DD149159A1 - METHOD FOR PRODUCING INFLUENZAVIRUS SUBUNITS VACCINE - Google Patents

Abstract

Cleavage and separation of influenza whole virus in subunits and production of a subunit vaccine. Selectively obtaining the Protextiv immunogenically active envelope proteins haemagglutinin and neuraminidase from influenza viruses by acting on a protease with subsequent separation from the remaining viral substance. The immunogenic envelope proteins haemagglutinin and neuraminidase from influenza viruses can be selectively released from whole virus particles with a protease (eg BROMELAIN) and then separated from the remaining viral substance (eg by ultracentrifugation) to give, with the addition of an adjuvant, a subunit vaccine for immunoprophylaxis of the influenza manufacture. Application: Development and production of inactive influenza virus vaccines.

Description

Title of the invention

Verfaliren for the preparation of IniQusnsavirus subunit iliapfstoff

Field of application of the invention

The invention relates to a process for the preparation of inactive influenza virus Isipfstoffen containing only the protective imniunogen acting structural proteins (envelope proteins) hemagglutinin and neurarninidase of human or animal Influensaviren, for parenteral administration in humans, wherein from a prepared by a known method, purified Yiruskonaentrat with a content of viral protein av / ischen 2 and 40 reg / ml is assumed. The application of the invention is possible in all establishments and companies working in the field of development and production of Vixus vaccines, and is particularly useful in manufacturing plants producing Influenza vaccines.

Characteristic of the known solutions

It is known that

The protective effects of inactive influenza virus vaccines are responsible for the eight known structural proteins of the eight known structural proteins.the influenza virus is responsible for the two egg proteins, hiraagglutinin and keuraoinidase, whereas the internal proteins, including the viral genome, do not exert a protective immunogenic effect;

all components of the influenza virus which do not confer protective immunity (non-protective immunogenic proteins, lipids) and, if present in the vaccine, organic components from the respective propagating substrate for the virus (k ₄ B _s egg protein ) and thieving additives for inactivating the

Virus genome (ζ.Β _β formalin) is a dietary fiber which, when used parenterally, leads to undesired local and / or systemic realities;.

'-' proteolytic enzymes and / or detergents break down the influenza virus partially or completely into its structural units'

The EisJQgel the known methods for the preparation of inactive Influenzavirus-Irüpf material in which no more complete virus is present, that

Split vaccines produced by treatment of the influenza virus with reagents which leach out the lipid portion of the virus envelope or split vaccines which the full virus decomposes into subunits by the detergents in the preparation of the virus, but which is not further worked up and separating, apart from the protective immunogenic structural proteins kaemagglutinin and neuraminidase, nor the internal proteins including the viral genome, and the latter requires inactivation to preclude vemiehrungsfähigkeit,

- after treatment of the influenza virus with proteolytic enzymes, there is a mixture of all the structural proteins from which the two envelope proteins, which are to constitute the constituents of subunit vaccines, must each be isolated by appropriate methods,

- by means of a special Eetergens that it is possible for the two proteins Hill Häsagglutinin and neuraminidase of the virus selectively body (core) to replace _"o

Object of the invention. ·. ,

The aim of the invention is to produce an influenza virus subunit vaccine using a proteolytic enzyme in a simple technological process, ie a vaccine which, as a protective principle, contains only the envelope proteins, chiaagglutinin and lieuraminidase, and therefore is free of fiber, including the viral genome, which may cause adverse reactions and which, because of its good local and systemic tolerance, is also desirable in children and in those at risk who contraindicate the use of whole virus vaccines or split vaccines is to be used for immunization against influenza »- -

Explanation of the essence of the invention

It is an object of the present invention to selectively detach the glycoproteins haemagglutiiiin and ^ euraminidase present on the surface of the influerisavirus particle while retaining their protective irinogenic activity, to separate them from the other viral components and to make a vaccine (subunits vaccine) with the addition of adjuvants ) It was found that

the bull proteins of the influenza virus, hiraagglutinin and ileum urinidase, can be selectively separated from the virus surface in the appropriate range of pH by enzymatic action;

- The remaining, in their structure remaining virussubstans (core) can be removed without residue from the reaction mixture, e.g. by sedimentation in the ultracentrifuge,

- the envelope proteins hemagglutinin and keuraminidase can be enriched, for example, by ultracentrifugation,

The technology according to the invention thus makes it possible to produce an inactive vaccine for the iramun prophylaxis of influenza, which

is equivalent or superior in its protective immunogenic activity to whole virus and / or split vaccines,

- is superior in its local and systemic compatibility Vollvirus- and / or Spalt-Irnpf substances,

- In contrast to Yollvirus and / or Spaltcipfstoffen can also be used in children and at-risk individuals,

- which is characterized by an extremely high stability in terms of the content of haemagglutinierender activity.

AusfüfcruTigsbeispiele

1 "1" virus concentrate with 2 - 40 mg virus protein / ml 1 _e 2 "to 1/10 of the total volume

Buffer, pH 7.2

Che laplex »solution

mercaptoethanol

1 "3β with 0.2 - 2.0 mg enzyme (ζ, B. Broinelain from pineapple stalks) per mg virus protein

1.4 «incubation at 37 ° C for 16-30 hrs with stirring 1 ₆ 5 _e centrifugation for 90 min at 100 000 χ g

1 _e 6 * supernatant in the sucrose gradient (5 - 60%) 4 - 18 h, centrifuged at 100 000 x ·

1 _β 7 · Fractionation to obtain fractions with high hemagglutinin and neuraminidase content

1 _Ö 8 * Adsorption of Kämagglutinin and Neuraminidase on A1 (OH) _- gel and sedimentation of the complex by centrifugation; if necessary adsorption and sedimentation 2 - 4 times v / repeat

1.9, adjustment of the Al (OH) virus protein complex to a specific haemagglutinin content by dilution with buffered saline,

2 _e column method (soli d-phase method) 2 «1« according to 1.1 «2.2. apply 1 to 2 mg of enzyme per 100 μg of virus protein to a suitable carrier, such as glass, polystyrene latex, ion exchange resins, agarose, cellulose

2 "4" introducing the Enzyme TrägeriDaterials (solid-phase) and the mixture of 2.2, in a suitable column

2.5 "incubation of the column at 37 ° C with vibration for 16-30 hrs.

2.6 «Recovery of the reaction mixture 2.7 · Centrifugation of the reaction mixture for 90 Eon at 100,000 g

2 »S« Adjustment of the supernatant (combagglutinin and l-uraminaminidase mixture) with buffered saline solution to a definite Haasagglutlningehalt with the addition of adjuvant.

3.1. corresponding to 1.1 * 3e2, corresponding to 1 _β 2 _{Φ 3β3} "0.2 to 4.0 ing of enzyme per mg of virus protein to a suitable membrane

to raise ''

3 «4β Applying the mixture of 3» 2 «to the membrane 3.5« Incubation of the over-coated membrane at 37 ° C under vibra. tion f-llr 16 ~ 30 hours *

3 * 6 «Filtration of the incubation gels through a membrane filter with a pore size which is permeable only to substances of the order of the enveloping proteins hemagglutinin and neuraminidase

3E7. according to 2 »8,

Claims (1)

- 6 - 2 I -7 · claims A process for the preparation of Influensavirus-Sufceinheiten vaccine characterized in that "the protease immunogenically active structural proteins hemagglutinin and neuraminidase with a suitable enzyme selectively released from the Yoll virus, from the non-protective immunogenically effective Yirussubstanz '(cores ) and, with the addition of a suitable adduct, be processed into a vaccine suitable for parenteral use also in children and at risk, and characterized by a long-term , in terms of endurance activity. 2. A method according to item 1, characterized in that viral material in a percenter is mixed with a suitable buffer and a suitable enzyme and incubated, the reaction mixture is centrifuged, the supernatant is gradient centrifuged and fractionated and the isolated protective protective proteinic immunoglobulin kagagglutinin and heuraminidase, adsorbed to a suitable adgoivan, used as starting material for vaccine production " 3e method according to item 1, characterized in that viral material introduced with a suitable buffer is introduced into a column filled with a suitable Enzvffi carrier material, and the reaction mixture is centrifuged, and the active protective structure which is effective as proton is proteins added with a suitable adjuvant can be used as starting material for the production of lipids 4 "Method according to" Punlct 1 ", characterized in that virus material mixed with a suitable buffer, applied to a membrane coated with a suitable enzyme and incubated, the reaction mixture is filtered through a suitable membrane filter and the isolated, proticively immunogenic structural proteins, with a suitable Adjuvant used as 'source material for the production of vaccine / ground' 5β Method according to item "U, 2", 3 and 4 "characterized in that the plant protease bromelain is used as an enzyme for selective cleavage of the protective immunogenic / active coat proteins haaagglutinin and lieuraminidase of whole virus particles of üdEluensaviruses