DE2449530C2 - Virus subunit vaccine - Google Patents

Description

The invention relates to the production of pyrogen-free virus subunit vaccines, in particular to Influenza vaccine and the vaccines that can be obtained.

The influenza vaccines generally used hitherto are deactivated whole-virus vaccines which In addition to the influenza viruses, they contain some Elproteln, which is derived from the Allantols liquid. In the the virus was harvested. When injected, this type of vaccine can cause hypersensitivity, which can be caused by the egg proton and the pyrogenic effects caused by the vialons.

The influenza virus and similar viruses of the same type, i.e. H. Orthomyxoviruses and paramyxoviruses, are characterized by an outer membrane, the "spikes" of antigen with haemagglutinating effect and wear Neuramlnldase effect. This outer membrane can be caused by solvents such as ether or by Surface-active agents are cleaved, with antigens being released as subunits, the one Have a haemagglutinating effect and a neuraminalase effect. The so-called »Spllt-Vlrus« - Vaccines which have hitherto been produced on these catfish showed a lower pyrogenicity than corresponding ones Whole virus vaccine, although no attempt was made to target the antigens with neuraminalase activity and haemagglutinative effects of pyrogens and other undesirable materials, many other antigens and to separate nucleic acids.

It has now been found, however, that the antigens with a haemagglutinating effect and with a neuraminalase effect are not themselves pyrogenic or toxic in any other way and that therefore still another Reduction of pyrogenicity by obtaining the desired antigens, essentially free of other things undesired material can be achieved.

An attempt has now been made to separate the components of a split virus preparation (Spllt virus) by zone ultracentrifugation. However, it was found that batch loading of an ultracentrifuge rotor for Large-scale productions was not feasible and it was found necessary to run one continuously To use a loadable zone centrifuge, in which the relatively dilute suspension of the Vlrus subunits In passes the rotor and flows through the centripetal end of a concentration gradient. It did, however found that the very small particles of the desired antigens with haemagglutinin and neuramlnidase activity could only be reluctantly introduced into the gradient at flow rates that corresponded to A large-scale production Consistent.

It has now been found, however, that viruses of the influenza-type and related types which easily enter In the gradient. :) A continuously loadable zone ultracentrifuge can be introduced successfully can be broken down as they go down the gradient if a surfactant is In the gradient solution is present. This makes the production of Unterelnhelten vaccines, which Im are essentially free of Elproteln, pyrogens and Vlrlon nucleic acids and essentially only those contain desired protective antigens with haemagglutinin and neuraminldase activity, on a large scale relieved. The haemagglutin found in the so-called "spikes" becomes itself surface-active

Cleaved agent to a monovalent form which has no further haemagglutination, but rather the has desired similar properties.

In aligemeinen can all ether-empfindUchen viruses are cleaved by treatment with detergents, and in addition to the above ortho- and paramyxoviruses following viruses can subunit vaccines showed the Schutzantlgenekomponenten analogous to Haemagglutlnln and Neuraminldase yield, ^s namely:

a) Togaviruses

b) rhabdoviruses

c) Leukoviruses iu

d) Coronoviruses

e) arenoviruses 0 herpesviruses g) smallpox viruses

According to a feature of the present invention, a method is therefore provided for the production of Vlrus lower elbow vaccines derived from ether-sensitive viruses which are free from pyrogens, Virlon nucleic acids and undesired antigenic material, ether-sensitive virus suspensions optionally after inactivating the viruses and cleaning the suspensions by centrifugation, Gel filtration or Adsorption are subjected to continuous zone ultracentrifugation, and is characterized by this net that a density gradient solution is used for the zone ultracentrifugation, which is a hemolytic Surfactant In a total concentration In the gradients of at least 0.5 to 1% (VoI / VoI) contains, whereupon the parliamentary groups or the parliamentary group that contain (contains) the Schutzariilgen-Unterelnhelten Isolated, freed from the surface-active agent and made up with adjuvants for the vaccine. The invention also relates to the virus subunit vaccines so obtainable.

It is possible to obtain the entire Vlrlonen directly from a relatively dilute culture medium, such as. Allantol liquid, but it is generally preferred! Some cleaning of the whole Carry out virus preparation in the ultracentrifuge before splitting.

Any intact disactivated virus can be used for such purification using conventional methods Measures to be isolated. For example, the influenza virus can be grown in chicken egg envelopes that are 10 to 11 days old. The harvested Allantols-FIOsslgkelt can then be treated e.g. B. with / i-propolactone and / or formalin to inactivate or "kill" the virus and then through Centrifugation e.g. B. To be clarified in a Wesiphalla centrifuge (trade name) with continuous flow.

The virus can then be cleaned again. This can be done by using continuous Sedlmentatlonszentrlfugation, treatment with fluorocarbons or fluorocarbons, gel filtration or Adsorption on red blood cells or minerals like barium sulfate and subsequent eluleren take place there however, because of the speed and ease of handling in the bulk, it is preferred to use continuous zone centrifugation, for example using a KI1 apparatus described by Anderson et al. In Analytical Blochem. 32, 400-494 and 495-5U (1969).

A KII rotor with a capacity of 3.2 l with a sucrose gradient element is particularly 4Π effective. The method of Reimer et al (Journal of Virology Vol. 1, No. 6, Selten 1207-1216) can be used. After purification, the fractions containing vialons, as determined by the haemagglutination titer, can then be combined and diluted and analyzed to bring the density to one for the Incorporation in a gradient in the second ultracentrifugation stage to reduce suitable scale.

For the cleavage stage of the virus, the. Virions preferably after purification as above described on a density gradient solution of a zone ultracentrifuge with continuous load capacity upset. As for the purification of the virus, a suitable medium exists for the gradient In a solution of a sugar, in particular sucrose, preferably in buffered saline at a for the Virus suitable pH. In general, a pH of a little above 7 is desirable and it is convenient to Use 0.01 m-phosphate as a buffer with a pH of 7.2. A density of about 1.02 to about 1.24 g / ml is suitable. Other solutions used to form a density gradient of this magnitude include polyols such as glycerin and salts such as tartrates or cesium chloride. The gradient can by filling the rotor halfway with a solution of the lowest density of the desired gradient and Adding an equal volume of a solution with the highest density can be prepared. By rotating the The gradient is then set in the rotor at moderate speeds.

As stated above, the Dlchtegradlent solution contains a surfactant to kill the virus cleave as it traverses the gradient. In general, the surfactant can be aqueous Solution In the Gradienter «are introduced and the centrifuge rotor is rotated a certain amount of time around the Ensure penetration of the surfactant into the Gradient solution. Alternatively, the surfactant can be present in the Gradient solution when it is loaded into the rotor. Many of the haemoly- μ Table surfactants which can be used in the present invention form different ones Bands in the gradient, although banding is not an essential feature. The location the band in the gradient is not critical since the virtons, which are relatively dense, traverse the gradient until they reach this band; after splitting, subunits can be placed in zones of suitable density migrate, which are on the side of the surface active band with low density. e, 5

As stated earlier, the surfactant should be a hemolytic surfactant. This is to be understood as a surface-active agent that gives a positive result in the following examination:

Red blood cells from chicks (0.5% vol / vol in 0.01 m-phosphate-buffered saline, pH 7.0; 0.25 ml) become a 1% w / v !. aqueous solution of the surfactant (0.25 ml) added. For If the result is positive, complete hemolysis must occur within 5 minutes at 22 ° C.

This category includes the following types of surfactants: Common names:

ζ. B. Nonldet P40 Tergitol NPX Triton NlOl Renex 698

W ⁵

Il Belloid EMP

Adinol C0630

Non-toxic aryl ether adducts of ethylene oxide Alkylphenol-ethylene oxide condensates

or

Alkylphenol-polyoxyethylenes

or

Polyethoxyalkyl phenols Octylphenol-polyoxyethylene

or

Polyethoxyoctyl phenol Nylphenol-10,5 -oxyethylene

or

10,5-ethoxynonyl-phenol

Nonylphenol-9-10-oxyethylene

or

9-10-ethoxynonyl-phenol

Nonylphenol-9-9,5-oxyethylene

or

9 -9,5-ethoxynony I -phenol

Ociyiphenol-polyoxyethylene

or

Polythoxyoctyl phenol Polyoxyethylene nonyl phenol Common names:

? g z. B. BrIj 96

; ■ * »Tergitol TMN10 ν Tergitol 15-S-7

Allphatlsche ether adducts of ethylene oxide

aliphatic ^ alcohol-polyoxyethylene

or

Polyethoxy-aliphatic alcohols Oleyl alcohol-10-oxyethylene

or

10-ethoxy-oleyl alcohol

Trlmethyl nonanol-10-ethylene oxide Linear alcohol Cn-Ci ₅ 7-Äthylenoxld

■; ■■■ »5

ύ Common names:

_; 'z. B. Clthrol 4ML 400

. ^ Ester adducts of ethylene oxide

Fatty acid ester-polyoxyethylene

or

Polyethoxy esters of fatty acids Polyethylene glycol monolaurate

or

Lauric acid 400 polyethylene glycol ester

Common names: z. B. Ethomeen 18/25

Ethomeen C / 25 «5 Ethomeen S / 15

Amine adducts of ethylene oxide Fatty amine polyoxyethylene

or

Polyethoxy fatty amine Stearylamine-15-oxyethylene

or

15-ethoxystearyl amine

Coconut oil ine-15-oxyethylene

or

15-Äthoxykokosfettamlne (average ^ utl. Mol-Gew. 860)

Soy fatty amine-5-oxyethylene

or

5 ethoxy soy fat bottles

Alkanolamldaddukie of ethylene oxide

Common names: Alkanolamld-polyoxyethylene

or

Polyethoxyalkanol amides

e.g. Conox J 754 exact composition not known

Anionic

z. B. Texapon N25 / 5 sodium salt of sulfated lauryl (Dehydag) alcohol-dloxyethylene i < >

or

Dloxyethylene sodium lauryl sulfate

Adlnol T Sodium N-methyl-N-oleyltaurine

Solumln T 45 S natural salt of sulfonated! Fatty alcohol-45-oxyethylene

or is

45-ethoxy fatty alcohol sodium sulfonate
Teepol 610 sodium secondary alkyl sulfate

Sodum deoxycholate anlonal bile salt or bile acid salt

Pentrone A4 Amine Sal7 vnn A | ky | aryl sulfure

Different

z. B. Belloid M3 Polyoxyethylene-3-condensate with an aliphatic alcohol

Galoryl MT5

In general, the minimum effective amount of surfactant should be used to reduce subsequent distance problems. For the preferred surfactant, an aryl ether adduct of ethylene oxide, especially Triton N101, is preferably the total concentration In the gradient at least 0.5% (vol / vol), advantageously 1.0% (vol / vol). The concentration in the localized band In that Gradient is of course higher. Some surfactant is usually found throughout Gradients in front of where the subunits of the Vlrlonenband are desired, resulting in the reaggregation of the subunits prevented or reduced to a minimum. If such aggregation occurs, there is separation of undesirable anligenous material is low.

The medium containing the purified vialons is then passed through the zone ultracentrifuge in such a way that that they enter the gradient and are broken down by the surfactant and each other possibly form bands of separated subunits. In general, the Vlrlonen should be in one Extent of about 2.8 rotor volumes per hour, e.g. B. about 10 1 per hour, with a 3.6-1 KH rotor be charged. (If the vialons are cleaned without splitting, the loading rate can be higher, for example 30 liters per hour). In general, the speed of rotation during the loading of the gradient at least 20,000 revolutions / min., expediently 35,000 revolutions / min, amount, which results in 90,000 g in the KII centrifuge. After complete “loading”, the Uitra centrifuge rotor becomes during a further period, e.g. B. 2 to 3 hours, rotated to separate the gangs welter and solidify and after the rotor has been slowly slowed down and brought to a standstill the contents of the gradient are drained in fractions. Fractions containing the desired subunits can then be combined or stored.

In the case of viruses that release neuraminidase or analogous material, this can be determined by standard test methods, e.g. B. by the method of Warren (J. Blol. Chem. 234, 1959, page 1971). In the case of viruses which release monovalent hemagglutinin or analogous material, this cannot be identified by examining the hemagglutinization, since the hemagglutinin obtained is in the monovalent form and does not develop such activity. It is therefore necessary to determine the antigenic effect of the samples (e.g. through the inhibition of the inhibition of radiation or the "single radis!" Diffusion test) or a physical characteristic such as the optical density, UV -To measure absorption, etc. In some cases the haemagglutinin is found in the same fractions as the neuraminidase.

The virus used is ether sensitive and thus has the necessary strippable shell that contains antigenic material. The preferred virus is influenza virus, however other suitable viruses include measles and human mumps and Newcastle disease viruses (NDV) of poultry (which are all myxoviruses) and bovine rhinotracheitis virus (which is a herpes virus 1st) a.

For use as a vaccine , the pooled fractions of antigenic subunits must be essentially free of any surfactant and , in the case of nonionic surfactants such as Triton N101, this can be achieved by lowering the flocculation point by adding ionic material to the dilute aqueous medium so that the surfactant separates out as a new phase. However, it has proven advantageous to separate the subunits by selective adsorption on colloidal aluminum hydroxide (e.g. "Alhydrogel"), the surface-active agent remaining in solution. This process, where applicable, may desirably be carried out as a second stage after the flocculation point lowering. The use of aluminum hydroxide has the advantage that the antigens are obtained in an adjuvant form which can be used as such as a vaccine. Alternatively, after removal of the surfactant, the antigens can be mixed with other suitable adjuvants, such as oil emulsions, for example emulsions of water in an oily fatty acid glyceride, such as ErdnttBol, In

Adjuvantsm to be transferred. Such emulsions can contain non-ionic emulsifiers such as sorbitan troleate and a stabilizer such as aluminum monostearate.

The effectiveness (potency) of the influenza subunit vaccines adsorbed on "Alhydrogel" was measured with compared to an aqueous whole virus influenza vaccine. The results are in the table below listed. Efficacy was assessed by using intramuscular inoculation of groups of 5 chicks in the leg Rated 0.5 ml vaccine dilutions on day 0 and day 14 and bleeding out on day 28. There were the inhibition of hemagglutinatlon and the antineuraminidase antibodies are determined.

Tabel

vaccine (Influenza virus strain A / Hong Kong / l / 68X)

Hemagglutination inhibition titer

geometric mean titer (GMT)

2 weeks 4 weeks

Antineuraminidase titre

G. M. T. 4 weeks

aqueous total virus singie strength

aqueous total virus 1: 4 dilution

total aqueous virus 1:10 dilution

total aqueous virus 1: 100 dilution

adsorbed subunit single strength

adsorbed subunit 1: 4 dilution

adsorbed subunit 1: IQ dilution

adsorbed subunit 1: 100 dilution

2 027

582

291

1536

1536

1 163

18th

75

27

137

26th

36

The aqueous single strength virus, based on the haemagglutination factor, contained 400 I.U./mi (i.u./mi). The single strength Unterelnhelten vaccine contained an equivalent amount. These results show that the adsorbed subunit vaccine has an antigenic activity equal to or better than that of the aqueous whole virus vaccine.

According to a further feature of the invention, there is therefore a method for producing an adjuvanted Vlrus subunit vaccine that contains antigenic protective ingredients and is substantially free from Pyrogens, nucleic acids, and undesirable antigens is the stage of adsorbing the required protective antigen component (s) obtained by the foregoing invention Workers' catfish, preferably after an initial removal of most of any remaining surfactants, including colloidal aluminum hydroxide. Non-ionic surfactants Funds can be removed, for example, by lowering the flooding track.

The colloidal aluminum hydroxide used advantageously contains about 2% as Al (OH) ₃ , e.g. B. Alhydrogel. The final concentration of the 2% aluminum hydroxide gel in the vaccine should generally preferably be 6.0 to 18% (vol / vol), preferably about 12.5 * (vol / vol). The adsorbed vaccine can, if desired, be washed free of any associated contaminants, e.g. B. residual surfactant, by centrifuging the aluminum hydroxide and resuspending in fresh phosphate buffered saline. The optimal pH for the final vaccine pH is 7 to 8, advantageously around 7.6.

The following examples serve to further illustrate the invention: example 1

Influenza virus strain A / Hong Kong / 1 / 68X was raised in 11 day old embryonic chicken eggs. The allantol liquid was harvested after 48 hours, inactivated overnight at room temperature with /? - propolactone (1 ml per 1000 ml allantol liquid) and then clarified with a centrifuge with continuous flow (Westphalia, trade name). The clarified inactivated allantol liquid was given 101 per hour over one of 60 »(w / w) sucrose in 0.01 m-phosphate-buffered saline solution of pH 7.2 (1.81) and pure 0.01 m-phosphate buffered saline solution of pH 7.2 (1.41). In a KII rotor

instilled. The rotor speed was 35,000 revolutions / min. (90,000 g) and it became 2 hours rotates after the entire Allantol liquid has been introduced. Those containing the influenza virus Fractions, as determined by their haemagglutinin titer, were pooled [13,500 IU (lu.) per ml; 1500 ml in total].

The virions were in the 40% w / w. Fractions containing sucrose were present and were 1: 8 In 0.01 m-phosphate-buffered saline solution of pH 7.2 to a sucrose concentration of 5% (W / w) diluted. The diluted virions were in a sucrose gradient containing 1% Triton N101 Filled into the KIl rotor at 10 liters per hour. The sucrose grade was lent by filling up the stationary rotor with 1.81 60% lger (w / w) sucrose In 0.01 m-phosphate-buffered saline solution from pH 7.2, which also contained 15b (vol / vol) Triton N101 and 1.41 0.01 m-phosphate-buffered saline at pH 7.2 on the upper part and then accelerating the rotor to 90,000 g (35,000 revolutions / min.) manufactured. The rotor was at 35,000 revolutions / min. Rotates 2 hours after the entire Influenza virus had been brought in. The large neuramlnidase and ΤγΙιοα-Ρ'ή'-. -.- holding fractions were united; Haemagglutinin was also present (50% of the original content due to the haemagglutinin titer). 1 part of 1.6 M phosphate buffer with a pH of 7.6 was added to 3 parts of this solution. The mixture became cloudy, and the phases separated in 20 minutes at 2000 g. The lower phase was collected and contained the neuramlnidase and the haemagglutinin with only about 0.05% (vol / vol) Triton N101 (Yield 40%). An equal volume of "Alhydrogel" was added to an aliquot of 10 ml. and the mixture was allowed to be adsorbed at 4 ° C overnight. Dilutions were made, and the Efficacy of the dilutions assessed on chickens. After determining the dilution to form a: n Vaccine with adequate effectiveness, the entire vaccine was diluted to this strength, but the Final concentration of "Alhydrogel" kept at 12.5%. Before the adjuvant formation of the vaccine, the Efficacy according to the "single radial-Dlffuslonsmethjde" by Schild, J. Gen. Virology, 16, (1972), 231-236 certainly.

Example 2

The procedure of Example 1 was repeated, but replacing In for the Triton N101 in the gradient was made by Ethomeen 18/25.

Example 3

The procedure of Example 1 was repeated, except that the Triton N101 was not reduced by lowering the flocculation point was separated. Parts of it was added to the fractions from the KII centrifuge, the haemagglutin, Neuramlnidase and Triton N101 contained, 12.5% (vol / vol) "Alhydrogel" added. The mixture was left to stand at 4 ° C. overnight and the "Alhydrogel" was spun off. The supernatant liquid contained the Triton N101. The pellet was made to its original volume with 0.01 m-phosphate buffered saline Rotated suspended from pH 7.2, and the pellet again in its original volume of 0.01 m-phosphate-buffered Suspended saline solution. The effectiveness of the vaccine was determined as in Example 1 and the Vaccine diluted accordingly, keeping the final concentration of »Alhydrogel« at 12.5% (vol / vol) became.

Example 4

The procedure of Example 1 was followed on influenza virus strain B / Hong Kong / 8/73 to form a effective vaccine repeatedly.

Example 5

The procedure of Example 1 was repeated with the 70 Influenza virus strain B / Vlctorla / 98926 / form a so effective vaccine.

Example 6

The procedure of Example 1 was repeated with influenza virus strain A / England / 42/72 to produce an effective vaccine.

Example 7

"Newcastle-Dlsease" -Vlrus was raised in 10-day-old embryo-isolated hen's eggs. The Allantols Rivers- «0 Slgkelt was harvested after 96 hours and inactivated with iS-propolactone (1 ml per 1000 ml liquid) for 2 hours at 37 ° C. The solution was at 10,000 g with a flow rate of 201 per hour centrifuged to clear them and remove urates from the solution.

The clarified allantol liquid was prepared via a sucrose gradient as in Example 1, In the KII rotor is filled with 101 per hour. The rotor was at 35,000 revolutions / min for 2 hours centrifuged after the introduction of all of the allantoic fluid. The fractions containing the subunits, determined by the neuraminldase content, were combined. It was continued as in Example 1 described, proceeded.

5 24 49 530 Example 1 were after
m-phosphate buffered
Haemagglutlnln per ml IO Example 8 39.5 ml
53 ml
5 ml
2.5 ml 15th Influenza, Neuramlnldase and Haemagglutinln containing fractions of
Removal of the Triton N101 by lowering the flocculation point combined and with 0.0
Saline solution of pH 7.2 diluted to form a liquid that is 1600 IU (lu)
contained. It was then homogenized in the following proportions: and the sorbitan troleate 2 (1 Vlrus-Unterelnhelten-Liquid
Vegetable oil
Sorbitan troleate
Aluminum monostearate 25th (The aluminum monostearate was heated in the vegetable oil to release it
added before mixing with the aqueous phase).
The antigenic effect of this vaccine was confirmed in the chicken experiment. 30th 35 40 45 50 I. 55 SH I. 60 j 65 I.
A. ρ I.
I.
I. 8th

Claims (9)

Patent claims: 1. Process for the preparation of virus sub-unit vaccines derived from ethereal plants Viruses that are free from pyrogens, virus nucleic acids and the desired extraneous material, with respirable virus suspensions optionally after the virus has come into effect and the suspensions have been cleaned subjected to continuous zone ultracentrifugation by centrifugation, gel filtration or adsorption, characterized in that a density gradient solution is used for the zone ultracentrifugation which contains a haemolytic surface-active agent in a total concentration in the gradients of at least 0.5 to 1% (vol / vol), whereupon the parliamentary groups or tion that contain (contains) the protective antigen sub-entities isolated, freed from the surfactant and are (will) be made up with adjuvants for the vaccine. 2. The method according to claim 1, characterized in that an aryl ether adduct of ethylene oxide is used as the surface-active agent. 3. The method according to claim 2, characterized in that the surfactant is nonylphenol-9-10-oxyethylene. 4. The method according to any one of claims 1 to 3, characterized in that an influenza, herpes, mumps, measles or Newcastle disease V Ims virus is used. 5. The method according to any one of the preceding claims, characterized in that the non-ionlsche surface-active agent from the suspended protective antigen sub-units by adjusting the ionic strength of the suspension is removed to the flocculation point of the surfactant. 6. The method according to any one of claims 1-5, characterized in that the protective antigen sub-units are both purified and adjuvanted by selective adsorption on colloidal aluminum hydroxide will. 7. The method according to claim 6, characterized in that the colloidal aluminum hydroxide contains about 2% as Al (OH) ₃ and the concentration of the colloidal aluminum hydroxide in the vaccine is 6.0 to 18% (Vol / Vol) concerns. 8. The method according to claims 1-5, characterized in that the adjuvant is an emulsion of Water In peanut oil and sorbltantrloleate as an emulsifier and aluminum mnostearate as a stabilizer contains. 9. Virus subunit vaccine, obtainable according to the measures of claims 1-8.