medium for both growth and maintenance of the cells.
In addition, just before use, this medium contained 7% (w / v) NaHCO ₃
An aqueous solution is added and mixed to adjust the pH to 7.0 in the growth medium and to 7.5 in the maintenance medium. Commercially available fetal calf serum was then added to a final concentration of 10% (w / w) in growth medium.
v) In addition, in the maintenance medium, it is additionally mixed to be 3% (w / v) before use.

Reference Example 2 Culture of Varicella Virus: The MRC-5 cell culture obtained in Reference Example 1 was mixed with the varicella virus Oka strain (WHO Technical).
Report Series, No. 275, pp.
102-124, 1985; ATCC VR-795).
Seed virus is inoculated at MOI (multiplicity of infection) of 0.03, and then cultured at 37 ° C. for 2 days. The maintenance medium described in Reference Example 1 is used as the medium. During virus culture, the infected cell area gradually expands as the virus grows. Virus-infected cells exhibit rounding under a microscope and can be detected as so-called CPE (cytopathic effect). Therefore, the extent of virus proliferation can be determined by observing the spread of infected cells based on such CPE under a microscope. The virus culture is terminated when CPE is observed throughout the cell culture monosheet. A roux bottle having a culture area of 210 cm ² is used as a culture container.

Reference Example 3 Culture of measles, mumps and rubella viruses: After culturing chicken embryo cells and quail embryo cells in MEM medium in the same manner as in Reference Examples 1 and 2, measles and mumps were cultured. The respective viruses are cultured in chicken embryo cells, and the rubella virus is cultured in quail embryo cells.

[0025] Reference Example 4 M / 100 PBS (-) [Ca 2 + ions and Mg ² + phosphate buffered saline containing no ion] Preparation of: Na
The Cl 8.0 g, the _{KCl 0.2g, Na 2 HPO 4 ·} 1
2.9 g of 2H ₂ O and 0.2 g of KH ₂ PO ₄ are dissolved in distilled water to a total volume of 1,000 ml. This PBS
The pH of (-) becomes about 7.4.

Reference Example 5 Preparation of Virus Suspension Liquid (A): Saccharose 50 g,
L-sodium glutamate 1.0 g, and EDTA-
PBS (−) obtained in Reference Example 4 sequentially with 0.1 g of 3Na salt
Add to 800 ml and dissolve each. On the other hand, 50 ml of distilled water was added to 2.0 g of gelatin, and 100 ml of distilled water was added to 25 g of gelatin hydrolyzate (manufactured by BBL [USA]).
Are added and each is heated in an autoclave to dissolve. Mix all these solutions, add distilled water,
Bring the total volume to 1,000 ml. Then, after filter sterilization, 1
Part is sampled and a sterility test is performed to confirm sterility. The composition of 1 liter of the virus suspension (A) is shown in Table 1 below: Table 1 ─────────────────────── --NaCl 6.4 g KCl 0.16 g Na ₂ HPO ₄ / 12H ₂ O 2.3 g KH ₂ PO ₄ 0.16 g Saccharose 50.0 g Sodium L-glutamate 1.0 g Gelatin 2.0 g * Gelatin hydrolyzate 25.0 g * EDTA-3 sodium salt 0.1 g ─────────────────────────── <Note> * Those are referred to in Reference Example 8.

Reference Example 6 Preparation of Virus Suspension Solution (B): In the same manner as in Reference Example 5, a virus suspension solution (B) was prepared with the amounts of components shown in Table 2 below. Gelatin and gelatin hydrolyzate were dissolved in distilled water, and the remaining other components were PBS (-) 8.
After dissolving in 00 ml, the whole solution was mixed, distilled water was added to make the total volume 1,000 ml, and then the solution was sterilized by filtration and used as a virus suspension (B) for subsequent use: Table 2 ─────── _{──────────────────── NaCl 6.4g KCl 0.16g Na 2 HPO} 4 · 12H 2 O 2.3g KH 2 PO 4 0.16g lactose 50.0 g D-sorbitol 20.0 g L-cysteine 2.0 g sodium L-glutamate 1.0 g gelatin 7.5 g gelatin hydrolyzate 25.0 g EDTA-3 sodium salt 0.1 g ───── ─────────────────────

Reference Example 7 Preparation of Virus Suspension Liquid (C): In the same manner as in Reference Example 6, a solution containing each component of the virus suspension liquid (B) at double concentration is prepared. This is used as a virus suspension (C) for subsequent use. Reference Example 8 Preparation of Experimental Solution: Each concentration (g / l) of gelatin hydrolyzate and EDTA-3 sodium salt described in (*) in Reference Example 5 was changed as follows. Each component other than the two substances and the concentration thereof are the same as in Reference Example 5, and the experimental solutions 1 to 8 shown in Table 3 below are prepared in the same manner as described in Reference Example 5: Table 3 ──────────────────────────────── Gelatin hydrolyzate EDTA-3 sodium salt (g / l) (g / l) ) ────────────────────────────────── Test solution 1 0 0 2 10 0 3 3 25 0 4 50 0 − −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− Experimental liquid 5 0 0.1 6 10 0.1 7 25 0.1 8500 2 ──────────────────────────────────

Reference Example 9 Preparation of gelatin hydrolyzate purified by removing Ca ² + ion and Mg ² + ion: gelatin hydrolyzate (BBL
(Manufactured by [USA]), weigh 120 g, and add distilled water 180
After adding ml, heat in an autoclave to dissolve. Then, add distilled water to a total volume of 300 ml, and add 40
% (W / v) gelatin hydrolyzate solution is prepared. After injecting the solution into a dialysis tube made of cellulose ester, Spectra / Pore (fraction molecular weight 1,000; manufactured by Funakoshi Co., Ltd. [Japan]), dialyzed at room temperature for 48 hours with distilled water as an external solution. After completion of dialysis, Ca ² + ion and Mg ² + ion were not detected by chelate titration method, and after confirming that they were substantially removed, they were sterilized by filtration and purified for a live vaccine stabilizer. It is used as a gelatin hydrolyzate for subsequent use. The chelate titration method is performed by colorimetric titration using EDTA-2Na salt as a chelating reagent and Eriochrome Black T as a metal indicator. The measurement system uses the color change from magenta to blue as the end point of the titration.

Reference Example 10 Varicella virus plaque assay: MRC-5 cell monosheets cultured in a petri dish were inoculated with a 10-fold serially diluted specimen virus solution, and then inoculated in a 5% (v / v) carbon dioxide incubator. Incubate at 37 ° C. 1 as basal medium
Reference Example 1 using 99 medium (manufactured by Difco [USA])
Agarose is added to and mixed with the maintenance medium prepared in the same manner as described above, to give a final concentration of 0.8% (w / v) to prepare a solid medium. This medium is used by overlaying on the infected cell monosheet. For staining infected culture cells, neutral red was added to the above solid medium at a final concentration of 0.01% (w /
v) The medium prepared by adding and mixing is further layered, and the plaques that have appeared are counted. The number, PFU
(Plaque forming unit) means the virus content in the live vaccine and represents the titre of the live vaccine with PFU. For example, after inoculating 1 ml of a 10 ⁴ -fold diluted vaccine solution and culturing, when 10 plaques appear, the total plaque is calculated as 10 ⁵ PFU / ml from 10 × 10 ⁴ .
Usually, it is expressed as 5.0 using its common logarithm log (10 ⁵ PFU / ml).

Reference Example 11 Measurement of titers of live measles, rubella and mumps vaccines: For both measles and rubella vaccines, refer to plaque assays of measles virus by Vero cells and rubella virus by RK-13 cells. The titer is measured in the same manner as described in Example 10. For mumps vaccine, TC of mumps virus by Vero cells
ID ₅₀ (media tissue culture
It is measured by infective dose. T
For CID ₅₀ , Vero cell sheets cultured in a 5 ml test tube were inoculated with a 10-fold serially diluted sample vaccine solution, and then cultured at 37 ° C, and CPE was observed in 50% of infected cells under a microscope. Calculated based on the dilution factor of the vaccine ("Review of Medical
Microbiology ", 13th ed.,
pp. 344 to 345, Lange Medical
Publications, published in 1976). For example, as a result of culturing by inoculating 10 cell tube test tubes with 10 ⁴ -fold diluted vaccine, 5 of these 10 cells (50
%) With CPE, TCID ₅₀ is log
Than 10 ^4, is referred to as 4.0.

[0032]

【Example】

Example 1 Preservation test of live vaccine (effect test of stabilizer): MRC-5 cell culture of 20 Roux bottles with a culture area of 210 cm ² was inoculated with varicella virus Oka strain seed virus, and then in Reference Example 2 Culture as described. After the culture is completed, the culture medium is discarded and the infected cells in each roux bottle are treated with 200 ml of PBS.
Wash twice with (-). Then 20 ml of 0.05
% (W / v) EDTA-3Na is overlaid on the infected cells in each roux bottle, and the cells are detached from the inner wall surface of the roux bottle and floated. Infected cell suspension in each roux bottle is pooled for a total of 40
0 ml of infected cell suspension is obtained. Then, divide this equally into 8 centrifuge tubes (T1 to T8) and divide into 8 equal parts,
1 / Make the infected cell suspension in a centrifuge tube. The infected cell suspension in each centrifuge tube is centrifuged at 2,000 rpm for 10 minutes at 4 ° C., the supernatant is discarded, and the pellet of infected cells at the bottom of each centrifuge tube is collected. The test liquids 1 to 8 prepared in Reference Example 8 were added to a centrifuge tube T1.
20 ml of the experimental liquid 1 in the
Add 0 ml, ...
After resuspending the infected cells in each centrifuge tube, freeze and thaw once,
To do. Next, ultrasonic treatment (20 kHz, 15
0 mA, 0.3 sec / ml), followed by centrifugation at 1,000 rpm for 10 minutes at 4 ° C. to collect the supernatant containing the cell-free virus. Prepare the vaccine solution. 0.5 ml of each live vaccine solution was dispensed into 3 ml vial bottles, freeze-dried, filled with nitrogen gas and sealed with a rubber stopper to hermetically seal the inside of the vial bottle. Keep these small pieces of live vaccine at 37 ° C,
Randomly extract 5 samples each week and use them for the effect test of the stabilizer. Immediately before the test, the dry contents were diluted with distilled water for injection at 0.
Add 7 ml and dissolve completely before use. The effect test was carried out by the plaque assay described in Reference Example 10, and each vaccine titer is represented by PFU. The results are shown in Table 4. The stabilizing effect observed in test liquid 7 was the best.
Table 4 Preservation test of live vaccine (effect test of stabilizer) ─────────────────────────────────── ──Potency = log (PFU / ml) Stabilizer ───────────────────────────── Before freeze-drying After freeze-drying 37 ℃ Storage days ───────────────────────── Experimental solution No. 0 7 14 21 28 ───────────── ──────────────────────── 1 5.3 5.3 4.3 3.5 2.7 2.5 2.1 2 5.1 4.6 3.7 3.3 3.2 3.2 3 5.0 4.6 3.9 3.6 3.4 3.2 4 5.0 4.5 3.7 3.7 3.1 3.1 3.0 −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− 5 5.2 4.4 3.4 2.8 2.4 2.2 6 5.3 4.7 4.7 4.0 3.5 3.5 3.3 7 5.4 4.8 4.3 4.3 4.1 4.0 3.8 8 5.3 4.7 4.2 4.2 3.8 3.8 3.6 ──────────────────────────────── ─────

Example 2 Production of stabilized live vaccine containing live attenuated varicella virus as an active ingredient: MR of 20 roux bottles having a culture area of 210 cm ^2.
Varicella virus Oka strain seed virus is inoculated into C-5 cell culture, and then cultured in the same manner as in Reference Example 2. After the culture is completed, the culture solution is discarded and the infected cells in each roux bottle are filled with 200 cells.
Wash twice with ml PBS (-). Then 20m
1 of 0.05% (w / v) EDTA-3Na is overlaid on the infected cells in each Roux bottle, and the cells are detached from the inner wall surface of the Roux bottle and floated. The infected cell suspension in each roux bottle is pooled and centrifuged at 2,000 rpm for 10 minutes at 4 ° C. to collect a pellet of infected cells. After resuspension in 100 ml of the live virus suspension (A) prepared in Reference Example 5, freeze-thawing is performed once. Next, sonicate in an ice water bath (20k
Hz, 150 mA, 0.3 sec / ml), then 1.00
Centrifuge at 0 rpm for 10 minutes at 4 ° C., collect the supernatant containing cell-free virus, and use this as the live vaccine stock solution.
30 ml was sampled from this stock solution for assay,
To the remaining 70 ml of the stock solution, the live virus suspension (A) prepared in Reference Example 5 is added and mixed to prepare 140 ml of the final bulk of the live vaccine. After sampling 30 ml from this final bulk for assay, 0.5 ml each of the remaining bulk was dispensed into a 3 ml vial bottle, and after freeze-drying,
Fill with nitrogen gas and seal with a rubber stopper to hermetically seal the inside of the vial bottle. This small portion of the live vaccine is stored at 4 ° C., and 0.7 ml of distilled water for injection is added immediately before use to completely dissolve the dried contents for use. On the other hand, for the above-mentioned vaccine stock solution and final bulk sampled, and 20 small parts,
Perform a certification test. In order to confirm safety, efficacy and homogeneity and to confirm eligibility as a live vaccine, such an assay test is based on the biologics standard (1989) stipulated in the Ministry of Health and Welfare Notification No. It will be carried out in accordance with the "Varicella vaccine". As a result of such certification test,
The virus content was 2 × 10 ⁴ PFU (plaque forming unit) /0.5 ml, and since it passed various tests prescribed in the above criteria, it was determined to be a live vaccine with qualification. In addition, as a result of subjecting it to the same storage vaccine test as described in Example 1, the experimental solution 7 described in Example 1 was obtained.
It was confirmed that this live vaccine is an excellent stabilized live vaccine because the stabilizing effect equal to or higher than that was recognized.

Example 3 Preservation test of 4 kinds of mixed live vaccine: Using the virus suspension liquid B obtained in Reference Example 6, the stock solution of varicella vaccine (V) was prepared in the same manner as described in Example 2. To prepare. On the other hand, in the same manner as described in Reference Example 3, measles,
Incubate rubella and mumps viruses. After completion of the culture, for measles virus, the culture solution in the culture container is discarded, PBS (−) is added to wash the virus-infected cells, and Ca ²⁺ and Mg ²⁺ are removed. Then 1 of the culture
After adding / 20 volume of 199 medium (manufactured by Difco [USA]) or MEM medium containing neither Ca ²⁺ nor Mg ²⁺ ion, the infected cells are detached from the inner surface of the culture vessel by pipetting to infect Collect the cell suspension. After freeze-thawing once for the suspension, low-speed centrifugation (4 ° C,
2,000 rpm, 10 minutes) and collect the supernatant to make measles virus suspension, and for both rubella and mumps virus, low-speed centrifugation (4 ° C, 2,000 rpm, 10 minutes) of each virus culture solution. ), Each of the supernatants was ½ on a cellulose membrane (manufactured by Millipore [USA]) for 300-kilodalton fractionation.
Concentrate to 0 volume. Then, the concentrate is subjected to ultracentrifugation (4 ° C.,
20,000 rpm, 90 minutes for Mumps, 18 for Rubella
After 0 minutes), the supernatant is discarded, and the pellet is suspended in 199 medium or MEM medium containing neither Ca ²⁺ nor Mg ²⁺ ion to obtain each virus suspension. The virus suspension C obtained in Reference Example 7 was mixed in an equal volume ratio to each of the virus suspensions to prepare the following three stock solutions of live vaccines for measles, rubella and mumps:

(1) Measles (MI), rubella (RI) and mumps (MU) were prepared by adding and mixing an equal amount of the virus suspension C to each virus suspension prepared in 199 medium.
Prepare each live vaccine stock solution of I); (2) Add ED to each virus suspension prepared in 199 medium.
The final concentration of TA-3 sodium salt was 0.15% (w /
v), and then mixed and mixed with an equal amount of the virus suspension liquid C to prepare live stock solutions of measles (M2), rubella (R2) and mumps (MU2); and

(3) An equal amount of virus diluent C was added to and mixed with each virus suspension prepared in MEM medium containing neither Ca ² + nor Mg ² + ions, and measles (M3) and rubella (R)
3) and Mumps (MU3) live vaccine stock solutions are prepared. Then, each of these live vaccine stock solutions is shown in Table 5 below.
Each is mixed as shown in to knit 3 final bulks: Table 5 ────────────────────────────────── ─── Final bulk No. Live vaccine mix (ml) ─────────────────────────── 500 100 200 200 200 ───── ────────────────────────────────────── 1 V M1 R1 MU1 2 V M2 R2 MU2 3 V M3 R3 MU3 ──── ────────────────────────────────

Then, in the same manner as described in Example 1, each bulk was dispensed in 0.5 ml portions into a 3 ml vial bottle, lyophilized, and divided into four kinds of mixed live vaccine aliquots. , 37 ° C. to be subjected to a stabilizer effect test. The effect test is carried out by the plaque assay and the force value measurement described in Reference Examples 10 and 11. The results are shown in Table 6. Final bulk No. 3 was the best as a stabilized live vaccine with a mixture of 4 kinds. Table 6 Preservation test results of four kinds of mixed stabilized live vaccines ───────────────────────────────────── Potency = log (PFU / ml) or TCID ₅₀ stabilizer ────────────────────────────── Addition Before freeze-drying Days of storage at 37 ° C after freeze-drying Final bulk ──────────────────────── 0 7 14 21 28 ──────────── ───────────────────────── Bulk No.1 Chickenpox 5.0 3.6 3.0 2.7 2.7 2.2 2.1 Hemp Rubella 6.1 5.6 5.2 4.9 4.9 4.9 4.8 Rubella 4.8 4.5 4.5 4.3 4.2 4.1 4.1 Mumps 6.1 5.7 5.2 4 9.9 4.9 4.7 Bulk No. 2 Chickenpox 5.1 4.2 4.2 3.6 3.3 3.3. 3.0 Measles 6.1 5.6 5.1 5.1 5.0 4.8 4.7 Rubella 4.8 4.5 4.4 4.4 4.2 4.2 4.1 Mumps 6.1 5.6 5.1 4.9 4.8 4.8 Bulk No. 3 Chickenpox 5.1 4.6 4.6 4.1 3.9 3.7 3.6 Measles 6.1 6.1 5.6 5.2 5.0 4.9 4.8 Rubella 4.8 4.5 4.5 4.3 4.2 4.2 4.1 Mumps 6.1 5.7 5.2 5.2 5.0 4.8 4.6 ────── ───────────────────────────────

Example 4 Vaccine stabilizing effect test of purified gelatin hydrolyzate: E
A virus suspension (A1) having the same concentration as the components of the virus suspension (A) described in Reference Example 5 except that the DTA-3 sodium salt is removed is prepared. Further, by using the purified gelatin hydrolyzate obtained in Reference Example 9, EDTA-
A virus suspension (A2) having the same concentration as the components of the virus suspension (A) described in Reference Example 5 except that the trisodium salt is removed is prepared. Then, using the virus suspensions (A), (A1) and (A2), a dried chickenpox live vaccine was prepared in the same manner as described in Example 2, and each live vaccine was stored at 37 ° C. Conduct a sex test.
The results are shown in Table 7. The virus stabilizing solution A2 showed the same virus stabilizing effect as A. That is, when a purified gelatin hydrolyzate containing neither Ca ² + ion nor Mg ² + ion is used as one component of the stabilizer, ED is used.
It was confirmed that TA-3 sodium salt was unnecessary. Table 7 Vaccine stabilization effect of purified gelatin hydrolyzate ───────────────────────────────────── = Log (PFU / ml) Stabilizer ────────────────────────────── Before freeze-drying 37 ° C after freeze-drying Days of storage Virus ──────────────────────── Suspension liquid 0 7 14 21 28 28 ─────────────── ───────────────────── A 5.4 4.9 4.5 4.3 4.3 3.9 A1 5.1 4.6 3.9 3.6 3.3 3.1 A2 5.3 4.9 4.4 4.4 4.1 4.0 3.8 ─────────────────────── ──────────────

Example 5 Production of Stabilized Live Vaccine Using Recombinant Attenuated Varicella Virus as an Active Ingredient: Attenuated Varicella Virus Recombinant attenuated varicella virus produced by inserting and linking the hepatitis B virus gene into the oka strain genome rVH17-5 Oka strain
(ECACC Accession Number V
Production of a live vaccine, an assay test, and a storage test are performed according to the procedure described in Example 2 except that 92041523) is used as a seed virus. As a result, it was confirmed that the produced live vaccine was a stabilized live vaccine having the same qualification as the live vaccine described in Example 2.

[0040]

EFFECTS OF THE INVENTION (1) A stabilized varicella vaccine containing varicella virus and / or recombinant varicella virus, which has excellent storage stability and stability of heat resistance, is provided. (2) A stabilized mixed live vaccine containing varicella virus and / or recombinant varicella virus as one of virus components is put to practical use. (3) gelatin was improved by Ca ² + ions Oyobi and Mg ² + ions to purify the masking or removal, and gelatin derivatives such as hydrolyzed gelatin, are provided as useful stabilizers for live vaccines. (4) Contribute to the spread, expansion, and labor saving of vaccination at the global level, such as the WHO immunization expansion plan. (5) Contribute to the improvement of human health and prevention of epidemics.

Claims (25)

[Claims] 1. A Ca ² + containing a viral component comprising at least one varicella virus selected from a live attenuated varicella virus and a attenuated recombinant varicella virus, and a stabilizer.
A stabilized live vaccine substantially free of Mg and Mg ² + ions. 2. The stabilized live vaccine according to claim 1, wherein the stabilizer is at least one selected from gelatin, gelatin derivatives and chelating agents. 3. The amount of gelatin and gelatin derivative is about 0.02 to about 1% (w / v) and about 0.5 to about 10% (w / v), respectively, as the concentration in the stabilized live vaccine. ) Is a stabilized live vaccine according to claim 2. 4. The stabilized live vaccine according to claim 2, wherein the gelatin derivative is a gelatin hydrolyzate. 5. The amount of the chelating agent is about 0.001 to about 0.1% (w / v) as a concentration in the stabilized live vaccine.
The stabilized live vaccine according to any one of claims 2 to 4, which is 6. The stabilizing agent, as a concentration in a stabilized live vaccine, is about 0.01 to about 5% (w / v) sodium glutamate and about 0.5 to about 10% (w / v) saccharose. , About 0.02 to about 1% (w / v) gelatin, about 0.
The stabilized live vaccine according to claim 4, which contains 5 to about 10% (w / v) gelatin hydrolyzate. 7. The stabilizing agent, as a concentration in a stabilized live vaccine, is about 0.01 to about 5% (w / v) sodium glutamate, about 0.5 to about 10% (w / v) saccharose. , About 0.02 to about 1% (w / v) gelatin, about 0.
The stabilized live vaccine according to claim 4, which contains 5 to about 10% (w / v) gelatin hydrolyzate, and about 0.001 to about 0.1% (w / v) chelating agent. 8. The stabilizer as a concentration in a stabilized live vaccine is about 0.5 to about 10% (w / v) lactose, about 0.2 to about 6.0% (w / v). Sorbitol, about 0.02 to about 1.0% (w / v) cysteine, about 0.0
1 to about 5.0% (w / v) sodium glutamate, about 0.02 to about 1.0% (w / v) gelatin and about 0.5
A stabilized live vaccine according to claim 4, which contains ˜about 10% (w / v) gelatin hydrolyzate. 9. The stabilizer as a concentration in a stabilized live vaccine is about 0.5 to about 10% (w / v) lactose, about 0.2 to about 6.0% (w / v). Sorbitol, about 0.02 to about 1.0% (w / v) cysteine, about 0.0
1-about 5.0% (w / v) sodium glutamate, about 0.02-about 1.0% (w / v) gelatin, about 0.5-
About 10% (w / v) gelatin hydrolyzate and about 0.00
The stabilized live vaccine of claim 4, which contains 1 to about 0.1% (w / v) chelating agent. 10. The stabilized live vaccine according to claim 2, 5, 7 or 9, wherein the chelating reagent is ethylenediaminetetraacetic acid or a salt thereof. 11. The gelatin and the gelatin derivative are:
The stabilized live vaccine according to claim 2 or 3, which is substantially free of Ca ² + ions and Mg ² + ions, respectively. 12. The stabilized live vaccine according to claim 4, 6 or 8, wherein the gelatin hydrolyzate is substantially free of Ca ² + ions and Mg ² + ions. 13. Gelatin and gelatin derivatives substantially free of Ca ² + ions and Mg ² + ions, respectively,
Chelate reagent-treated gelatin obtained by treating gelatin and a gelatin derivative containing Ca ² + ion and / or Mg ² + ion with a chelating reagent to mask the ion, and a purified product thereof and a chelate reagent-treated gelatin derivative or the same The stabilized live vaccine according to claim 11, which is a purified product. 14. A gelatin and a gelatin derivative substantially free of Ca ² + ions and Mg ² + ions, respectively.
Ca ² + ions and / or Mg ² + dialyzed gelatin and gelatin derivatives containing ions, gel filtration treatment, treatment with a cation exchange resin, Ca ² + ions and toward at least one processing selected from treatment with a chelating resin The stabilized live vaccine according to claim 11, which is a purified gelatin or a purified gelatin derivative obtained by substantially removing Mg ² + ions. 15. The stabilized live vaccine according to claim 11, 13 or 14, wherein the gelatin derivative is a gelatin hydrolyzate. 16. The stabilized live vaccine according to claim 1, wherein the virus component further contains at least one live attenuated virus other than varicella virus. 17. The stabilized live vaccine according to claim 16, wherein at least one live attenuated virus other than chickenpox virus is selected from live attenuated viruses of measles, rubella, mumps, Japanese encephalitis, influenza and hepatitis. 18. The stabilized live vaccine according to claim 16, wherein the viral component further contains a live attenuated measles, rubella, and mumps virus. 19. The stabilized live vaccine according to claim 1, which is a liquid product or a freeze-dried preparation. 20. A live attenuated varicella virus and a live attenuated recombinant varicella virus which comprise at least one selected from gelatin and gelatin derivatives substantially free of Ca ² + ions and Mg ² + ions. A stabilizer for a live vaccine containing at least one chickenpox virus as a virus component. 21. A gelatin and a gelatin derivative substantially free of Ca ² + ions and Mg ² + ions, respectively.
Chelate reagent-treated gelatin obtained by treating gelatin and gelatin derivatives containing Ca ² + ions and / or Mg ² + ions with a chelating reagent and masking the ions and purified products thereof and chelate reagent-treated gelatin derivatives or purification thereof The stabilizer according to claim 20, which is a product. 22. The stabilizer according to claim 21, wherein the chelating agent is ethylenediaminetetraacetic acid or a salt thereof. 23. A gelatin and a gelatin derivative substantially free of Ca ² + and Mg ² + ions, respectively.
Ca ² + ions and / or Mg ² + dialyzed gelatin and gelatin derivatives containing ions, gel filtration treatment, treatment with a cation exchange resin, Ca ² + ions and toward at least one processing selected from treatment with a chelating resin agent according to claim 20 mg ² + ions in purified gelatin and purified gelatin derivative formed by substantially removed. 24. The stabilizer according to claim 23, wherein the chelating resin contains ethylenediaminetetraacetic acid or a salt thereof. 25. The stabilizer according to claim 20, wherein the gelatin derivative is a gelatin hydrolyzate.