Title:  Method to produce inactivated w/o emulsion adjuvated vaccines

United States Patent:  6,458,363

Issued:  October 1, 2002

Inventors:  Schrier; Carla Christina (Boxmeer, NL); Rijke; Eric Onno (Boxmeer, NL)

Assignee:  Akzo Nobel N.V. (Amhem, NL)

Appl. No.:  868818

Filed:  June 21, 2001

PCT Filed:  June 29, 2000

PCT NO:  PCT/EP99/10178

371 Date:  December 15, 1999

The present invention is directed to a method to prepare an inactivated w/o emulsion adjuvated vaccine, wherein an aqueous solution comprising one or more inactivated angigens is mixed under mild conditions with a ready-made w/o emulsion. Preferably the vaccine is prepared just prior to vaccination. The aqueous solution and w/o emulsion are stirred or shaken by mechanical means or by hand. The present invention also relates to a kit of parts for use in a method according to the invention.

Description of the Invention

The present invention is related to inactivated vaccines and a method to produce said vaccines.

Vaccines have been widely used for the prophylaxis and treatment of infectious diseases in both animals and man. The vaccines used can be divided in two main categories: live vaccines and inactivated vaccines. Live vaccines make use of naturally occurring mild strains or attenuated strains of live pathogens. Inactivated vaccines comprise antigens constituting whole inactivated micro-organisms or specific components (subunits) of said micro-organisms. In case of the latter, two types of inactivated vaccine are distinguished: subunit vaccines in case of components that have been obtained via biochemical purification, or recombinant vaccines in case the isolated components of the microorganism have been prepared via recombinant technology.

Inactivated vaccines have the advantage over live vaccines in that this way of immunisation against pathogens has no risk of infection. The general problem perceived with the use of inactivated vaccines is their inability to raise an immune response that is sufficient for protection. As a consequence, inactivated vaccines are often combined with an adjuvant, i.e. a compound or composition that is capable to increase the general or specific immune response in the vaccinated subject. The administration of inactivated micro-organisms or components thereof and the adjuvant leads to a strong, effective and generally protective immune response. One of the most commonly used adjuvants is a water-in-oil (w/o) emulsion. W/o emulsions provide a two phase system for the vaccine: an aqueous phase in which the antigen can be dissolved or suspended and an oil phase in which the aqueous phase is dispersed as small droplets.

Inactivated vaccines that make use of a w/o emulsion as adjuvant are usually prepared by emulsifying an aqueous solution comprising the inactivated antigen, a suitable oil and emulsifying agents until a w/o emulsion is obtained in which the antigens are homogeneously distributed over the aqueous phase. The production of these inactivated w/o emulsion adjuvated vaccines takes considerable time and costs, but is generally regarded as necessary: only thorough emulsification of the antigen would lead to a homogeneous distribution of said antigen in the w/o emulsion. This is generally considered to be necessary for effectively stimulating the immune response. The emulsification process however is a highly energetic process carried out under vigorous process conditions including high temperatures and/or strong shear forces. For some antigens, these vigorous emulsification conditions can alter the structure or conformation of the antigen and as such reduce the efficacy of the resulting vaccine. Storage in emulsified form can decrease the stability of the antigen because the antigen is present in a dissolved or suspended state. Furthermore, during storage the chemical components that are. present in the emulsion can reduce the stability of the antigen, as is the case for example with enveloped viruses: prolonged contact with the emulsifying agent destroys the viral envelope resulting in a decreased efficacy of the vaccine.

Surprisingly it was now found that inactivated, w/o emulsion adjuvated vaccines can be prepared by simply mixing of an aqueous solution comprising the inactivate,d antigen and a ready-made w/o emulsion, said mixing carried out under mild conditions. The vaccines thus obtained were found to be homogeneous compositions, which remained homogeneous for extended periods of time, despite the fact that the aqueous solution comprising the inactivated antigen had not been subjected as a whole to an emulsification process.

As already indicated emulsification is a highly energetic process in which an aqueous phase, an, emulsifying (surfactant) agent and a non-aqueous phase, preferably an oil phase, are mixed under vigorous process conditions. The mixing process according to the present invention however is carried out under extremely mild process conditions compared to the process conditions of the emulsification process, said mild conditions including low or no shear forces and ambient temperatures. Preferably the mixing process according to the present invention, is carried out by hand at ambient temperature.

Mixing of said aqueous antigen solution and said ready-made w/o emulsion according to the invention can be carried out by mere shaking, stirring or any other way of combining the two constituents without the excessive process parameters that are necessary for emulsification. Compared with the time needed for emulsification, mixing according to the invention can be carried out in a short time period. Preferably the mixing is achieved in a few seconds or minutes. More surprisingly, it was found that the mere shaking by hand of said aqueous solution and the already prepared w/o emulsion was sufficient to obtain a stable and homogeneous inactivated vaccine composition with an efficacy similar to an inactivated vaccine prepared according to a standard emulsification process.

The aqueous solution according to the invention comprises one or more inactivated antigens. An aqueous solution comprising two or more different inactivated antigens can be used in the method according to the invention to obtain multivalent inactivated vaccine emulsions.

The method of preparation according to the invention has several advantages over standard methods of preparation: it is easy to carry out, less time consuming and a more economical process. An important advantage of the method according to the present invention is the fact that the inactivated antigen is not exposed to the damaging shear forces and/or high temperatures that occur during the emulsification process: the chemical structure of the antigen is better preserved.

In a specific embodiment of method according to the invention the w/o emulsion adjuvated vaccine is prepared in the field prior to vaccination. The method provides a veterinarian a more flexible and adequate reaction upon field circumstances: in case of an outbreak the necessary inactivated antigens can be dissolved in an aqueous solution, mixed by hand with a suitable amount of a stable, ready-made w/o emulsion, and subsequently administered to the subject animals. Thus the method according to the invention provides for a quick and effective preparation of inactivated vaccine emulsions in the field just prior to use.

A further advantage of the method according to the invention is that in this way inactivated antigens which are normally not compatible, can be mixed together prior to vaccination to obtain a multivalent inactivated vaccine emulsion. Since the incompatible inactivated antigens are combined just prior to vaccination, they are only present in the emulsion for a short-lived period during which they cannot exert their adverse effect on each other.

Another advantage of the method according to the invention is that the inactivated antigens can be stored separately in dried form, preferably in lyophilised form, to prevent deterioration of the components during storage. The stability of the inactivated antigens declines much faster during storage in solution or suspension than in dried form. Just prior to vaccination the dried components are re-constituted with water into an aqueous solution. The dried vaccine components furthermore need less storage capacity than their corresponding solutions or suspensions.

Thus, the present invention provides for a method to prepare an inactivated, w/o emulsion adjuvated vaccine, wherein an aqueous solution comprising one or more inactivated antigens is mixed under mild conditions with a ready-made w/o emulsion. Preferably the aqueous solution and the w/o emulsion are stirred or shaken. The aqueous solution and the w/o emulsion can be mixed, preferably stirred or shaken, by mechanical means or by hand, more preferably by hand. The ready-made w/o emulsion may additionally comprise one or more antigens.

In a further embodiment the invention provides for a method to prepare an inactivated, w/o emulsion adjuvated vaccine, wherein an aqueous solution comprising one or more inactivated antigens is mixed with an already prepared w/o emulsion just prior to vaccination. Preferably the aqueous solution and the w/o emulsion are stirred or shaken, more preferably shaken. The aqueous solution preferably comprises a combination of inactivated antigens. The ready-made w/o emulsion may additionally comprise one or more antigens.

In another embodiment the invention provides for a kit of parts that can be used for the preparation of an inactivated, w/o emulsion adjuvated vaccine, said kit comprising at least one dosage of inactivated antigen and a separate dosage of a stable w/o emulsion. Preferably said kit comprises two or more dosages of inactivated antigen, each dosage constituting a different inactivated antigen. The inactivated antigen in a kit according to the invention is presented in the form of an aqueous solution or suspension or in a stable freeze-dried form. Preferably the antigen is presented in a stable, freeze dried form such as e.g. lyosphere, powder or tablet, which has to be dissolved in a small volume of water before use in a method according to the invention. In addition the separate dosage of w/o emulsion may comprise one or more antigens.

A kit according to the invention is suitable for use in the field and provides for tailor-made vaccines. Depending on the field circumstances, a proper selection of inactivated vaccines is dissolved in a small volume of water, stirred or shaken with a suitable amount of w/o emulsion and subsequently administered to the subjects. Thus a kit according to the present invention enables an adequate and quick reaction to an outbreak in the field.

A preferred kit according to the invention comprises inactivated IBDV antigens and optionally one or more inactivated antigens of chicken pathogens, preferably NDV and IBV.

For the purpose of the invention, the inactivated antigens are defined as immunogenic material derived from infectious micro-organisms (pathogens), which immunogenic material is non-replicative as opposed to live antigens. Inactivated antigens that are suitable for use in the method or kit according to the invention include inactivated whole micro-organisms of viral, bacterial or parasital origin, extracts of said micro-organisms or purified subunits of said micro-organisms. Examples of suitable micro-organisms include but are not limited to Newcastle Disease Virus (NDV), Infectious Bursal Disease Virus (IBDV), Infectious Bronchitis virus (IBV), E. coli, CM, Reo virus, coccidial agents and the like. The micro-organisms can be inactivated by chemical or physical means following conventional methods. Suitable chemical agents are for example formaldehyde, glutaraldehyde, .beta.-propiolactone, ethyleneimine and derivatives. Suitable physical agents for inactivation of micro-organisms are for example UV radiation, .gamma.-radiation, "heat-shock", and X-radiation. The inactivated antigens can also be isolated (recombinant) components or subunits or extracts of a pathogen, e.g. purified protein, protein-polysaccharide, protein-lipopolysaccharides, lipopolysaccharides and the like.

The inactivated antigens according to the invention may be produced by conventional methods known in the art including biochemical purification or recombinant DNA technology or may be purchased from commercial sources. In case of the latter, the inactivatede antigens are often present as a freeze-fried or lyophilised powder, tablet or lyosphere, and have to be dissolved in a small volume of water before use in a method according to the invention.

W/o emulsions that are suitable for use in a method according to the invention can be produced by methods known in the art or may be purchased commercially.

The oil component of the w/o emulsion will be present in an amount from 40% to 90% by weight, preferably 50% to 80% by weight. Especially preferred are w/o emulsions in which the oil component is present in an amount of 55% by weight.

The oil component of the w/o emulsion includes mineral oils such as Bayol.RTM. and Drakeol.RTM. and metabolizable oils. Suitable metabolizable oils are vegetable oils, fish oils, animal oils, tocopherol and tocopherol derivatives, and synthetically produced oils which can be metabolised and which are not toxic to the subject's body. Sources for vegetable oils include nuts, seeds, and grains. The most commonly available nut oil are peanut oil, soybean oil, coconut oil and olive oil. Seed oils include safflower oil, cottonseed oil, sunflower oil, sesame seed oil, and the like. Grain oil includes oil from cereal grains such as corn, wheat, oats, rye, rice, and the like. Fish oils include oil which can be readily recover led from fish, such as cod liver oil, shark liver oil, and the like. Suitable animal oils include whale liver oil. Synthetic oils include a number of branched chain oils that are synthesised biochemically in 5-carbon isoprene units and which are referred to as terpenoids. Squalene is a branched unsaturated terpenoid that is present in shark liver oil. Both squalene and its saturated analogue squalane are preferred oils and like the other fish oils are readily available from commercial sources or may be produced by methods known in the art.

The aqueous component of the w/o emulsion includes water, a buffer, saline, and the like.

W/o emulsions may be. prepared by conventional methods using emulsifying agents and/or emulsifying surfactant agents including Span 80, Span 85, Arlacel 80, Tween 80 and the like.

The particle size of the droplets of the dispersed phase is less than 20 .mu.m, preferably less than 1 .mu.m, more preferably less than 0.5 .mu.m in diameter. Preferably the w/o emulsion according to the invention is a stable emulsion.

Optionally, the w/o emulsions according to the invention comprise one or more antigens.

The inactivated, w/o emulsion adjuvated vaccines thus prepared by the method according to the invention can be used to vaccinate animals and man against disease causing pathogens. The vaccines can be administered per oral or via parenteral routes such as for example subcutaneous (sc.) injection, intramusculair (im) injection, intraperitoneal (ip) injection. Other administration routes that are favoured are application via the respiratory tract, e.g. eye drops, nasal drops, aerosol spray.

Claim 1 of 8 Claims

What is claimed is:

1. A method for preparing an inactivated, water-in-oil emulsion adjuvated vaccine, comprising mixing an aqueous solution comprising one or more inactivated antigens under mild conditions with a ready-made water-in-oil emulsion.
 

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