JPS6035326B2 - Manufacturing method for influenza vaccine - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to influenza vaccines, particularly influenza subunit vaccines, and methods for their production by selective solubilization and separation of the immunogenic components of influenza viruses.

FIG. 1 is a graphical representation of influenza virus particles.

Riboacid (RNA), which is a genetic material, is surrounded by a double membrane consisting of the inner layer of protein and the outer layer of lipid material from the host king, in association with the nuclear protein unique to the sheep. Two glycoproteins, hemagglutination table and neuraminidase, exist on the surface of the viral envelope in the form of projections or spikes. It was confirmed that two glycoproteins, hemagglutinin and neuraminidase, are the main immune components of the influenza virus, and that all other components, including other viral proteins, nucleic acids, and lipids, are not essential for immune induction. .

The presence of such non-essential substances in influenza vaccines can cause undesirable side effects, limit the amount of vaccine administered, and limit the level of immunity achieved. Therefore, the ideal influenza vaccine should be free or substantially free of non-essential components of the virus particle and should contain the two essential immunoxins, hemagglutinin and neuraminidase.

Therefore, the first methods attempted to isolate influenza immunogens used anionic surfactants, such as sodium desoxycholate or sodium dodecylate, to substantially destroy the virus particles in the first step. The virus was completely destroyed or solubilized, and all or most of the virus components were released and transferred into the solution together with the immunogen. However, such methods require purification or partial purification of the immunogen of interest, which requires a great deal of effort, and the yield is usually low. The present invention provides a method for the separation of hemagglutinin and neuraminidase, which selectively solubilizes these components and removes them from the intact lipid/protein membrane surrounding all other non-essential viral components. It is possible to leave it as a semi-viral particle consisting of

Because the solubilized immunogen and the remaining semiviral particles are different in size or density, the immunogen can be easily separated by conventional separation methods that take advantage of these differences in physical properties. We have found that selective solubilization of hemagglutinin and neuraminidase components can be achieved by treating influenza virus with a cationic surfactant.

The present invention therefore provides a method for separating hemagglutinin and neuraminidase from influenza viruses, which comprises treating influenza viruses in aqueous soot with a cationic surfactant to selectively solubilize such components. The method is characterized in that the resulting solubilized components are separated from the remaining half-virus particles.

The method of the invention may be suitably utilized against influenza A, AI, A or B influenza viruses or mixtures thereof. The strain used will, of course, depend on the immunity desired from the immunogen released, but examples include: - A2/Aichi/68, MRC-2 (recombination of type A2/England/42/72); , M old C-11 (A2
Type/Port Chalmers
)/73 recombination), A/Pasteur/3 [``Mutagrip''(''Yanautagi〆), Institut Pasteur] and B/Mass/67 strain.

The influenza virus to be treated is inoculated into 11-day-old fertilized chicken eggs, for example, and kept at a suitable temperature for a suitable period of time, for example at 370° C. for 2 days, to allow proper growth.

The obtained allantoic fluid is then collected appropriately, the virus is ultracentrifuged, and then, for example! Resuspend in phosphate-buffered saline or centrifuge in a continuous-flow zonal centrifuge using, for example, a sucrose gradient of phosphate-buffered saline, then resuspend as appropriate. The sucrose content is reduced to, for example, less than 5% by dialysis against normal saline, or suitably concentrated and purified by sepadex chromatography or dilution. The initial virus concentration is not critical and can be adjusted according to the desired amount of immunogen. It is appropriate to adjust the p-value of the virus concentrate to 6.5 to 8.5 using a buffer such as a phosphate buffer, if necessary, before adding the cationic surfactant. , the concentrate may also be inactivated, for example by addition of formaldehyde.

A cationic surfactant is then suitably added to the virus concentrate in the form of an aqueous solution. The amount of cationic surfactant to be added depends on its type, but generally the ratio of cationic surfactant to protein is 1:2 to 1:10, particularly 1:3 to 1:5.
It is appropriate to add something so that After the addition, the mixture is left for example at 4-37°C for 30 minutes to 1 hour. The higher the temperature, the shorter the standing time, preferably at room temperature for 30 to 6 minutes or at 4° C. overnight. The cationic surfactant used is one that has sufficient activity to solubilize the hemagglutinin and neuraminidase components, but not destroy all virus particles under the conditions used.

Such cationic surfactants may be of the formula [wherein R4 is alkyl or aryl, R1, R2, and R3 are the same or different and alkyl or aryl, or R, and R2 together with the nitrogen atom to which they are attached are 5 or 6 haze saturation is formed, R3 is alkyl or aryl, or R
, R2 and R3 together with the nitrogen atom to which they are bonded form a 5- or 6-membered heterocyclic ring unsaturated with the nitrogen atom, and x represents an anion.

] may be selected from the compounds shown below.

A representative compound represented by formula 1 is a compound represented by the formula [wherein, or alkyl having 8 to 22 carbon atoms, or R,
is methyl and R2 represents pendyl.

], especially compounds of the formula [wherein R'4 and
has the same meaning as above] or a compound represented by the formula [wherein R'4 and X have the same meaning as above].

A more typical compound represented by formula 1 is a compound represented by the formula [wherein, X has the same meaning as above, R'4 represents alkyl having 12 to 18 carbon atoms,
R5 represents hydrogen or methyl, preferably hydrogen.

].

Preferred alkyl groups having 8 to 22 carbon atoms are those having 12 to 18 carbon atoms.

Specific examples of alkyl groups having 12 to 18 carbon atoms include lauryl, myristyl, cetyl and stearyl. In the above formula, X is preferably an anion such as chloride, furomide, sulfate or acetate;
Especially chloride or bromide. Preferred among the compounds of formula laa are myristyltrimethylammonium salt and cetyltrimethylammonium salt, especially chloride or bromide, and even more particularly preferred is bromide.

Preferred among the compounds of formula lab are stearyldimethylbenzylammonium salts, especially chloride or bromide, especially bromide.

Preferred compounds of formula lb are cetylpyridinium salts, especially chloride or bromide, more particularly preferred bromide.

Other suitable cationic surfactants that may be used include benzalkonium.

Examples include lides and bromides such as penzetonium chloride or methylbenzenetonium chloride and decamethonium chloride. A preferred cationic surfactant for use in the method of the invention is cetyltrimethylammonium bromide.

After the above treatments have been completed, conventional methods employed to separate materials of different size or density from the remaining intact semiviral particles, e.g. It is separated by centrifugation using sucrose or sodium glutamate soot, fractionation of the slant, sedimentation, molecular chromatography, or compression in an ultracentrifuge.

The mixture of immunogens obtained by the method of the invention is suitable for use in influenza vaccines.

For this purpose, the hemagglutinin and neuraminidase components separated as described above are diluted with a conventional diluent, e.g.
% sodium chloride solution, optionally with a buffer such as phosphate buffer. Residual sucrose from the initial purification of the virus or from the separation of the solubilized components should suitably be less than 5% by weight in the vaccine, eg by dialysis. Furthermore, the content of residual cationic surfactant should be as low as possible, for example by dialysis or gel chromatography, to less than 0.01% in the vaccine.
If desired, a preservative or inactivating agent such as formaldehyde can be added to the vaccine in conventional amounts, eg, 1 part per 1000 B by weight.

The immunity of the vaccines of the invention may be enhanced by the inclusion of conventional immunological adjuvants such as aluminum hydroxide or aluminum phosphate in conventional amounts, eg 0.2% aluminum hydroxide.

The vaccine produced by the method of the present invention is useful as a vaccine against influenza viruses as described above. For example, groups of 30 mice each are administered intraperitoneally with 0.25 Kite of the whole virus vaccine and the subunit vaccine of the invention with a hemagglutinin content of approximately 3, 4 and 8 weeks after immunization. Eyes were infected by spray application of virulent virus, and protection was assessed for mortality and lung lesions on day 9 post-infection. When the test was repeated using vaccines with different antigen contents, it was confirmed that the subunit vaccine of the present invention has the same effect as the whole virus vaccine, except that it provides more lasting immunity against the infectious virus. Ta. The dosage used may be varied as appropriate, but generally satisfactory results are obtained by administering a single dose of about 9 to 43 international units per k9 of the animal's body weight. 600~ for larger horned mammals
A single dose of 3000 international units applies. Administration is suitably subcutaneous or intramuscular.

Next, the present invention will be explained with reference to Examples.

Example 1 Influenza virus of antigen type x-31 (recombination of N/Aichi/68 strains) was grown at 37C for 2 days during the fertilization experiment.

The eggs are then cooled to 4° C. and the resulting infected allantoic fluid is collected. The virus was then separated using a continuous flow zonal centrifuge (model RK, electronucreonics (model RK)).
．． Electro-Nucleon; cs)] from infected allantoic fluid by centrifugal separation using sucrose gradient phosphate-buffered saline. The virus concentrate obtained after reducing the sucrose content to less than 5% by dialysis against cold phosphate-buffered saline had a hemagglutination titer of 1:217 and a protein content of 0.7/9. It is cc. A volume of 1'50 of an aqueous surfactant solution (cetyltrimethylammonium bromide, 1% solution) is added to the virus suspension to separate the immunogen. After 30-6 minutes (at room temperature) the reaction mixture is subjected to zonal gradient centrifugation using a pre-prepared linear sucrose gradient and then fractionating the gradient with a oscillating pump.

Hemagglutinin and neuraminidase are quantitatively solubilized and are easily separated from the remaining particles of virus, which reside at the top of the gradient and precipitate more rapidly. Example 2 Virus propagation, concentration and preparation are performed as described in Example 1.

The treatment is carried out by equilibrium centrifugation on a prepared sucrose gradient. After adjusting the equilibrium, the gradient is fractionated and tested. Hemagglutinin and neuraminidase are present in the lighter part of the gradient and are easily separated from the remaining, more dense particles of the virus. Example
3 influenza strains MRC-2 (recombination of A2/England/42/72) or MRC-11 (A2/England/42/72 recombination)
The method described in Example 1 or Example 2 is carried out, except that the method described in Example 1 or Example 2 is used, except that Vote-Chalmers/73 recombination is used.

Example 4 The procedure is as described in Example 1 or 3, except that the reaction mixture is subjected to molecular chromatography.

Example 5 Formol-inactivated type A/Pasteur/Triple (“
An aqueous solution of cetylpyridinium bromide (0.5%
) is added.

The treatment is carried out in a manner similar to that described in Examples 1, 2 or 4. Example 6 The procedure was as described in Examples 1, 2, 4, or 5 except that influenza strain B/mass/67 was used. Example 7 The cleavage mixture was compressed and processed in an ultracentrifuge. The method described in Examples 1, 3, 5 or 6 is used.

This is for example Beckmann L-2-6
$ Centrifuge (Lower-60Ti, 3500 m.p.m.
9 beats).

Solubilized immunogen is present in the supernatant fluid fraction. Example
8 Instead of cetyltrimethylammonium bromide solution,
The method of any of Examples 1-7 was repeated using a 1% solution of myristyltrimethylammonium bromide, benzethonium chloride, methylpensethonium chloride, decamethonium chloride or stearyldimethylbenzylammonium bromide with similar results. can get.

Example 9 The influenza vaccine of the present invention is formulated as follows: Immunogen mixture: 700 International Units Thiomerosal: 1000
Kuni Ikari's 1 part phosphate buffer addition 0.9%: plus 0.
The physiological saline immunoprecipitate prepared in Example 3 may be prepared according to any of the previous examples, such as in Example 3, where influenza strain MRC-11 (type A2/Boat-Chalmers/73) was prepared. (recombination).

[Brief explanation of drawings]

FIG. 1 is a diagram of influenza virus particles.

Claims (1)

[Claims] 1. Influenza virus particles prepared by treating influenza virus particles with a cationic surfactant in an aqueous medium without disruption of the lipid/protein membranes containing the non-essential viral components of the virus. A method for producing an influenza vaccine, characterized in that the hemagglutinin and neuraminidase components of the influenza virus, which do not contain other components of the virus particles, are mixed with an inert liquid diluent.