NO855169L - OIL ADJUSTED VACCINE, AND PROCEDURE FOR ITS MANUFACTURING. - Google Patents

Description

The invention relates to an oil-adjuvanted vaccine, as well as a method for its production.

Many antigens are so poorly immunogenic that they do not trigger any measurable or only a small immune response upon a one-off injection into an animal, so that it cannot be used as an inoculant without further ado. The antigenic effect can be enhanced, as these antigens are used together with an immune stimulator or adjuvant. For this reason, most inactivated viral and bacterial vaccines contain adjuvants.

It is known to adjuvant aqueous antigen solutions by emulsification with mineral or vegetable oils.

However, such emulsions, especially mineral oil emulsions, are poorly measured locally. Therefore, such emulsions are often used intramuscularly to produce a better measurability.

However, as a sterile abscess develops at the injection site, this method is increasingly rejected for commercial animals for meat-related legal reasons. Furthermore, oil emulsion vaccines are relatively viscous, which makes handling them difficult. The vaccine content of such vaccines is usually between 30 and 50%. A reduction in the oil content is not possible, as the vaccine would then become pasty and could no longer be injected. Furthermore, a specific emulsifier suitable for this oil is required for each oil.

It was now surprisingly found that a combination of an oil with certain emulsifiers is suitable for adjuvant vaccines, as the mentioned disadvantages are not observed.

The object of the invention is therefore a vaccine containing an aqueous antigen solution, and an adjuvant emulsion, characterized by the content of an oil, a sorbitan oleate, a compound of formula I with a molecular weight from 4000 to 5500, a and c being statistically equal, and b means such a number that the weight ratio between the oxyethylene part and the oxypropylene part is 7:3 to 9:1, optionally a polyoxyethylene sorbitan monooleate, and optionally glycerol.

The oil can be a mineral or a vegetable oil. The adjuvant components are preferably mixed with each other before being added to the aqueous antigen solution, and emulsified with an aqueous phase before being added to the antigen solution.

The sorbitan oleate is a mono- or trioleate, preferably

a monooleate.

Preferably, such a vaccine contains an adjuvant emulsion, which contains 5-20 ml, preferably 8-12 ml of an oil, preferably mineral oil, 0.5-10 ml, preferably 1-5 ml of sorbitan oleate, and 0.5- 10 g, preferably 1-4 g of a compound of formula I in 100 ml.

Optionally, this further contains 0.5-10 g, preferably 0.5-4 g of a polyoxyethylene sorbitan monooleate, and optionally 0.5-10, preferably 0.5-3 ml of glycerol per 100 ml.

The volume ratio between aqueous antigen solution to adjuvant emulsion can amount to 0.5 to 2, preferably 0.8 to 1.2.

As mineral oil, "Bayol" F or "Marcol" 52 is preferred. However, paraffin oil, (pure, if Merck), "Drakeol" or "Miglyol" can also be used.

The antigen can above all be a virus, bacteria, cell or protein antigen.

Aqueous adjuvants such as saponin and further plant extracts, aluminum hydroxide, aluminum phosphate, kaolin, bentonite, diatomaceous earth or silicic acids can be added as additional immunostimulatory substances.

The adjuvant according to the invention causes a higher immune stimulation compared to the method according to the state of the art.

The required amount of adjuvant is less than known methods. Quantities are preferably used such that the vaccine contains up to 15 ml of adjuvant in 100 ml of finished vaccine. However, larger amounts of adjuvant can also be added.

Usually, it is enough to simply shake an aqueous antigen solution when adjuvant mixing according to the invention to

obtain a stable emulsion.

The vaccine may contain common auxiliaries or additives.

The invention will be explained in more detail with the help of some examples.

Example 1

Preparation of an adjuvanted vaccine

0.5 ml paraffin oil, boiling range 190-240°C ("Bayol" F),

0.1 ml sorbitan trioleate, molecular weight approx. 1050,

0.1 ml polyoxyethylene sorbitan trioleate, molecular weight approx. 1300, 0.2 ml of a solution of 40 g of the compound of formula 1, molecular weight 4750, weight ratio oxyethylene/oxypropylene part, 8:2, in 100 ml of water, and

0.4 ml of glycerol was mixed and 5 ml of antigen (virus diarrhoea/mucosal disease antigen) in water and 3.7 ml of phosphate buffered saline (PBS) were slowly added to the emulsion formed and mixed.

The formed water-in-oil emulsion remains stable after mixing.

When instead of "Bayol" F "Draceol", "Marcol" 52, paraffin oil, (pure, white Merck), or "Mygliol" was used, stable emulsions were likewise formed.

Effect and tolerability

The vaccine (each time 2 ml) was administered subcutaneously to rabbits. As a comparison, a vaccine containing 5 ml of antigen solution and 5 ml of Complete Freund's Adjuvant served

(FCA).

While these 3 weeks after application at the injection sites showed approx. 30 ml hardnesses, the hardnesses of the vaccine adjuvanted with "Bayol" F according to the invention were only pea-sized.

In the neutralization test, the antibodies were determined against VD/MD virus. The titer of sera from rabbits inoculated with FCA-containing VD/MD vaccine was 1:14 0 according to the invention with "Bayol" F 1:500.

Example 2

In example 1, FMD antigen was used instead of VD/MD antigen. The vaccine (each time 0.4 ml) was applied to mice. 3 weeks later, the animals were challenged with FMD virus.

Then the vaccine shows a 50% protective dose (PD^q) of 4.3. Comparison: FCA-containing FMD vaccine 0.2 PD D ,.U..

Example 3

Vaccine A 0.5 ml paraffin oil "Marcol" 52

0.1 ml sorbitan trioleate, molecular weight approx. 1050, 0.1 ml polyoxyethylene sorbitan trioleate,

molecular weight approx. 1300,

0.2 ml of a solution of 40 g of the compound of formula I, molecular weight 47 50, weight ratio oxyethylene/oxypropylene part 8:2 in 100 ml of water,

0.4 ml glycerol, and

0.5 ml saponin, 1% in water,

was mixed. Then 5.0 MKS antigen and 3.2 ml of PBS were mixed.

Vaccine B 5.0 ml FCA and

5.0 ml FMD antigen.

Vaccine C As vaccine A but without saponin.

Mice were immunized with these three vaccines and 3 weeks after inoculation they were infected with infectious FMD virus. The results were as follows:

This example shows that by adding saponin, the immunostimulatory effect of the oil vaccine can also be increased considerably.

Example 4

0.5 ml paraffin oil, boiling range 190-240°C,

0.1 ml sorbitan trioleate, molecular weight approx. 1050,

0.1 ml polyoxyethylene sorbitan trioleate, molecular weight approx. 1300, 0.2 ml of a solution of 40 g of the compound of formula I, molecular weight 4750, weight ratio oxyethylene/oxypropylene-

part 8:2, in 10 0 ml of water,

0.4 ml glycerol, and

8.7 ml of PBS

was mixed in an emulsion, and applied each time with 0.2

ml on mice s.c. 3 days later, these mice as well as untreated mice were challenged with a lethal dose of FMD virus or V. cholerae. While all untreated animals died, those that had received the oil emulsion by administration survived

V.cholerae 50% and off

FMD virus 60%.

This shows that with these preparations an immune stimulation is achieved which does not depend on a formation of antibodies which, however, manifests itself in an increased resistance to other viral and bacterial infections (paraimmunity).

Example 5

Preparation of an adjuvanted vaccine

0.5 ml paraffin oil, boiling range 190-240°C ("Bayol" F),

0.1 ml sorbitan monooleate, molecular weight approx. 1050.

0.1 ml of polyoxyethylene sorbitan monooleate, molecular weight approx. 1300, 0.2 ml of a solution of 50 g of the compound of formula I, molecular weight 4750, weight ratio oxyethylene/oxypropylene part 8:2 in 100 ml of water,

was mixed and to the formed emulsion was added 5 ml of antigen (virus diarrhoea/Mucosal disease antigen) in water and 4.1 ml of phosphate buffer in a saline solution (PBS) and mixed.

The water-in-oil emulsion formed remained stable after centrifugation.

When instead of "Bayol" F "Drakeol" was used, "Marcol"

52, paraffin oil (pure, white, Merck) or "Mygliol", likewise formed stable emulsion.

Effect and durability

The vaccine (each time 3 ml) was administered subcutaneously to rabbits. As a comparison, a vaccine containing 5 ml of antigen solution and 5 ml of Complete Freund's Adjuvant served

(FCA).

While this 3 weeks after application, the injection site showed approx. 30 ml large hardness formations, the hardness formation in the vaccine adjuvanted with "Bayol" F according to the invention was only the size of a pea.

In a neutralization test, the antibodies were determined against VD/MD virus. The titer of the serum from rabbits inoculated with FCA-containing VD/MD vaccine was 1:14 0 according to the invention with "Bayol" F 1:500.

Example 6

In example 5, FMD antigen was used instead of VD-MD antigen. The vaccine (each time 0.4 ml) was applied to mice. 3 weeks later, the animals were challenged with FMD virus. Thereby, the vaccine shows a 50% protective dose (PD5Q) of 4.3 Comparison: FCA-containing FMD vaccine 0.2 PDb ,.U-.

Example 7

Vaccine A 0.5 ml paraffin oil "Marcol" 52,

0.1 ml sorbitan monooleate, molecular weight approx. 1050, 0.1 ml polyoxyethylene sorbitan monooleate, mol wt

about. 1300,

0.2 ml of a solution of 40 g of the compounds of formula I, molecular weight 4750, weight ratio oxyethylene/oxypropylene part 8:2 in 100 ml of water, and

0.5 ml saponin, in % in water,

was mixed. Then 5.0 ml FMD antigen and 3.6 ml PBS were mixed.

Vaccine B 5.0 ml FCA and

5.0 ml FMD antigen.

Vaccine C: Like vaccine A but without saponin.

With these three vaccines, mice were immunized and 3 weeks after inoculation were challenged with infectious FMD virus.

The results were as follows:

This example shows that by adding saponin, the immunostimulating effect of the oil vaccine can also be increased considerably.

Example 8

0.5 ml paraffin oil, boiling range 190-240°C,

0.1 ml sorbitan monooleate, molecular weight approx. 1050,

0.1 ml polyoxyethylene sorbitan monooleate, molecular weight approx. 1300, 0.2 ml of a solution of 40 g of the compound of formula I, molecular weight 4750, weight ratio oxyethylene/oxypropylene part, 8:2 in 100 ml of water, and

9.1 ml of PBS,

was mixed into an emulsion and applied to mice each time 0.2 ml s.c. 3 days later, these mice as well as untreated mice were challenged with a lethal dose of FMD virus, or V. cholerae. While all untreated animals died, those that had received the oil emulsion by administration survived

V cholerae 50%, and of

FMD virus 60%.

This shows that even with these preparations an immune stimulation is achieved which does not depend on the formation of antibodies,

which, however, manifests itself in an increased resistance to deadly viral and bacterial infections (paraimmunity).

Example 9

Vaccine A 0.5 ml paraffin oil, "Marcol" 52,

0.025 ml sorbitan monooleate, milk weight approx. 1050, 0.025 ml polyoxyethylene sorbitan monooleate, molecular weight approx. 1300,

0.5 ml of a solution of 40 g of the compound of formula I, molecular weight 4750, weight ratio oxyethylene/oxypropylene part, 8:2 in 10 0

ml of water,

was mixed, then 5.0 ml FMD antigen and 3.95 ml PBS were added.

Vaccine B 0.5 ml paraffin oil, "Marcol" 52,

0.025 ml sorbitan monooleate, molecular weight approx. 1050, 0.025 ml polyoxyethylene sorbitan monooleate, molecular weight approx. 1300,

0.5 ml of a solution of 40 g of the compound of formula I, molecular weight 4750, weight ratio oxyethylene/oxypropylene part 8:2 in 100 ml of water.

0.4 ml of a suspension of 20 g diatomaceous earth ("Filtercel") in 100 ml 0.1 molar phosphate buffer pH 7.3.

was mixed, then 5.0 ml FMD antigen and 3.2 ml PBS were added.

With these two vaccines, mice were immunized and 3 weeks after inoculation were challenged with infectious FMD virus.

The results were as follows:

This example shows that by adding diatomaceous earth, the immunostimulatory effect of the oil vaccine can also be increased.

Claims (7)

1. Vaccine, containing an aqueous antigen solution, and an adjuvant emulsion, characterized by a content of an oil, a sorbitan oleate, a compound of formula I with a molecular weight from 4000 to 5500, with a and c being statistically equal, and b means such a number and the weight ratio between the oxyethylene part and the oxypropylene part is 7:3 to 9:1, optionally a polyoxyethylene sorbitan monooleate, optionally glycerol, and optionally additional adjuvants. 2. Vaccine according to claim 1, characterized in that it contains an adjuvant emulsion containing 5-20 ml, preferably 8-12 ml of an oil, preferably mineral oil, 0.5-10 ml, preferably 1-5 ml of sorbitan oleate, and 0 .5-10 g, preferably 1-4 g of a compound of formula I in 100 ml. 3. Vaccine according to claim 1, characterized in that it contains an adjuvant emulsion containing 0.5-10 g, preferably 0.5-4 g of a polyoxyethylene sorbitan monooleate, and optionally 0.5-10 ml, preferably 0, 5-3 ml glycerol; of 100 ml. 4. Vaccine according to claim 1, characterized in that the volume ratio between aqueous antigen solution and adjuvant emulsion amounts to 0.05 to 0.3, preferably 0.1 to 0.2. 5. Vaccine according to claim 1, characterized in that it further contains a mineral adjuvant, saponin, and/or a plant extract known as an adjuvant. 6. Process for producing a vaccine according to claim 1, characterized in that an oil, a sorbitan oleate, a compound of formula I, optionally a polyoxyethylene sorbitan monooleate, optionally glycerol and optionally a mineral adjuvant, saponin are added to an antigen solution , and/or a plant extract known to activate. 7. Process for producing a vaccine according to claim 1, characterized in that the oil sorbitan oleate, the compound with formula I, optionally polyoxyethylene sorbitan monooleate, optionally glycerol and optionally in the mineral adjuvant saponin and/or a plant extract known as an adjuvant are mixed with each other and emulsified with an aqueous base before adding to the antigen solution.