JP2841592B2 - Virus particle disruption method - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for disrupting virus particles.

Conventional Techniques and Problems to be Solved by the Invention As virus particles and crushing methods, chemical crushing methods such as ether treatment and physical crushing methods such as ultrasonic treatment have been conventionally used. It was not possible to disrupt virus particles while retaining the partial structure of the virus particle envelope.

This can be explained by taking the virus disruption method in the manufacturing process of influenza vaccine as an example. The current influenza HA vaccine (see Fig. 1) developed instead of the whole particle vaccine (see Fig. 1), which had side effects such as fever. In the production of (see FIG. 2), influenza virus particles are treated with ether to crush the virus particles, and the protective antigens of influenza virus, haemoglutinin (HA) and neuraminidase (NA), are used.
Has been used as an immunogen. in this way,
The outer layer of the influenza virus particles, that is, the outer layer of the lipid bilayer, and the inner layer of the matrix protein that is in contact with the outer layer, have the structure of the envelope composed of HA and NA bound from the outside destroyed, and are manufactured using this method With the current influenza HA vaccine, although the pyrogenicity disappears, it is known that the reduction of the immunogenicity (titer) of HA and NA is caused by the dissociation of the lipid bilayer and the binding to the matrix protein. At present, enhancement of the originality (titer) is desired. In addition, a method of physically crushing influenza virus particles by sonication has also been attempted, but, similarly to the above ether treatment, even if it is suitable for collecting HA and NA, the structure of the envelope portion of influenza virus particles is It is impossible to crush without damaging.

In addition, as a measure to enhance the immunogenicity (titer) of current influenza HA vaccines, adjuvants (immunostimulants) are added to HA and NA that have been separated by ether treatment.
There have been various attempts, such as research on the addition of HA and NA, and the reconstitution of a whole-particle vaccine type by engaging HA and NA separated by ether treatment with an artificial membrane made of lipid components. However, it has not reached a practically satisfactory range.

On the other hand, the principle of high-speed collision treatment has been applied to the crushing of cells having a diameter of about 10 to 30 μm, but the pressure, flow rate, speed, etc. during the process of the apparatus are limited, and the diameter is about
It was never considered for use in disrupting 0 nm virus particles.

Means for Solving the Problems Under such a background, the present inventors apply high pressure to cause influenza virus particles to collide with each other at a high speed, and in addition, the virus particles do not impair the partial structure of the envelope. If the virus is found to be crushed (see Fig. 3) and an influenza vaccine is produced using the obtained crushed virus particles, the vaccine has the same immunogenicity (titer) as the whole particle vaccine and has lost pyrogenicity. Were found to be able to be produced, and further research was conducted based on these findings, and as a result, the present invention was completed.

That is, the present invention is a method for crushing virus particles, which comprises subjecting virus particles to high-speed collision treatment.

Examples of the virus particles used in the present invention include influenza virus having a diameter of about 20 to 300 nm, rubella virus, Japanese encephalitis virus, mumps virus,
Examples include measles virus, adenovirus, herpes simplex virus, rabies virus, vaccinia virus, hepatitis B virus, hepatitis A virus, adult T-cell leukemia virus, human immunodeficiency virus, and the like.

The above-mentioned virus particles are prepared as a material to be subjected to high-speed collision processing together with an appropriate medium, preferably a liquid. For example, when influenza virus particles are used as virus particles, a material to be subjected to high-speed collision processing is used. The method for adjusting is described below.

Influenza virus strains for vaccine production [eg, A / Yamagata / 120/86 (H1N1) strain, A / Yamanashi / 2/77 (H3N2) strain, A / Fukuoka / C
29/85 (H3N2) strain, A / Sichuan / 2/87 (H3N2) strain, B / Gifu / 2/73
Strain, B / Kanagawa / 3/76 strain, B / Nagasaki / 1/87 strain, etc.] are inoculated into the chorioallantoic space of embryonated chicken eggs, and the virus is cultured to obtain a virus suspension. The obtained virus suspension may be used as it is as a material for the high-speed collision treatment, but it is preferable to use a virus fraction obtained by purifying and concentrating the solution by sucrose density gradient centrifugation or the like. Further, formalin is added to the above-mentioned virus suspension or virus fraction so that the final concentration is about 0.001 to 0.5 v / v%, preferably about 0.02 v / v%, and thimerosal is added to a final concentration of about 0.01 v / v. / v%
Immediately after addition, or at about 4 ° C, about 1-14.
An inactivated virus suspension or an inactivated virus fraction that has been inactivated for a period of days may be used as a material for high-speed collision treatment. Further, the above-mentioned material for high-speed collision treatment may be added with a surfactant (eg, polysorbate 80 or the like) as needed, so that the final concentration is about 0.01 to 1.0 v / v%, preferably about 0.1 v / v%, and immediately added. Alternatively, it may be used after standing at 4 ° C. for about 1 to 7 days.

As a device used for high-speed collision processing, any device having a principle of causing virus particles to collide with an object at high speed may be used, and in the collision area of the device, for example, virus particles may collide at high speed, Although virus particles may collide with the wall of the device at high speed, it is desirable to use a device having a principle of causing virus particles to collide with each other.

The above principle will be described in more detail below.

The material for high-speed collision treatment is guided to the collision area at a high pressure, preferably by an air pump, and the pressure can be converted to a flow velocity by reducing the diameter of the flow path. Further, after the flow path is branched into two flow paths and then formed into a facing shape, it becomes possible for the virus particles to collide in the collision area. In the collision area, the virus particles are crushed by the interaction between the impact force at which the virus particles collide with the object at high speed and the cavitation due to the vacuum action generated when the high-speed collision processing material (fluid) passes at high speed. However, at this time, it is desirable to maintain a constant flow rate at high pressure, and for this purpose, it is preferable to use a pressure pump capable of continuous operation at high pressure. If desired, the material for high-speed collision treatment may be continuously circulated to crush the virus particles.

 Devices having the above principle include, for example, microphones
Rofluidizer (Microfluidics, rice
Country), Nanomizer (Manufactured by Pressure Flow Industries, Japan)
I can do it.

The pressure in the apparatus used for high-speed collision treatment and the flow rate of the high-speed collision treatment material containing virus particles vary depending on the type of virus particles used for crushing and the degree of crushing of the target virus particles, but usually about 1500 to 3,000. kg/
Virus particles can be disrupted at a pressure of cm ² and a flow rate of about 500-800 m / sec. When crushing the influenza virus particles in Influenza vaccine production purposes, for example, pressurization of about 1500~2500kg / cm ² and 500 to 700 m /
Influenza virus particles collide with each other at a flow rate of sec, and the target crushed product can be obtained by continuously circulating the device for about 0.5 to 5 hours. If the influenza virus particles collide with each other at a higher speed by reducing the diameter of the particles, the processing time can be reduced.

According to the method for crushing virus particles of the present invention, it is possible to crush virus particles while maintaining the partial structure of the envelope of virus particles. In the case of influenza virus particles, for example, hemoaglutinin (HA),
It is possible to disrupt influenza virus particles without impairing the partial structure of the envelope consisting of neuraminidase (NA), lipid bilayer and matrix protein. By using the resulting disrupted product to produce an influenza vaccine, The exothermicity of the reaction disappears,
The adjuvant effect of retaining the lipid bilayer and binding to the matrix protein makes it possible to produce an influenza vaccine in which the immunogenicity (titer) of HA and NA is equivalent to that of a whole particle vaccine.

Examples Hereinafter, the present invention will be described more specifically with reference to Examples and Examples, but the present invention is not limited thereto.

Example 1 A / Yamagata / 120/86 (H1N1) strain [influenza virus strain for vaccine production] was inoculated into the chorioallantoic space of embryonated chicken eggs.
The infected chorioallantoic fluid obtained by culturing was collected, purified and concentrated by density gradient centrifugation using purified sucrose, and the virus fraction was collected.

 Formalin was added to the virus fraction at a final concentration of 0.02 v /
v% and stored in a refrigerator at 4 ° C for 1 week (7 days
(Pauses) Leave to inactivate virus. This inactivation interface
75 ml of the virus fraction of virus (all particles) (virus titer: 7,7
About 12CCA / ml) By vessel
The virus particles were crushed.

 Microfluidizer The processing conditions of the vessel are sample
The micro full diizer Transfer to the container of the device and
Pressure of 7.5 ~ 8.90kg / cm^TwoAdjusting and pressurizing pump
Pressure of 1,800kg / cm^TwoAnd the virus fraction
Sample into the interaction chamber,
Virus particles collide with each other at a flow rate of 600 m / sec, and
Crushing of the ground. Sample after chamber is again
Return to the original container,
The pull was circulated, and the virus was crushed. This c
The processing time required for crushing the ils was 120 minutes.

This sample was used as a microfluidizer-treated vaccine stock solution. In order to investigate the quality as an influenza vaccine, as controls, (1) a stock solution of the current ether-treated vaccine (HA vaccine) and (2) an inactivated intact vaccine (whole-particle vaccine) in which the virus before ether treatment was inactivated with formalin ) Was prepared from the above virus fraction.

Example 2 A / Yamanashi / 2/77 (H3N2) strain [influenza virus strain for vaccine production] was inoculated into the chorioallantoic space of embryonated chicken eggs, and the infected chorioallantoic fluid obtained by culturing was collected and purified. Purification and concentration were performed by density gradient centrifugation using sucrose, and a virus fraction was collected.

 Formalin was added to the virus fraction at a final concentration of 0.02 v /
v% and stored in a refrigerator at 4 ° C for 1 week (7
Day) and left to inactivate the virus. This inactivation interface
105 ml of virus fraction (whole particle) (virus titer: 1
6,372 CCA / ml) for microfluidizer vessel
To crush the virus particles.

 The processing conditions for disruption of virus particles are as follows:
Rofluidizer Transfer to equipment container
The pressure of 7.5-8.0kg / cm^TwoAnd adjust the pressure of the pressure pump to
1,704kg / cm^TwoAnd run a sample of the virus fraction
Send to Interaction Chamber, about 600m / sec
Virus particles collide with each other at the flow rate of
Awakened. The sample after the chamber is again
The sample is circulated by the continuous operation of the pressure pump.
The cells were ligated and the virus was disrupted. This virus breaks
The processing time required for crushing was 60 minutes.

This sample was used as a microfluidizer-treated vaccine stock solution. In order to investigate the quality of the influenza vaccine, (1) the current ether-treated vaccine (HA vaccine) and (2) the inactivated intact vaccine (whole-particle vaccine) in which the virus was inactivated with formalin before ether treatment were used as controls. A vaccine stock solution was prepared from the virus fraction.

Example 3 B / Gifu / 2/73 strain [influenza virus strain for vaccine production] was inoculated into the chorioallantoic cavity of embryonated chicken eggs, and the infected chorioallantoic fluid obtained by culturing was taken and the density was determined by purified sucrose. Purification and concentration were performed by gradient centrifugation, and the virus fraction was collected.

 Formalin was added to the virus fraction at a final concentration of 0.02 v /
v% and stored in a refrigerator at 4 ° C for 1 week (7
Day) and left to inactivate the virus. This inactivation interface
60 ml of the virus fraction (whole particle) (virus titer: 13,
080CCA / ml), microfluidizer In a bowl
The virus particles were crushed.

 The processing conditions for disruption of virus particles are as follows:
Rofluidizer Transfer to equipment container
The pressure of 7.5-8.0kg / cm^TwoAnd adjust the pressure of the pressure pump to
1,800kg / cm^TwoAnd run the virus fraction sample
Send to Interaction Chamber, about 600m / sec
Virus particles collide with each other at the flow rate of
Awakened. The sample after the chamber is again
The sample is returned by the continuous operation of the pressurized sample.
Circulation was performed to crush the virus. This virus
The processing time required for crushing was 80 minutes.

This sample was used as a microfluidizer-treated vaccine stock solution. In order to investigate the quality of influenza vaccine, (1) the current ether-treated vaccine (HA vaccine) and (2) the inactivated intact vaccine (whole-particle vaccine) in which the virus before ether treatment was inactivated with formalin were used as controls. A vaccine stock solution was prepared from the virus fraction.

Example 4 A / Fukuoka / C29 / 85 (H3N2) strain [an influenza virus strain requiring vaccine production] was treated in the same manner as in Example 2 to obtain a microfluidizer-treated vaccine stock solution and an ether-treated vaccine (HA vaccine) stock solution. And an inactivated intact vaccine (whole particle vaccine) stock solution was prepared.

Example 5 Preparation of influenza vaccine (final bulk) Each virus strain obtained in Examples 1 to 3 (A / Yamagata / 120/8)
6 (H1N1), A / Yamanashi / 2/77 (H3N2) and B / Gifu / 2/73
The stock vaccine solution was treated with phosphate buffered sodium chloride solution in Table 1.
Influenza vaccine (final bulk) of 3 strains mixed according to the formulation shown in Table 1
(300 ml) was prepared.

As a control, a mixture of three strains of influenza vaccine (final bulk) (300 ml) was prepared in the same manner from the current stock solution of ether-treated vaccine (HA vaccine) and stock solution of inactivated intact vaccine (whole particle vaccine).

Experimental Example 1 Influenza vaccine quality test results (1) Influenza vaccine pyrogenicity Rabbit pyrogenicity test was performed on the vaccine stock solutions of each virus strain obtained in Examples 1 to 3 to examine viral pyrogenicity. 1.0, 1.5, 2.0, 2.
Body temperature was measured after 5,3.0,4.0 and 5.0 hours. The results were as shown in Table 2.

As is clear from Table 2, the inactivated intact vaccine before ether treatment (whole-particle vaccine) slightly differs depending on the virus strain, but shows a viral fever at an inoculation dose of 30 to 300 CCA / kg, and shows the current ether treatment. The vaccine (HA vaccine) was negative for all virus strains at a dose of 1000 CCA / kg. In contrast, in the microfluidizer-treated vaccine, no viral fever was observed even at inoculation doses of 1000 CCA / kg and 3000 CCA / kg, which was negative.
The fever decreased more than two-fold, and the viral fever of the influenza vaccine disappeared.

(2) Influenza Vaccine Titer An influenza vaccine (final bulk) having the same composition as in Example 5 was prepared from the stock solutions of the respective virus strain vaccines obtained in Examples 1 to 3. As a control, an influenza vaccine (final bulk) was prepared in the same manner from a current stock solution of an ether-treated vaccine (HA vaccine) and a stock solution of an inactivated intact vaccine (whole particle vaccine). For each influenza vaccine (final bulk), the ability to produce neutralizing antibodies in mice was measured over time. That is, after 1, 2, 3, 4 and 5 weeks after immunization of mice (vaccine diluent: M100 phosphate buffer), egg neutralization test (neutralization time:
30 minutes) to determine the ED ₅₀ value. The results are shown in Table 3- (1)-
As shown in (3).

As is clear from Table 3- (1), A / Yamagata / 120/86 (H
The antibody production of the microfluidizer-treated vaccine of the 1N1) strain peaked in almost two weeks, and the titer was equal to or higher than that of the inactivated intact vaccine. In contrast, the titers of the ether-treated vaccine were somewhat lower, with peak antibody production after three weeks.

As is clear from Table 3- (2), A / Yamanashi / 2/77 (H3N
2) The antibody production of the microfluidizer-treated vaccine of the strain peaked in 3 weeks, and the titer was almost the same as that of the inactivated intact vaccine. In contrast, the titers of the ether-treated vaccine were low, with peak antibody production after 4 weeks.

As is clear from Table 3- (3), the absolute value of the antibody production of the microfluidizer-treated vaccine of the B / Gifu / 2/73 strain was low, and the peak was 5 weeks later. It was almost equivalent to the titer. In contrast,
The ether treatment vaccine ₅₀ value ED even after 5 weeks is 5 ^{1 - 5,} the antibody titers were very low.

In addition, each microfluidizer-treated vaccine (final bulk) met all the test items of the current biological standards for ether-treated vaccine (HA vaccine).

Effect of the Invention According to the method for crushing virus particles of the present invention, virus particles can be crushed without impairing the partial structure of the envelope of virus particles, and intermediate between all the particles of virus particles and each component. A crushed material that is in charge of the form and has both characteristics can be obtained.

[Brief description of the drawings]

FIG. 1 shows an electron micrograph showing the form of a virus (organism) in the stock solution of the inactivated intact vaccine (whole-particle vaccine) prepared in Example 4. FIG. 2 shows the ether-treated vaccine (HA) prepared in Example 4.
(Vaccine) An electron micrograph showing the morphology of a crushed virus (organism) in the stock solution. FIG. 3 is an electron micrograph showing the morphology of a crushed virus (organism) in the microfluidizer-treated vaccine stock solution prepared in Example 4. The white line shown at the lower right of FIGS. 1 to 3 indicates that the length is 200 nm.

Continued on the front page (58) Fields investigated (Int. Cl. ⁶ , DB name) A61K 39/12 B02C 19/12 A61K 39/145 CA (STN) WPI (STN) BIOTECHABS (STN) MEDLINE (STN)

Claims (1)

(57) [Claims] 1. A method for crushing virus particles, comprising subjecting the virus particles to high-speed collision treatment.