NO142024B - PROCEDURE FOR THE PREPARATION OF A VACCINE, SPECIFIC INFLUENCE VACCINE, BY AA CULTURE A VIRUS - Google Patents

Description

The present invention relates to a method

for the manufacture of a vaccine, in particular

flu vaccine.

The influenza vaccines that are currently used are inactivated, whole virus vaccines which, in addition to influenza virions, contain some egg protein obtained from the allantoin fluid from which the virus is harvested. When injected, this type of virus can cause hypersensitivity due to the egg protein and pyrogenic effects caused by the virions.

The influenza virus and similar viruses of the same type,

i.e. orthomyxoviruses and paramyxoviruses, are characterized by an outer membrane that has "spikes" of antigens with hemagglutinating and

neuraminidase activity. This outer membrane can be broken up by solvents such as ether or by surface-active agents to release as (sub)sub-units antigens that have hemagglutinating and neuraminidase activity. The so-called "cleaved virus" vaccines which have so far been prepared in this way have been found to have lower pyrogenicity than corresponding whole virus vaccines, although no attempt has been made to separate antigens with neuraminidase-

and hemagglutinating activity from pyrogens and other unwanted materials such as other antigens and nucleic acids.

However, it has been found that the antigens with hemagglutinating and neuraminidase activity are not in themselves pyrogenic or otherwise toxic and that therefore a further reduction in pyrogenicity can be achieved by obtaining the desired antigens essentially free of other unwanted material.

We have attempted to separate the components of a cleaved virus preparation by zone ultracentrifugation. However, batch loading into an ultracentrifuge rotor was found impractical for large-scale production and it was found necessary to use a zone centrifuge for continuous loading where the relatively dilute suspension of virus subunits is fed into the rotor and flows across the centripetal end of concentration gradient. However, it was found that the very small particles of the desired antigens with hemagglutinating and neuraminidase activity

was difficult to transfer to the gradient at the flow rates required for large-scale production.

However, we have now found that viruses of influenza and similar types, which can easily be introduced into the gradient in a zone ultracentrifuge with continuous replenishment, can be successfully cleaved when passed through the gradient if a surface-active substance is present in the gradient solution. This facilitates the large-scale production of subunit vaccines which are substantially free of egg protein, pyrogens and virion nucleic acids and which essentially contain only the desired protective antigens with hemagglutinin and neuraminidase activity. The hemagglutinin found in the so-called "spikes" is split by the surface-active agent into a monovalent form that no longer exhibits hemagglutination but possesses the required antigenic properties.

In general, all ether-sensitive viruses can be cleaved by wood

treatment with detergents and in addition to the above ortho-

and paramyxovirus, the following viruses can provide subunit vaccines containing protective antigenic components analogous to hemagglutinin and neuraminidase, namely:

a) Togavirus

b) Rhabdovirus

c) Leukovirus

d) Coronovirus

e) Arenovirus

f) Herpes viruses

g) Poxvirus

According to the present invention, a

method for producing a vaccine, in particular an influenza vaccine, by cultivating a virus, in particular influenza virus, in a veal culture or in the allantois from poultry eggs with embryos, collecting the liquid containing virions, and optionally inactivating, clarifying and concentrating the fraction(s) containing the virions. The method is characterized by the fact that the fractions containing the virions are introduced into an ultracentrifuge with continuous filling which is equipped with a density gradient solution containing a hemolytic surfactant, whereby the virions pass into the density gradient and are cleaved by the surfactant, and the antigenic sub-units form isopycnic bands, and the fractions or the fraction containing protective antigen subunits are collected and transferred in a known manner in an administration form suitable as a vaccine.

While it is possible to fill whole virions directly from a relatively diluted culture medium such as allantoin liquid, it is usually preferred to carry out some purification of the whole virus preparation before it is digested in the ultracentrifuge.

For such purification, the whole, intact, inactivated virus can be isolated in the usual way. Influenza virus can thus, for example, be grown in 10 to 11-day-old chicken eggs with embryos.

The harvested allantoin fluid can then be processed, e.g. with p-propiolactone and/or formalin, to inactivate or "kill" the virus and then clear by centrifugation, e.g. in a "Westphalia" (trademark) continuous flow centrifuge.

The virus can then be further purified. While this can be carried out by using continuous sedimentation centrifugation, treatment with fluorocarbons, gel filtration or adsorption on red blood cells or minerals such as barium sulphate and subsequent elution, it is preferred to achieve rapid and simple treatment in larger quantities to use continuous zone centrifugation , for example by using a "KII" apparatus (manufactured by the Molecular Anatomy Program, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830, USA and described by Anderson et al. in Analytical Biochem. 32, 400-494 and 495-511

(1969) and available from Electro-Nucleonics Inc., 368 Passaic Avenue, Fairfiels, New Jersey, 07006, USA.)

A 3.2 liter capacity "KII" rotor with a sucrose density gradient is particularly effective. The method used may be that of Reimer et al. (Journal of Virology, Vol 1, No. 6, pages 1207 - 1216). After purification, the fractions containing the virions as determined by hemagglutination titer can be pooled and diluted or dialyzed to reduce the density to a level suitable for introduction to a gradient in the second ultracentrifugation step.

For the cleavage step, the virions are loaded, preferably after purification as described above, onto the density gradient solution in a zone ultracentrifuge for continuous loading. As with virus cleaning, it is the appropriate medium for

the gradient a sugar solution, especially sucrose, preferably in buffered saline at a suitable pH for the virus. Usually a pH slightly above 7 is desirable and it is appropriate to use 0.01m phosphate as a buffer at pH 7.2. A density gradient of 1.02 to 1.24 g/ml is suitable. Other solutions that can be used as a density gradient of this magnitude include polyols such as glycerol and salts such as tartrates or cesium chloride. The gradient can be prepared by half-filling the rotor with a solution of the lowest density of the desired gradient and adding an equal volume of the highest density. Driving the rotor at moderate speed will then complete the gradient.

As indicated above, the gradient solution contains

a surfactant to cleave the virus as it passes through the gradient. Usually, the surfactant in water solution can be introduced into the gradient and the centrifuge rotor run for such a long time that the surfactant penetrates into the gradient solution. Alternatively, the surfactant may be present in the gradient solution when it is topped up on the rotor. Many of

the hemolytic surfactants that can be used in this invention form distinct bands in the gradient, although banding is not a critical property. The position of the band in the gradient is not critical since the virions, which are relatively heavy, will pass through the gradient until they reach this band. After cleavage, subunits may move to zones of appropriate density which may be on the low density side of the surfactant band.

As indicated, the surfactant should be a hemolytic surfactant. By this is meant a surfactant that will give a positive result in the following test: Red blood cells from chicken (0.5% volume/volume in 0.01m phosphate-buffered saline with pH 7.0, 0.25 ml) are added

to a 1% w/v aqueous solution of the surfactant (0.25 ml). For the result to be positive, complete haemolysis must occur within 5 minutes at 22°C.

Included in this category are the following types of surfactants:

Nonionic compounds.

Mixed Compounds:

Generally, the minimum effective amount of surfactant should be used to reduce the problem of removal afterwards. For the preferred surfactant, an arylether adduct of ethylene oxide, particularly "Trton N101", the total concentration in the gradient is preferably at least 0.5% v/v, advantageously 1.0% v/v. The concentration in the localized band on the gradient will naturally be higher than this. Some surfactant will normally be present throughout the gradient where desired subunits of the virions form bands and this prevents or reduces reunification of the subunits. If such an association occurs, the separation from unwanted antigenic material is poor.

The medium containing the purified virions is then passed through the zone ultracentrifuge so that they penetrate the gradient and are cleaved by the surfactant and form bands as subunits. Typically, the virions should be replenished with an amount of 2.8 rotor plumes per hour, for example approx. 10 liters per hour for a 3.6 liter "KU" rotor. (When virions are to be purified without cleavage, the filling quantity can be larger, for example 30 litres/hour). Typically, the speed of rotation during filling of the gradient is at least 20,000 rpm, conveniently 3,500 rpm giving 90,000 g in the "KII" centrifuge.

After the "filling" is complete, the ultracentrifuge rotor is run for a further time, say 2 to 3 hours, to further separate and consolidate the bands and, after the rotor has been slowly slowed and stopped, the gradient is separated into fractions. The fractions containing the desired sub-units can then be collected.

In the case of viruses which secrete neuraminidase or analogous materials, this can be determined by standard test methods, for example the method of Warren (J. Biol. Chem. 234, 1959, p. 1971). In the case of viruses that release monovalent hemagglutinin or analogous material, this cannot be identified by testing for hemagglutination, as the hemagglutinin obtained is in monovalent form and does not exhibit such activity. It is therefore necessary to determine the antigenic effect of the samples (for example by hemagglutination inhibition titre or the simple radial diffusion test) or measure a physical property such as optical density, ultraviolet absorption etc. In some cases, hemagglutinin will be found in the same fractions as neuraminidase.

The virus used is ether-sensitive and as such will have the necessary removable coating containing antigenic material. The preferred virus is an influenza virus, but other suitable viruses include measles and mumps viruses in humans and Newcastle disease virus (NDV) in poultry (all of which are myxoviruses)

and bovine rhinotracheitis virus (which is a Herpes virus).

For use as a vaccine, the collected antigenic sub-unit fractions must be substantially free of surfactant and this can be achieved by, in the case of non-ionic surfactants, such as "Triton N101",

lowering the cloud point by adding ionic material to the dilute aqueous medium, so that the surfactant

stands out as a new phase. However, it is advantageous to separate the sub-units by selective adsorption on colloidal aluminum hydroxide (for example "Alhydrogel") and let the surfactant remain in the solution. This method can preferably be carried out as a second step after lowering the cloud point, in the cases where it can be used. Use of the aluminum hydroxide has the advantage that the antigens are obtained in a form where they are present in combination with additives so that they can be used as a vaccine in the present form. Alternatively, after the surfactants have been removed, the antigens can be added to other suitable additives such as oil emulsions, for example emulsions of water in an oily fatty acid glyceride, such as peanut oil. Such emulsions may contain non-ionic emulsifiers such as sorbitan trioleate and a stabilizer such as aluminum monostearate.

The potency of "Alhydrogel" adsorbed influenza subunit vaccine was compared with aqueous whole influenza virion vaccine. The results are reproduced in the following table. The strength was determined

by injecting groups of 5 chickens intramuscularly in the leg with 0.5 ml vaccine dilutions on day 0 and day 14 and bleeding on day 28. Haemagglutination inhibition and antineuraminidase antibodies were determined.

Aqueous virus of single strength, based on hemagglutination titer, contained 400 I.U./ml. The single strength subunit vaccine contained an equivalent amount of antigen. These results show that the adsorbed sub-unit vaccine has equal or greater antigen content than aqueous whole virus vaccine.

According to the invention is obtained

a method for producing an adjuvant subunit virus vaccine containing protective antigenic components and it is substantially free of pyrogens, nucleic acids and unwanted antigens, comprising the step of adsorbing the desired protective antigenic component obtained by the preceding method according to the invention on colloidal aluminum hydroxide, preferably after first removing most of any remaining surfactant. Non-ionic surfactants can, for example, be removed by lowering the cloud point.

The colloidal aluminum hydroxide used appropriately contains approx. 24 as A1(OH)3, for example "Alhydrogel". The final concentration of the 2% aluminum hydroxide gel in the vaccine should usually preferably be from 6.0 to 18% volume/volume, preferably approx. 12.5% volume/volume. The adsorbed vaccine can, if desired, be washed free of possible contaminants, for example residues of surface-active agents, by centrifuging the aluminum hydroxide and resuspending it in fresh phosphate-buffered saline solution. Optimal pH in the final vaccine is from 7 to 8, advantageously approx. 7.6.

The following examples further illustrate the invention.

Example 1

Influenza virus type A/Hong Kong/l/68X was cultured in 11-day-old embryonated chicken eggs. The allantoin fluid was harvested after 48 hours, inactivated with 6-propiolactone (1 ml/1000 ml allantoin fluid) overnight at room temperature and then clarified using a "Westphalia" (trademark) continuous flow centrifuge. The clear, inactivated allantoin liquid was allowed to flow at 10 l/hour over a gradient prepared from 60% w/v sucrose in 0.01 m phosphate buffered saline pH 7.2 (1.8 liters) and pure 0.01 μm phosphate buffered saline pH 7 .2 (1.4 liters) in a "KII" rotor. The rotor speed was 35,000 rpm. (90000g) and it was run for 2 hours after all the allantoin liquid had been added. The fractions containing influenza virions, as determined by their hemagglutinin titers, were collected (13500 I.U./ml, total 1500 ml).

The virions were found in the fractions containing 40% w/v sucrose and were diluted 1:8 in 0.01m phosphate-buffered saline with pH 7.2 to a sucrose concentration of 5% w/v. The diluted virions were flowed over a sucrose gradient containing 1% "Triton N101" in the "KII" rotor at 10 liters/hour. The sucrose gradient was prepared by filling the stationary rotor with 1.8 liters of 60% w/v sucrose in 0.01 M phosphate buffered saline solution of pH 7.2 also containing "Triton NlOl" (1% v/v ), and 1.4 liters of 0.01m phosphate buffered saline with pH 7.2 at the top and then accelerate the rotor to 90000g (35000 rpm). The rotor was run at 35,000 rpm. for 2 hours after all the influenza virus had been added. The fractions containing the large neuraminidase and "Triton" peaks were collected. Hemagglutinin was also present (504 of the starting amount according to hemagglutinin titers). To 3 parts of the collected fractions, 1 part of 1.6m phosphate buffer with pH 7.6 was added. The mixture became cloudy and the phases were separated by spinning at 2000g for 20 minutes. The lower phase was collected

and contained the neuraminidase and hemagglutinin with only approx. 0.054 vol/vol "Triton N101" (yield 40%). To an aliquot (10 ml)

an equal volume of "Alhydrogel" was added and the mixture was allowed to adsorb overnight at 4°C. Dilutions were made and the strength of the dilution determined on chickens. After the dilution that produced an adequate vaccine was determined, all the vaccine was diluted to this strength, but the final "Alhydrogel" concentration was kept at 12.54. Before adjuvants were added to the vaccine, potency was determined by the single radial diffusion method of Schild (J. Gen. Virology, 16, (1972) pp. 231-236).

Example 2

The procedure of Example 1 was repeated except that "Ethomeen 18/25" replaced "Triton N101" in the gradient.

Example 3

The procedure of Example 1 was repeated, but "Triton N101" was not separated by cloud point lowering. Instead, 12.54 vol/vol "Alhydrogel" was added to the fractions from the "KII" centrifuge containing the hemagglutinin, neuraminidase and "Triton N101". The mixture was kept at 4°C overnight and the "Alhydrogel" centrifuged out. The supernatant liquid contained "Triton N101". The pellet was suspended in its original volume of 0.01m phosphate-buffered saline at pH 7.2, centrifuged and the pellet resuspended in its original volume of 0.01m phosphate-buffered saline. The strength of the vaccine was determined as in example 1 and the vaccine diluted in a similar manner, keeping the final concentration of "Alhydrogel" at 12.5% volume/volume.

Example 4

The procedure from example 1 was repeated with influenza virus strain B/Hong Kong/8/73 so that a potent vaccine was obtained.

Example 5

The procedure from Example 1 was repeated with the influenza virus strain B/Victoria/98926/70 so that a potent vaccine was obtained.

Example 6

The procedure from example 1 was repeated with influenza virus strain A/England/42/72 so that a potent vaccine was obtained.

Example 7

Newcastle disease virus was cultured in 10-day-old embryonated chicken eggs. The allantoin liquid was harvested after 96 hours, inactivated with γ-propiolactone (1 ml per 1000 ml liquid) for 2 hours at 37°C. The solution was centrifuged at 1000g at a flow rate of 20 liters/hour to clarify and remove urates from the solution.

The clarified allantoin solution was flowed over a sucrose gradient prepared as in Example 1 in the "KII" rotor at 10 liters/hour. The rotor was centrifuged at 35,000 rpm. for 2 hours after all the allantoin liquid had been added.

The fractions containing the subunits, determined by the neuraminidase content, were collected. The rest of the procedure was as in example 1.

Example 8

The fractions/which contained influenza neuraminidase

and -hemagglutinin from Example 1 after "Triton N101" had

had been removed by cloud point lowering, was collected and diluted with 0.01 M phosphate-buffered saline at pH 7.2 to obtain a liquid containing 1600 i.e. hemagglutinin/ml. It was then homogenized in the following proportions.

(The aluminum monostearate was heated in the vegetable oil to be dispersed and the sorbitan trioleate was added before mixing into the aqueous phase).

The antigenicity of this vaccine was confirmed in chickens.

Claims (3)

1. Method for producing a vaccine, in particular an influenza vaccine, by cultivating a virus, in particular influenza virus, in a tissue culture or in the allantois from poultry eggs with embryos, collecting the liquid containing virions, and possibly inactivating, clarifying and concentrating the fraction(s) ) containing the virions, characterized in that the fractions containing the virions are introduced into an ultracentrifuge with continuous filling which is equipped with a density gradient solution containing a hemolytic surfactant, whereby the virions pass into the density gradient and are cleaved by the surfactant, and the antigenic subunits form isopycnic bands, and the fractions or the fraction containing protective antigen subunits are collected and transferred in a known manner in an administration form suitable as a vaccine. 2. • Method according to claim 1, characterized in that a sucrose solution is used as the density gradient solution. 3. Method according to claim 1 or 2, characterized in that nonylphenol-9-10-oxyethylene is used as surfactant.