JPH01279841A - Hepatitis vaccine - Google Patents

Abstract

PURPOSE:To provide a hepatitis vaccine composed of a specific subunit of hepatitis B virus surface antigen (HBsAg), free from contamination of virus and purifiable by easily separating and removing serum protein component. CONSTITUTION:A hepatitis B virus composed of a glycopeptide originated from HBsAg. It has the following physical and chemical properties. Molecular weight, 24,000-28,000 (by electrophoretic analysis with SDS-polyacrylamide); solubility, about 15% under nearly neutral condition; ultraviolet absorption, molecular extinction coefficient at 280mm is 35; temperature stability, the antigenicity is maintained even by heating the aqueous solution at 60+ or -0.5 deg.C for 1hr; color reaction, exhibiting the color reaction of peptide by Lowry Folin reaction, of amino acid by ninhydrin reaction of hydrolysis and of sugars by alpha-naphthol sulfuric acid reaction and phenol sulfuric acid reaction; amino acid composition, shown in the Table; ratio of protein and sugar; sugar is 10-13wt.%; color and shape; amorphous material having almost white color; etc.

Description

[Detailed description of the invention] The present invention relates to hepatitis vaccines.

Hepatitis B virus (HBV) is known as a virus that causes serum hepatitis in humans, and often infects patients who require blood transfusions and those involved in clinical tests and plasma preparations, causing a truly troublesome problem. Today, IBV is a core antigen (H8CAC).
J), surface antigen (HBSAQ), and e antigen, with a diameter of 40 nm and a DNA polymerase in the core.
It is known that it is a large particle ((1110Te
Chnical Report 5eries Ha
570, Viralhepatitis ) 1975
), and it has already been revealed that Anti-IBs (8-anti-HBS) can prevent HBV infection.

Conventionally, l-IBsAa is obtained by dividing plasma or plasma fraction containing it into a sample having a density of 1.2 by density gradient ultracentrifugation.
How to obtain both parts of 0-1.23 ((J, L.

Gerin, P., V., Holland and R.
It, Purcell): (Journal o
7, pp. 569-576 (1971) and (N, 5ukeno, R.

5hirachi, R, 5hirachi, J,
Yasaguchi and N, l5hida):
The same magazine, Volume 9, Pages 182-183 (1972)
] was obtained in relatively pure form.

Furthermore, a new method of graphing affinity cloak 1 has been proposed (Hiroyuki Shiraishi, Kagawa Ishida, Nori Sukeno, Japanese Society of Virology Abstracts, November 4, 1970, Tokyo). The outline of this method is as follows.

Human serum that does not contain I-IBsAa is added to goat anti-HBs serum obtained by immunizing an animal, such as a goat, with purified H[3SAQ to absorb and precipitate the antibodies against the mixed human serum components, The supernatant was fractionated with γ-globulin anti-HBs (antiHBs) by ammonium sulfate precipitation.
get. Sepharose-48 activated with cyanogen bromide (
Sepharose-4B) Toko (DantiH
88 and prepare a column of this product. Human plasma containing human 1B5Ag, or α- and β-globulin fractions fractionated from it by a conventional method, was
．． 01M phosphoric acid loose! The suspension is suspended in Li solution, centrifuged, and the resulting supernatant is subjected to chromatography using the above column. HBsAa bound to the column was 0.4M-
Elute with glycine-)I(,J (11H2,5)!l buffer, and dialyze the eluate against 0.01M phosphorus!!21!li solution to obtain an HBSAG solution. This solution is applied to the column described above. , except that instead of using goat anti-HBS serum, horse anti-human plasma serum obtained by immunizing an animal, such as a horse, was used. The plasma components remain in the column and the HBsAa solution is collected as a flow-through.

Regarding the subunits constituting HBsAq, 1-I
Searching for peptides found by cleaving BSAQ, many authors have reported that the peptides contain polypeptides of various molecular weights. That is, Guerin (supra) reports that HBSAQ consists of polypeptides with molecular weights of 26.000.32.000 and 40,000. Besides, it is a polypeptide with a molecular weight of 25.000 and 32.000 ((G).

N., Vyas, E., H., Williams, GG.
B Klaus and H, E, Bond)
: (The Journal of Ig+
nunology) Volume 108, No. 1114-11
18 pages (1972')), 39,000.32
,000.27°000 and 22.000 polypeptides and those of 16,000 and 10.000 (
(G, R, Dressmann, F, B, tl
Ollinger, J.R.

5uriano, R.S., Fujioka,
J, P, 8runschwig and J,
L, He1nik): Journal of V
irology, Vol. 10, pp. 469-476 (1972)] and polypeptides of molecules m1oo, ooo as main components, 65,000.36,000 and 2.
0゜OOOO (C, R, lower and A,
J.

Tuckerman) : (l1cpatitis
Memoranda), l-1-576, 197
October 3, U, S, A,) 17) It has been reported that it is composed of ++71 nits. Also N, 5uk
eno, S, 8ikawa'I3 and N, l5hi
da: Same magazine, H-292, April 1972, tl, s
, A,) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis wJP where Lt, HBSAQ is cleaved by sodium dodecyl sulfate in the presence of urea! Easy molecular analysis method (Colowick-Ni aplan: Me
thod inEnzylogy Vol. 26, Page 3 (1972), it is the molecule ff12.
It has been reported that the polypeptide consists of 5,000.28°000 and 33.000 polypeptide nab units.

The search for such subunits of H[3S A O is as follows:
The aim is to clarify the relationship between subunits and antigenicity, and to extract more specific antigenic sites, which in turn can be used as raw materials for efficient vaccines and reagents, and
The aim is to utilize BSAQ for research on antigenic groups.

The co-researchers of the present invention have previously proposed a method for recovering antigenic subunits of HBsAq, including heat treatment in the presence of a certain type of surfactant that can be used to delipidate HBSAG. By doing so, HBSA (about 5 intermolecular
Uniform spherical particles approximately 18-20 nl in size consisting of 5.000 single polypeptide-only subunits (
1-IBAa55) (Japanese Unexamined Patent Publication No. 160420/1983).

The present inventors also invented a new method for collecting particles with HBSAQ antigenicity, and by this method, they have novel properties, namely, isoelectric point pl + 5.5-6.5,
Molecule R2-4 million, specific gravity 1.80-1.24. diameter 18
We have also succeeded in providing uniform modified HBSAG particles that retain the high antigenicity of spherical particles of 0 to 220 people (
JP-A No. 53-104724).

Following these successes, the present inventors have continued research to obtain peptides that can actually be used as vaccines.

As a vaccine, purified HBSAQ particles can be used as they are, but in that case, infectious He
It is almost impossible to completely remove the virus, and therefore methods of inactivating the He virus that is expected to be contaminated by formalin or heat treatment are being considered. However, as a method to prove whether or not it has been inactivated, a test using chimpanzees is necessary since chimpanzees are currently the only animals known to be infected with the ToIB virus. However, the number of chimpanzees in the world is currently limited, and testing is economically expensive.
There is no guarantee that tests can be continued without any inconvenience in the future, and it is extremely difficult to completely remove human serum proteins with HBSAG particles as they are, and if these proteins are denatured through inactivation treatment, it may cause side effects. There is.

On the other hand, if peptides can be obtained by separating rib units with a smaller molecular weight than refined tJHBsAq and used as a vaccine, there is no risk of contamination with large He virus particles, and HBSA 0 particles Serum protein components that were more difficult to separate due to some affinity can also be easily separated and removed.

The peptide used as the HB vaccine is HB S
It is essential to have ACJ antigenicity. As previously explained, slight differences in the chemical treatment of H[3SAQ particles can separate them into subunits of various molecular weights, but this should not change their safety and efficacy when used as a vaccine. Also, since stability as a vaccine agent must be ensured, a certain polypeptide is most desirable.

The present invention was made against the background of the above, and is directed to a molecule f having antigenicity of J to HBSA.
Invented a method to obtain a glycopeptide consisting of f124,000 to 28,000, and developed a method to obtain a safe, effective and stable H B
successfully provided a vaccine.

The present invention relates to 11[3sAQ obtained by N production from human plasma.
By heating in the presence of a surfactant and a reducing agent, HBsAa is cleaved into glycobebutide of 100% by weight, and the surfactant and reducing agent are present to prevent this from repolymerizing. The mixture is fractionated by gel filtration method to separate two fractions consisting of glycopeptides with a molecular fit of 24,000 to 28,000, and an adsorption aid for removing the surfactant used and maintaining the solubility of the glycopeptides. The present invention provides an HB vaccine consisting of a glycopeptide recovered as a physiologically acceptable near-neutral solution by dialysis using a solubilizing agent, and a method for producing the same.

The HBS A Q used in the present invention can be obtained purely from plasma or serum by a known method. Also, instead of plasma or serum, α-
Plasma fractions containing β-globulin and β-globulin, such as the N-1 fraction in Cohn's alcohol fractionation method, are also preferred as raw materials.

The surfactant used in the present invention is an anionic surfactant selected from alkyl sulfate salts such as sodium dodecyl sulfate, alkyl phosphate salts, and the like. The amount of surfactant added is 0.1-10%, preferably 0.5-2.0%. It is acceptable to add a large amount of anionic surfactant, but if the concentration is high, it tends to solidify, which is not preferable since it is necessary to maintain a high temperature.

The reducing agent used in the present invention is a thiol-type reducing agent selected from 2-mercaptoethanol, dithiothreitol, glutathione, and the like. The reducing agent addition chamber is 0.1~
10%, preferably 0.5-2.0%.

Heating is near neutral! 2 to 6 at 100 to 40℃ in buffer solution
Glycopeptides produced by treatment for 0 minutes, preferably at 100-90°C for 1-5 minutes or at 70-50°C for 20-40 minutes recombine in the absence of surfactants and reducing agents. Therefore, gel filtration is performed in the presence of an anionic surfactant and a reducing agent having a thiol group, and the various bebutides and sugar bebutides produced are fractionated according to their molecular weights.

Gel filtration uses 0.05 to 2%, preferably 0.1 to 0.5% of sodium dodecyl sulfate as a surfactant.
, and as a reducing agent, for example, 2-mercaptoethanol is present at 0.05-2%, preferably 0.1-0.5%, using a near-neutral Vll liquid, at 15-45°C, preferably at 35-40°C. Perform at ℃.

Gel filtration agents are polymeric polysaccharide granules such as cellulose, agarose, cross-linked dextran, or cross-linked polyacrylamide.Products include Sephacryl S1 Sephadex, Sepharose, and Pyogel (Blo-
Examples include Get ) P, Bio-Gel A, etc.
The carrier is not limited to these as long as it has a molecular sieving action.

As a result of gel filtration, the molecular weight was approximately 68.000 and approximately 27.0.
00 and about 22,000 others, among which molecular weight 24,000-28
,000.

The two components obtained in this way are concentrated to an appropriate concentration, and the anionic surfactant and thiol group-containing reducing agent contained in both components are removed to ensure physiological acceptability. However, if both the surfactant and the reducing agent are completely removed, the solubility of the sugar peptide will deteriorate, and if the solution is to be stored for a long period of time, there is a risk of precipitation. , Pluro
A solubilizing agent selected from non-ionic field 1III activators such as nic) F1a, Tween 80, etc. is preferably kept in solution.

The solution after concentration is near neutral l! The surfactant and reducing agent used in the heating reaction are removed by dialysis against a buffer solution, but complete removal is difficult.

Therefore, in the external dialysis fluid, Plu 0nik F68 and Twineen 8 were added.
Bio-Rad contains 0.01 to 0.1% of a solubilizing agent selected from nonionic surfactants such as 0, and is used as an adsorbent for adsorbing and removing anionic surfactants and reducing agents. A basic patch containing an ion exchange adsorbent such as AG-2 (trade name) is used.

The solution obtained in this way is physiologically isotonic around neutrality and prepared to have a constant concentration of glycobebutide,
After sterilization and filtration, it is sent to the authorities for a few minutes to complete the production of He vaccine.

The effectiveness of a He vaccine can be determined by injecting it together with its HBsAg antigen titer into experimental animals such as rabbits and guinea pigs and observing the production of antibodies against HBsAq.

The physicochemical characteristics of the sugar bebutide of the present invention were determined using the one obtained in Example 1.

(1) Molecular Weight The molecular weight of the glycopeptide of the present invention was measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis and was found to be approximately 27,000. In addition, in gel filtration analysis using Sephadex G-100, the molecule was 124.0.
It was 00-28.000. Therefore, the molecular weight is 24,0
00 to 28.000 is considered to be the most reliable range.

(2) Solubility The solubility of the glycobebutide of the present invention in water is approximately 5% F or less in the vicinity of neutrality, and above that, the glycobebutide exhibits a protein milky color. This solubility is chloride]-lium solution,
Rin/i! There is not much difference between the 2-salt MS solution and the Tris-HCIMWR solution, but the solubility is improved by the presence of a solubilizing agent selected from ion surfactants such as Pluronic F68 or Tween 80 at about 0.01 to 0.1%. increases.

(3) Ultraviolet absorption The molecular extinction coefficient F1%CM 280 nl of the aqueous solution of the peptide of the present invention determined from the absorbance at 28 On1 m was 35.

(4) Temperature stability 0.05% Pluronic F6 of the glycopeptide of the present invention
Al17.2 phosphorus N salt buffer containing 8 was heated at 60°C±
Incubation at 0.5° C. for 1 vg did not destroy the HBSAO antigenicity.

(5) Color reaction When the aqueous solution of Bebutide of the present invention was tested by the Lowry-Forin method, it exhibited a blue color characteristic of Bebutide. Further, as a result of hydrolysis in a 6N hydrochloric acid solution at 110° C. for 22 hours and then tested by ninhydrin reaction, it exhibited a purple-blue color due to α-amino acid. In the α-naphthol sulfuric acid reaction (Holish reaction) for the saccharide test, it turned purple, and in the phenol sulfuric acid reaction, it turned brownish red, both of which were positive! 6) II Quaternary Amino Acid The glycopeptide of the present invention was hydrolyzed by a conventional method, and the amino acid composition was determined using an automatic amino acid analyzer.

The results are shown in Table 1.

Table 1 The analysis results for the amino acid composition of purified IBsAQ are also shown in Table 1, and when these results are compared with the results for the glycopeptide of the present invention, no major differences are observed.

From this, it is inferred that this sugar bebutide constitutes the main part of HBsAo.

(7) The constituent sugar sialic acid of the carbohydrate moiety was determined using the thiobarbic acid method (Dey, Amic (0)
The sample was methanolysed and then trimethylsilylated using the 8-m1noff) method, and the composition of the sugars contained in the sugar peptide of the present invention was analyzed by gas chromatography mass fragmentography. The results are shown in Table 2.

Table 2 Fucose Not detected Mannose 3.0% (2.4-3.5%)
Galactose 2.3% (1.2-3.2%
) Glucose 3.6% (2,8-4.4
%) N-Ahiti/L/ 2.5% (2,2
~2.7%) Glucoamine N-acetyl Not detected Galactosamine Sial M 0.2% Total sugar content 11.6% (10~12.
5%) (8) Constituent ratio of protein and carbohydrate The protein quantitative results of the glycobebutide of the present invention by the semi-microkerbourg method were 87 to 90%. The remaining 13-10%
The carbohydrate content matches well with the 12.5 to 10% described in (9) above.

(9) Color and shape When the sugar peptide of the present invention was dried, it became an almost white amorphous powder.

Examples are given below to explain the present invention more specifically, but the description of the Examples is not intended to limit the present invention in any way.

Example 1 Human pool plasma 11 determined to be HBSAQ positive was
Anti-Has antibody globulin (goat)-conjugated sepharose 4
B (anti-H8s alobulin coupe
HBSACI is adsorbed onto the column. After washing the adsorption column with 0.15M cesium chloride solution,
．． Elute H[3SAq with 4M glycine-HC1* buffer.

After the eluate was dialyzed against 0.15M sodium chloride solution,
Anti-human plasma antibody globulin (goat)-conjugated sepharose 4
It passes through the column prepared in step B, and the human plasma components are adsorbed onto the column. After concentrating the solution that passed without adsorption,
Perform linear gradient ultracentrifugation using cesium chloride, collect both fractions with a density of 1.19 to 1.23, and concentrate to 2I! I did it with ll.

As a result of immunoelectrophoresis on anti-human plasma antibody globulin (goat), the presence of human serum components was not detected, and electron microscopy showed that HBSAQ was uniform with a diameter of 2201 mm.
It consists of small spherical particles, no large particles are observed, and the antigen titer is 1 as measured by the RP 1-1A method.
It's 1.280.000. The recovery rate was 52%. This purified HBSAG was used as a material for producing HB glycobebutide vaccine.

Add t-thorium dodecyl sulfate and 2-mercaptoethanol to a solution of N-made HBSA Q at 1 W/V% each.
After heating at 100°C for 2 minutes, add 0.1% sodium dodecyl sulfate,
1/100M phosphorus containing 0.1% 2-mercaptoethanol and 0.15M sodium chloride! Gel filtration was performed at 37°C using a column of Sephacryl S-200 equilibrated with di-salt buffer, pl+7.2. The two divided images at that time are as shown in FIG.

The F3 fraction shown in Figure 1 was collected and gel-filtered again, resulting in 5 mixed F fractions (F3 fraction is mainly composed of F and F4.
(composed of both components) is completely separated, 0
．． Dialyze against phosphate buffer containing 5% Bio-Rad AG-2 and 0.02% Pluronic F68. The solution after dialysis contains 0.02% Pluronic F68.
Diluted by adding 5M thorium chloride solution so that the sugar peptide was 60 μ9/d, and after sterilization filtration, 1 td
A total of 510 l-(F3 vaccine preparations) were obtained, which were divided into individual containers.

The IB vaccine thus obtained was administered to three rabbits V. Injection is 0.5Ml subcutaneously on the back and sarcastically, 2'! Blood was collected two weeks after the injection was performed three times at intervals of A, and the 11BS antibody titer was measured using the PHA method for the plasma, which was 1:256.1:8.0, respectively.
00.1: 8,000)-IBs antibody production was observed.

Example 2 The viscous Has of Example 1 was heat-treated at 60°C for 30 minutes using dithiothreitol instead of the 2-mercaptoethanol used in Example 1, and then heated at 60°C for 30 minutes.
．． 1% sodium dodecyl laaate, 0.1% dithiothreitol and 0.15 MI! 1 containing sodium
/100M phosphate! The gel was passed through a Sephadex G200 column equilibrated with 1i solution, pH 7.2, at 37°C.

F3 itself was collected in the same manner as in Example 1, and gel filtration was performed again to completely separate the mixed F4 components. Vaccine formulation 1
I got 20 pieces.

This product produced 11Bs antibodies against two rabbits (t was 1:1.024 and 4.096F for -1'L, respectively).

Example 3 In Example 1, 13 fractions were collected and gel-filtered again to completely separate the mixed F4 components.
．． After dialyzing with 0.15 Mjm sodium solution containing 0.2% Tween 8O and diluting with EiJ solution, the glycopeptide concentration was 50 μg/lII! It was prepared as follows.

0.1% aluminum hydroxide was added to this solution, and after sterile filtration, 590 H8 vaccine preparations were obtained, which were divided into containers of 11 Ie each.

The HB vaccine obtained in this way was distributed to 34 guinea pigs]
・Injected. Injections were performed in the same manner as in Example 1. The results were 1:512.1:32 for each guinea pig.
000,1:32.000 (7) HBS antibody production was observed.

[Brief explanation of the drawing]

Figure 1 shows ii! obtained by gel filtration in Example 1! i
This is a fractional image graph of minutes. In the figure, fractions e of each of F, [, F, F and F5 are shown.

Claims (1)

[Scope of Claims] A glycopeptide derived from HB_sA_g having the following physical and chemical properties: (a) Molecular weight: 24,000 to 28,000 as determined by electrophoretic analysis using SDS-polyacrylamide;
b) Solubility: The solubility in water is about 15% near neutrality, and above that the protein becomes milky in color. (c) Ultraviolet absorption: molecular extinction coefficient at 280 nm (
E^1^%_1_c_m) is 35. (d) Temperature stability: Even if the aqueous solution is heated at 60°C ± 0.5°C for 1 hour, its antigenicity is not lost. (e) Color reaction: Color reaction of peptides by Lowry-Follin reaction, color reaction of amino acids by ninhydrin reaction after hydrolysis, and color reaction of amino acids by α-naphthol sulfuric acid reaction (
Moriciu reaction) and phenol-sulfuric acid reaction, which show purple and colored sugar reactions, respectively. (f) Constituent amino acids: aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine,
Cysteine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine, arginine, (g) Sugars constituting the carbohydrate moiety: Mannose (2.4-3.5%), Galactose (1.
2-3.2%), glucose (2.8-4.4%), N-acetylglucosamine (2%), glucose (2.8-4.4%),
．． 2-2.7%), sialic acid (0.2%), and does not contain fucose and N-acetylgalactosamine. (h) Constituent ratio of proteins and carbohydrates: 10 to 13% carbohydrates by weight, and (i) Color and shape: Hepatitis B vaccine that is almost white and amorphous.