CS272935B1 - Adjuvant for virus vaccines - Google Patents

Abstract

The solution concerns an adjuvant consisting of 1.5 to 4.5 percent by weight of water-soluble split bovine collagen with molecular weight of 30,000 to 40,000, in combination with 0.15 to 0.45 percent by weight of vegetable saponine in final vaccine form. This adjuvant boosts the immunogenic response to applied antigen and does not have negative effect on the organism.

Description

(54)

Adjuvant for Viral Vaccines (57) The present invention relates to an adjuvant consisting of 1.5 to 4.5 wt. % water-soluble split bovine collagen having a molecular weight of 30,000 to 40,000, in combination with 0.15 to 0.45 wt. plant saponin in the final vaccine form. This adjuvant enhances the immunogenic response to the applied antigen and does not adversely affect the body.

( ¹¹ ), (13) B1 (51) Int. Cl. ⁵ A .61 K 47 (OoZ) A 61 K 37/12

* - CS 272 935 Bl

The invention relates to an adjuvant for viral vaccines. They address the enhancement of the immunogenic response to the administered antigen. The adjuvant effect of the vaccines is 1.5-5.5% by weight. % water-soluble bovine collagen in combination with 0.15 to 0.45 wt. plant saponin.

An antigen administered parenterally induces a certain degree of immunity. Adjuvants are added to enhance its action (Ramon, G .: CR Acad. Sc. 181, (1925), 157; Ramon, G .: Bull. Soc. Cent. Med. Vet. (1925), 101, 227. ).

In particular, the use of aluminum hydroxide gel, mineral oils and saponin has stabilized worldwide (Ramon.G., Richon, R., Thiery, 3.P., Gerbeaux, Cl .: D Immunology (1943) 13, 301. Classical paraffin oil adjuvants is Freund's adjuvant (Freund, 3: Ann. Rev. Microbiol: 1 (1947) 291; Freund, 3: A.3. Clin. Path. 21 (1951) 645), which is enriched with optional emulsifiers. in addition to the adjuvant effect, some side-effects 3 are mainly reactions at the application site, but also in recent years - in connection with the deteriorating ecological situation and increasing susceptibility of animals - general anaphylactoid reactions, which mainly occur in cattle. death.

These drawbacks are overcome by the adjuvant for the viral vaccines according to the invention, which comprises 1.5 to 4.5 wt. % in soluble cleaved bovine collagen having a molecular weight of 30,000 to 40,000 and 0.15 to 0.45 wt. plant saponin.

The vaccine adjuvant of the invention has the following advantages over the adjuvants used hitherto:

It is non-antigenic in nature, parenterally tolerated in animals, potentiates the immunogenic effect of both live and inactivated antigens better than previously known adjuvants, is easily absorbable and does not leave residues and sequelae at the injection site.

The basic component is water-soluble split bovine collagen with a molecular weight of 30,000 to 40,000. At concentrations up to 10% by weight, it is liquid at temperatures up to -10 ° C. Antigenic particles bind relatively tightly to numerous binding sites. The second component is plant saponin.

The mixture of these components may be sterilized by boiling, autoclaving, radiation or, if necessary, addition of antiseptics. The mixture is prepared in distilled water, saline or buffered saline, optionally directly in the antigen solution.

The adjuvant with antigens slowly decomposes in the body and thus ensures a long-term adjuvant effect. It elicits an immune response that is equal to or higher than the oil adjuvant vaccines, without the risk of side reactions that sometimes occur after the oil adjuvant, and higher than the aluminum hydroxide adjuvant, after which aluminum residues and tissue damage remain.

The titers documenting efficacy are given in the examples.

Preferably, the appropriate ratio of collagen to saponin component utilizes the adjuvant effect in a complex action as a result of the depot and immunostimulatory effect.

The following examples illustrate but do not limit the invention.

Example 1

Preparation of water-soluble collagen kg of native bovine sausage collagen (Cutisin Oilemnice) was placed in a 50-liter flask, 10 liters of distilled water were added and dissolved at room temperature with stirring. The pH was monitored for 2 hours and 20% w / w added. sodium hydroxide to 7.0. After the pH had stabilized, the temperature was raised to 37 ° C and 1 g of trypsin (Bíoveta Terezín) was added. The vessel temperature was maintained at 37 ° C with stirring until all solids of collagen had disappeared (12 h). The temperature was then raised to 80 ° C for 20 minutes. thereby inactivating trypsin and the collagen cracking process stopped. After cooling down

The CS 272 935 B1 solution was filtered, filled into working vessels and collagen content determined. A solution containing 7 wt. collagen was adjusted to 6 wt. solution, which was used for further processing.

Example 2

Preparation of a liquid inactivated vaccine against infectious bovine rhinotracheitis

10,000 ml of a suspension of IBR strains were prepared at a concentration of 10 < 10 > / cm < ³ > and mixed with 10,000 ml of 6 wt. of collagen solution prepared according to Example 1. 60 g of soap saponin (i.e. to a final concentration of 0.3% by weight) were added and 2 g of merthiolate (i.e. to a final concentration of 0.01% by weight) were preserved. After homogenization, the prepared vaccine was dispensed into vials. The vaccine dose for skeleton was 5 cm ³ with revaccination at 21 days with the same dose. Verification of the vaccine prepared by VVVel Brno concluded that its administration caused the formation of specific antibodies whose serum concentrations peaked 5 weeks after vaccination. The level of antibody response and its dynamics was adequate for the immune response induced by the administration of an oil adjuvanted vaccine but without adverse side effects.

Tab.č. 1

Adjuvant type Resultant antibody titer

Oil adjuvant 45

Collagen adjuvant with saponin 48

Example 3

Preparation of lyophilized vaccine against infectious bovine rhinotracheitis

The vaccine prepared according to Example 2 and filled into vials was frozen to minus 40 ° C and lyophilized. The vials were sealed under vacuum. Before use, the contents of the vial were made up to the original volume with distilled water. The use of the vaccine was the same as in Example 2, and the results (shown in Table 1) were the same.

Example 4

Preparation of a liquid vaccine against rabies and myxomatosis in rabbits cm ^{3 of a} suitable organ suspension containing inactivated swine fever virus was mixed with 10 cm ^{3 of a} solution containing:

- 3 wt. water-soluble collagen according to Example 1 (0.3 g)

- 0.30 wt. primrose saponin (0.03 g)

- 100 vaccine doses of myxomatosis virus MXT (1.8 cm ³ )

- water up to 10 cm ³ .

This resulted in a 20 cm ³ vaccine in which a collagen concentration of 1.5 wt% and a saponin of 0.15 wt% were used as adjuvant. The dose for one rabbit was 0.2 cm ³ percutaneously. Verification of the vaccine thus prepared was carried out by direct demonstration of efficacy - challenge - against rabbit plague and myxomatosis. All vaccinated rabbits survived experimental infection, all control unvaccinated rabbits died. The result has shown that the adjuvant potentiates the effect of the antigen in the production of specific antibodies against rabbit plague and at the same time against myxomatosis as well as the previously used aluminum hydroxide gel with saponin, without adverse effects of damaged tissue and skin at the injection site.

Example 5

Preparation of vaccine against rota and corona infections of newborn calves

280 cm ^{3 of} inactivated rotavirus RVK 1: 16 were mixed with 280 cm ^{3 of} inactivated ko4

CS 272 935 B1 ronavir with a haemagglutination titre of 128. 200 cm ^{5 of} 6 wt. of collagen according to Example 1 and 1.14 g of chestnut saponin. Furthermore, 100,000 IU of Penicillin potassium salt, 0.1 g of streptomycin sulfate, 3 cm @ ^{3 of a} 1 wt. solution of fungicidin and 2.5 cm ^{3 of} 10 wt. sodium hydroxide solution. The vaccine was filled in 25 cm ³ . One vaccine dose was 2.5 cm ³ . In this case, the adjuvant consisted of 1.5 wt% collagen. and saponin at a concentration of 0.15 wt.

This vaccine was tested on experimental animals compared to an oil adjuvant vaccine with this result.

Adjuvans

Collagen with saponin

Oil

Experiment · Resulting average antibody titers ^c 'antirotavir anti-coronavirus

ELISA HIT

1: 16 1: 128

1: 16 1: 64

1: 16 1: -.64

1: 8 1: '64

The immune response of the collagen adjuvant is equal to or higher than that of the oily adjuvant used hitherto and is free of side effects.

Claims (1)

SUBJECT OF THE INVENTION Viral vaccine adjuvant comprising 1.5 to 4.5 wt. % water-soluble split bovine collagen having a molecular weight of 30,000 to 40,000 and 0.15 to 0.30 wt. plant saponin.