DE3028248A1 - EMULSIFIED PREPARATION OF ANTIGENS FROM BACTEROIDES NODOSUS AND ALUMINUM ADJUVANS AND THEIR USE AS A VACCINE - Google Patents

Description

DR. BERG DIPL.-ING. STAPF DIPL.-ING. SCHWABE DR. DR. SANDMAIR

PATENTANWÄLTE PO Box 860245 8000 Munich 86 3028248

Lawyer file: 31 036 25. Ju //

THE WELLCOME FOUNDATION LTD.

18 3-19 3 EUSTON ROAD LONDON N.W.l / UK

Emulsified antigen preparation from Bacteroides nodosus and aluminum adjuvant and its use as a vaccine

P (089) 988272 Telegrams: « AAAAA * λ μ α Λ Bank accounts: Hypo-Bank Munich 4410122850

988273 BERGSTAPFPATElWMuMhM '' 'ö / U / ββ (BLZ 700200 U) Swift Code: HYPO DE MM

988274 TELEX: Bayen Vereinsbank Munich 453100 (BLZ 70020270) 983310 0524560BERGd 'Postscheck Munich 65343-808 (BLZ 70010080)

Description

The invention relates to antigen preparations, vaccines obtained therefrom, their production and their use for prevention and treatment of soft leg (foot rot), particularly in sheep.

Mold leg in sheep is a widespread contagious disease that affects the epidermal tissues of the foot and due to the synergistic effect of two gram-negative anaerobic bacteria, namely Bacteroides nodosus (Fusiformis nodosus) and Fusobacterium necrophorum (Fusiformis necrophorum) will. The disease only occurs when these two organisms are present.

F. necrophorum is usually found in the digestive tract and is excreted with the faeces, so that the organism is usually in the immediate vicinity of the sheep's feet is present and can participate in the infection. B.nodosus, on the other hand, is unable to function under normal Conditions to survive outside of the deciduous limb lesions for more than a few days. The infected foot is his only one Natural habitat and B. nodosus is consequently often described as the specific cause of the disease. The elimination of B. nodosus in a flock of sheep by healing all existing limp cases or by the specific Destruction of the organism could be achieved,

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would lead to the eradication of the disease as F. necrophorum in the absence of B. nodosus does not cause the mold limb can.

In the past, the mustard leg was controlled by isolating the diseased animals and treating them kept what extensive peeling of the affected foot surfaces and external use of disinfectants or external or parenteral administration of suitable antibiotics. More recently made the successful Large culture of B. nodosus vaccination of sheep with vaccines with emulsion or aluminum adjuvant, see e.g. GB-PS 1,375,544. In many cases in which such vaccines were used, however, only an incomplete control was obtained of the disease and intensive efforts have been made to determine the cause of the failure and to improve the effectiveness of existing vaccines.

It has now been found that an antigen preparation according to the invention allows a higher antibody titer and better disease control than those previously used B. nodosus vaccine. The invention therefore relates to an antigen preparation from a 2-phase water-in-oil emulsion, those in the aqueous phase of the emulsion from Bacteroides nodosus organisms Contains antigenic material obtained or made therefrom and an aluminum adjuvant.

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The antigenic material of the preparation can be immunogenic Contain extract of the organisms, but preferably it contains the whole killed organisms in the form of an anaculture (whole cultures treated with formalin) or cells obtained from the culture. It can be any immunogenic strain or Variant or any combination of a number of strains and / or variants that are common to one or more B.nodosus serotypes are representative. In particular, satisfactory protection has been obtained through the use of organisms from fimbrated colonies in which the organisms are heavily piled (cf. J.A. Short et al., J.appl.Bact. 1976, 4Ό, 301-315).

Freshly isolated B. nodosus organisms on solid media are heavily piled up, and liquid culture conditions should be used in practicing the invention be chosen so that the maximum piling is ensured, so as to reduce the effectiveness of the resulting vaccine to promote. The heavily piled B. nodosus organisms described here are understood to mean organisms that per organism have at least 20 PiIi, preferably over 100 PiIi. For maximum effectiveness, the antigen preparations and the vaccines obtained from them should be completely off heavily piled B. nodosus variants exist, but in practice sufficient protection is obtained if 90% of the organisms are heavily piled variants. The heavily piled varian

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te of strain CSIRO No. 19 8 (Wellcome Laboratories Culture No 6476) (registered with American Type Culture Collection on 7/19/19 79 under No. 3145) is particularly valuable. Preferably, protection against homologous infection is provided (i.e. Infection with one or more serotypes that are homologues of the B.nodosus serotype (s) of the preparation), there the protection against heterologous infection is weaker. An infection with organisms that are responsible for more than one B. nodosus Are representative of the serotype, control is therefore preferably carried out with a preparation that contains more than one strain, these all serotypes of the infectious organisms contain.

Those to be used in the preparations according to the invention B.nodosus organisms can be grown by any known method or cultivated, for example with that described in GB-PS 1,375,544; also with the by Short et al. (see above) described methods with which heavily piled organisms are generated.

The aluminum adjuvant naturally promotes the antibody response on the antigen material and can be made from known substances such as potassium alum, aluminum hydroxide and aluminum phosphate to get voted. Particularly preferred are thixotropic β-type aluminum hydroxide gels which are produced in the absence of electrolytes are positively charged. Such a gel is for example "Alhydrogel" (trade name). Preferably contain the

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Preparations 0.25 to 4.0% by volume of the aluminum adjuvant.

The emulsion has a single continuous oil phase and a single dispersed aqueous phase (i.e., it is a 2-phase water-in-oil emulsion), and it can using one or more emulsifying agents and an oil. Of course they have to The emulsifier to be included in the preparations is non-toxic and compatible with the antigenic components of the vaccine be. Preferably they are non-ionic so that they are less likely to be caused by components of the antigen preparation are precipitated as if a cationic or anionic emulsifier is used. If a non-ionic emulsifier is used, the risk of denaturation of the antigen protein is also reduced. The combination, amount and ratio of the emulsifying agents should be chosen so that they are included in the vaccine effect maximum promotion of antigenic activity and stability of the emulsion, and preferably one should lipophilic and a hydrophilic emulsifier may be present.

The lipophilic emulsifying agents are preferably nonionic surfactants with a low level hydrophilic-lipophilic balance (HLB) between 8 and 20 (William C. Griffin, "Calculation of HLB values of non-ionic surfactants, "Journal of the Society of Cosmetic Chemists

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(1954) Vol. 5, pp. 24-9-256; Osipow, Surface Chemistry, ACS Monograph 153, pp. 295-314, Reinhold, 1962). this is the Upper limit of HLB values of emulsifiers that are best suited for inclusion in the oil phase; an optimal one The limit of the HLB values is 2 to 6. Suitable lipophilic emulsifiers include di- and tri-esters of polyvalent ones Alcohols and fatty acids, oxidized fatty oils, and partial esters of common fatty acids, e.g. palmitic, Lauric, stearic and oleic acids with hexitol anhydrides, which are obtained from sorbitol or mannitol. Specific Emulsifiers are e.g. mannitan and sorbitan fatty acid esters such as mannide monooleate (Arlacel A, HLB 4.3), sorbitan monooleate (Arlacel 80, HLB 4.3), sorbitan monopalmitate (Arlacel 40, HLB 6.7), sorbitan monostearate (Arlacel 60, HLB 4.7), and sorbitan sesquioleate (Arlacel C, HLB 3.7).

The hydrophilic emulsifying agents to be used in the preparations should preferably have an HLB value between about 9 and 20, optimally between 11 and 18. Suitable Substance groups are polyglycol fatty acid esters, modified polyoxyethylene fatty acid esters, polyoxyethylene polyol fatty acid esters, Polyoxypropylene fatty alcohol ethers and polypropylene glycol fatty acid esters. A particularly useful group of substances are polyoxyalkylene derivatives of polyhydric alcohol fatty acid esters, like the polyoxyethylene derivatives of partial Esters of lauric, palmitic, stearic and oleic acids

with hexitol anhydrides. Examples of emulsifiers are:

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Polyoxyethylene (20) sorbitan monolaurate (Tween 20, HLB 16.7), Polyoxyethylene (20) sorbitan monopalmitate (Tween 40, HLB 15.6), Polyoxyethylene (20) sorbitan monostearate (Tween 60, HLB 1H.9), Polyoxyethylene (20) sorbitan monooleate (Tween 80, HLB 15), polyoxyethylene (20) sorbitan trioleate (Tween 85, HLB 11.0) and Polyoxyethylene (8) laureate (G 2127, HLB 12.8).

In the preparations according to the invention, any non-toxic Vegetable or mineral oil that is compatible with the antigen material can be used. Pharmaceutical mineral oils Quality are particularly preferred as they are generally a much more stable emulsion and less tissue irritation produce. Light liquid paraffin oils (Bayol F and Drakeol No 6R) gave satisfactory emulsions and formulations.

The liquid of the aqueous phase can be obtained from the culture medium or when the cells are extracted or from the culture consist of aqueous saline or water.

The preparations according to the invention can be used with any known method for the production of emulsions of biological materials be prepared, e.g. by vigorously stirring an aqueous antigen / aluminum adjuvant mixture with the Oil and the emulsifying agent (s). The aqueous antigen / aluminum adjuvant mixture is prepared by adding the

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B. nodosus organisms e.g. inactivated with formalin, the culture or anaculture by one of the known methods to about 30% but not less than 10% of the volume of the original The culture is concentrated and the aluminum adjuvant is added to the aqueous antigen. The aluminum adjuvant can after can be added to the concentration, but an aluminum adjuvant can also be used for the concentration process itself will. An example of the former method is precipitation at the isoelectric point, an example of that second method is co-precipitation with a flocculant.

Precipitation at the isoelectric point is special for Antigenic material that contains heavily fungal B. nodosus organisms is suitable. In this process, the pH of the culture or anaculture lowered to about the isoelectric point of the cells by the addition of acid, although also a lower pH can be used. Suitable acids can be selected from mineral acids such as hydrochloric acid, phosphoric acid and, in particular, sulfuric acid, and strong organic acids such as acetic or lactic acid can be selected. The pH is suitably below 6.0, preferably below 5.6, best between 4.8 and 5.2. At a pH below 4.8, the precipitation should be complete, depending on the antigen material used, however, can damage the antigen. After lowering the pH, the temperature should the culture or anaculture can be lowered to 4 to 12 C.

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The co-precipitation using a flocculant can be achieved by adding a sterile solution of the flocculant to the anaculture and adjusting the pH e.g. Adjusts the alkali so that an optimal precipitation is obtained. After settling in the cold (about 2 days or more) the clear supernatant fluid is removed, creating a concentration of cells and proteinaceous material (including PiIi) is effected in anaculture. Even though if some flocculant is removed with the supernatant, the amount used for the precipitation is preferably too limit, because the larger it is, the more of it appears in the precipitate (cell concentrate) and the more irritation is produced by the later vaccine. Suitable flocculants include sulphates, chlorides and insoluble hydroxides with polyvalent cations, as well as polyanionic polymers such as Sodium carboxymethyl cellulose and hydroxypropyl methyl cellulose. Potash alum is particularly suitable for the present invention, since this can also perform the function of the aluminum adjuvant. It has been found that with the addition of 1 volume 10% w / v. Potash alum to 7 volumes of anaculture (1.25% w / v total alum) and when the pH is adjusted to 5.4 a precipitate with suitable properties and a clear supernatant result Develop fluid. The higher the pH (e.g. 5.8, 6, H), the longer it takes for the supernatant to clear completely. The lower the alum concentration, the longer the clarification time. The mass of Precipitat also depends on the

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Amount of alum added to the system; so the addition of 1 volume results in 10% w / v. Alum to 11 volumes of anaculture (0.83% Alum w / v overall) a barely sufficient Precipitat with an uncomfortably long precipitation period, while the Addition of 1 volume of alum to 4 volumes of anaculture (2.0% alum w / v) results in a very extensive precipitate, which does not settle sufficiently well without centrifugation to be useful for cell production. In a similar way the purified aluminum hydroxide substance "Alhydrogel" (trade name) remains when added to B.nodosus Anacultures are long in the dispersed phase, allowing the recovery of absorbed cells / protein by centrifugation must be done.

Another method of concentrating the culture, or anaculture In addition to centrifugation, there is also ultra-filtration, and either of these two methods can be used together with precipitation at the isoelectric point or co-precipitation will. If the antigen material contains more than one B.nodosus serotype, an aqueous antigen / Aluminum adjuvant mixture prepared as described above for each strain or each variant, then before the Emulsify approximately equal amounts of each mixture mixed together. The emulsion is preferably prepared by the oil with the lipophilic emulsifier and the aqueous antigen / aluminum component with the hydrophilic emulsifier

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mixes and then combines oil and aqueous phases. The aqueous phase is preferably closed slowly and with stirring added to the oil phase in order to keep any tendency towards the formation of an oil-in-water emulsion as small as possible. Finer ones Dispersions of the aqueous phase are obtained by homogenization or grinding in a colloid mill; This method can be carried out until there is no further reduction in particle size.

The optimal ratio of the oil to the water phase in a vaccine depends on a number of factors, e.g. the concentration of the antigen material in the aqueous phase, the desired viscosity of the vaccine, as well as the antigen effectiveness of the antigenic material. Preferably the aqueous phase makes up no more than 60% and no less than 10% of the total Total volume of the emulsion. A larger proportion of water is because of increased viscosity and instability of the emulsion undesirable, while a proportion of less than 10% the incorporation a sufficient amount of antigen is difficult without the need for unacceptably large dose volumes. It was found, that satisfactory vaccines are obtained when the aqueous phase makes up 20 to 50% by volume of the total vaccine. If a large proportion of water, e.g. over 40%, is required, precautions must be taken to prevent the formation of an oil-in-water emulsion and the instability of the emulsion. It has been found that such emulsions, the polyoxyethylene

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Contain (20) sorbitan trioleate by adding falba can be stabilized. It is this a stabilizer that is made from beeswax and paraffin oil of various viscosities Contains lanolin extracted oxycholesterols.

The amount of emulsifier required in each phase depends on a number of factors, in particular on the HLB values the emulsifier and the chosen oil; it is estimated that 2.5 to 15% by volume of a lipophilic emulsifier will result and 0.5 to 10.0% by volume of a hydrophilic emulsifying agent in the respective phases. Vaccines having the properties described here. A considerable increase in the antigenic activity of the antigens was obtained when the respective emulsifiers Represent 5 to 12% by volume of the oil phase and 0.2 to 7% by volume of the aqueous phase.

The preparations according to the invention can be used for administration Appropriate vaccine can be prepared by taking the preparations in an appropriate concentration as sterile Includes preparation in a container.

It is necessary to sterilize the preparation after emulsifying or to sterilize the individual components first and then emulsify under sterile conditions. The preparations according to the invention or their components can with the help of heat and filtration, or with gamma radiation

treatment is sterilized without loss of effectiveness or stability will. - / 18

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The finished vaccine is filled into sterile containers and sealed. It is desirable that the vaccine have a bacteriostat as commonly used in killed bacteria vaccines. For this purpose, in the aqueous phase of the preparation 0.01% (w / v) thiomersal (sodium ethyl mercurithiosalicylate) can be absorbed.

Appropriately, the finished vaccine preparation contains a total of 2.5x10 to IxIO ¹¹ , preferably IxIO ⁸ to 1. 25xl0 ⁹ killed organisms per ml of the preparation.

The vaccines of the invention are best administered to animals by subcutaneous, intramuscular, or intraperitoneal injection administered. It is advantageous to give the vaccine to a site where there is any local reaction to the breeder is harmless, e.g. on the anterior dorsal region of the neck or on the lower jaw. The most preferred dosage

8 11 range is between 1x10 and 1x10 killed organ-

8 10

nisms per dose, in particular between 1x10 and 1x10, where the dose is the total amount of antigenic material present in an animal such as a sheep at one time in an appropriate Amount of liquid, e.g. 1 to 2 ml, is administered. The vaccine dose can be given to an animal as a single dose or as a A variety of divided doses can be given, the latter over a period of time or by simultaneous Injections can be given at different sites.

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A suitable immunization schedule may include a single injection of the full dose or two injections of which each containing a partial dose, and which are administered simultaneously to different parts of the animal's body. The optimal one Dosing schedule will of course vary depending on the for that Based on the vaccine selected strains of organisms, the total number of organisms used, the type of adjuvant and the route of administration. It turned out to be particularly beneficial to have a vaccine containing 3x10 organisms / ml in a single one 1 ml or 2 ml dose to be administered subcutaneously and a second injection after at least 6, preferably 12 weeks to follow the same volume, the same strength and on the same path. Booster doses should be taken immediately before the expected seasonal spread of the mustard limb.

In addition to the antigen material obtained from B.nodosus, a vaccine according to the invention can also contain antigen material, that was obtained from other bacteria, the disease-causing one Organisms e.g. in sheep, so in particular from F. necrophorum and Clostridia. A particularly preferred combination is a multicomponent vaccine that is a preparation one or more Clostridia antigens as described in GB-PS 1 143 545 together with one as described herein manufactured B.nodosus antigen preparation contains.

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The invention thus relates to

a) an antxgen preparation which is a 2-phase water-in-oil emulsion contains, in the antigenic material obtained from or consisting of Bacteroides nodosus organisms as well as an aluminum adjuvant in the aqueous phase of the Emulsion are included.

b) A process for the production of an antxgene preparation according to a), characterized in that an aqueous medium, which is derived from or consisting of B. nodosus organisms Contains antigen material and an aluminum adjuvant, is emulsified with an oil.

c) A vaccine, characterized in that it contains a sterile preparation of a preparation defined under a).

d) Method for the prophylaxis or treatment of mildew infection in sheep, characterized in that the sheep are given an effective, non-toxic dose of a vaccine is administered according to c) above.

The following examples are intended to explain the invention in more detail.

Reference Example 1 Growth of Organisms

B.nodosus (strain CSIRO 19 8) organisms from sown cultures were inocculated into the medium (cf. Thorley, CM. (1976)) J.Appl.Bact. 40, 301-309, page 302), which is in bottles with

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Screw cap was included and the air was displaced by a mixture of 10% carbon dioxide and 90% nitrogen. ^{The cultures were kept at 37 °} C. for 20 to 40 hours, and the organisms were killed by adding formalin (0.5% by weight). Examination of the organisms under the electron microscope showed that at least 90% piled heavily (> 100 PiIi per organism).

Example 2
Antigen preparation

3200 ml of a formalin-treated anaculture of heavily piled B. nodosus organisms, which ge as described in Example 1 was treated with 320 ml of an aqueous solution (10% w / v) of potassium alum, and the pH of the mixture was adjusted to 6.2 by adding aqueous sodium hydroxide (40% w / v). After the mixture has been at for 24 hours Maintained 4 C, the supernatant over the precipitated organisms was removed until the volume was up 1200 ml was reduced.

30 ml Tween 80 (40% by volume aqueous solution) and 1.2 ml thiomersal (10% aqueous solution) were added and the mixture with 2.8 l of a mixture of Arlacel A (10% by volume) and Marcol (90 vol.%) Emulsified.

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Examples 3 to 6 Antigen Preparations

Heavily piled B. nodosus organisms were collected by precipitation at the isoelectric point by treating the anacultures with sufficient dilute aqueous sulfuric acid to lower the pH to 5.0. After standing for 3 days at 16 to 20 ^° C., the organisms were concentrated 9 to 10 times by pouring off 133.7 l of supernatant. The organisms were used in the preparation of the vaccine formulations of Examples 3-6, Table I.

In each case the concentrated anaculture (column 3) was diluted with aqueous sodium chloride solution and the aqueous mixture Thimerosal (column 7) was added to achieve a concentration of 0.01% before emulsification with the oil (column 8) Weight / volume to obtain. In Examples 4 to 6, the pH was adjusted after the addition of the hydrophilic emulsifier.

Example 7
Antigen preparation

With the method described in Example 2, another preparation was made using heavily piled B.nodosus organisms (obtained as in Example 1). For more details see Table I.

1 30008/0789

LO 4th O O O 5 00 O 6th 00 7th (D

Table I.

Column 1 Column 2 Column 3 Column 4 Column 5 Column 6 Column 7 Column 8

Ex. Anaculture Concentration Dilution Addition pH Hydrophi- Aqueous "Oil" -Vol. No. (volume, tration m. Aqueous of set les Emul- total volume (1) ml) (-fold) Sodium Chloride Aluminum Lumen (I)

Ride solution umadjuvans (0.9% w / v)

3,200

200 300 500

6 1

10 700 ml Potash alum
(154 ml;
10%
Weight / volume) 6.1 10 644 ml AH
(210 ml) 6.1 9.2 1.7 1 AH
(500 ml) 6.1 9.2 1.5 1 AH
(500 ml) 6.1 3 Potash alum
(1.2 1;
10%
Weight / volume) 6.2

T80 1.054 2,459 (13.3 ml) T80 1.054 2,459 (13.3 ml) T80 2.5 2.5 (62.5 ml) T80 2.5 2.5 (62.5 ml) T80 3.078 7.0 (75 ml)

Notes: T80 sorbitan monooleate ("Tween 80") solution (40% by volume) AH: Thixotropic 3-type aluminum hydroxide gel ("Alhydrogel")

"Oil": This contains a mixture of mannide monooleate ("Arlacel A") ,

(10 vol.%) And "Marcol" (90 vol.%) ^

Example 8 Vaccines

The preparations described in Examples 3 to 7 can be filled into sterile 1, 2 or 10 ml ampoules and sealed and sterilized with gamma irradiation.

Example 9 Effectiveness

In this experiment 4 groups of 5 sheep each were subcutaneously on the lower jaw with 2 ml vaccine prepared as in Example 2 (vaccine A) and 3 other vaccines (B, C and D) vaccinated. Six weeks later, all sheep were given a second dose of vaccine, the same as the first in all respects was the same, and five sheep from each of the vaccine groups were infected by using a live culture of the vaccine strain was applied directly to each (predisposed) foot. Each group of 5 sheep was then taken monthly So that the last infection took place 12 weeks after the 2nd vaccination. 3 weeks after each infection the feet were examined for the clinical picture of the limp. A result of 1 or more with an undercutted mold leg affected feet or severe inter-toe dermatitis on two or more feet of 2 or more of the 5 animals was classified as a failure of the vaccine due to insufficient protection, and this vaccine has not been tested any further.

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The 4 experimental vaccines A, B, C and D were run in parallel tested in comparison with a non-vaccinated control group of sheep to check the virulence of the infection culture. 5 groups of 4x5 sheep were used. Blood samples were taken at 0 and 6 weeks and at the time of the foot examination taken. Agglutination titers were determined and geometric mean titers found. The results are shown in Table II.

The piled alum / oil vaccine A according to the invention was accordingly the only one who gave protection to sheep during the fourth infection. 2 weeks after the foot examination they were Foot damage in the one sheep in this vaccine group that infected had healed, while the disease was as severe as before in the 5 control sheep.

There was also a clear difference between the serum agglutination titers from sheep immunized with vaccine A and those who received other vaccines, established.

Infection 2 was unusable as 3 control sheep did not become ill. Infection 4 was very severe, the control sheep were on a preliminary examination 10 days after infection seriously infected.

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Table II

Infection 1

Infection 2

Infection 3

Infection 4

vaccine

Infested Infested Infested Infested Sheep Titer Sheep Titer Sheep Titer Sheep Titer

A.
Piliert
(with alum /
Oil adjuvant) 0/5 59 020 0/5 B.
Not piled
(with alum /
Oil adjuvant) 0/5 33 630 0/5 I 300Oi C.
Piliert
(Oil adj
vans) 0/5 11 880 1/5 Ϊ / 0789 D.
Not piled
(with oil ad-
j uvans) 0/5 11 890 1/5 Not-
vaccinated
animals 5/5 80 2/5

124 ooo

17 800

8 900

7 759

0/5 40 960 1/5 13 510

2/5 3 880 not tested

1/5 13 490 4/6

560

1/5 1 940 6/6 320
4/5 80 5/5 160

K) OO K)

This experiment has shown for the first time the possibility to vaccinate sheep against dead limbs for a period of Protect 12 weeks after administration of the last dose.

Vaccines B, C, and D were identical to Vaccine A with the following exceptions:

Vaccine B: The organisms were not virulent and genetically not fungal, ie they did not have any bacterium
more than 20 piIi.

Vaccine C: The aluminum adjuvant was omitted.

Vaccine D: The aluminum adjuvant was dropped and the organisms were not fungal as in vaccine B.

Example 10

Effectiveness of the vaccines

In this experiment, vaccines from the preparations described in Examples 3 to 7 were compared with vaccines containing only alum as an adjuvant (Vaccines F and G). Fodder-free sheep were treated with either a single dose of a vaccine of Examples 3 to 7 or 2 doses of vaccines F or G every 6 weeks. At certain intervals after vaccination, groups of the vaccinated sheep were given non-vaccinated controls with regard to their resistance to infection with artificial infections
a B.nodosus live culture according to Example 9 compared. The results are given in Table III.

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3 vaccine
dose Table III after vaccination
Sheep) Weeks 4th (ml) infection
(infected 12 weeks 15 _ 5 1 8 weeks 0/4 - 6th 1 1/5 0/5 0/5 vaccine 7th 2 0/5 - 0/5 Ex. 7th 1 - - - Ex. 1 0/5 1/5 - Ex. 2 0/5 0/5 - Ex. Controls 2x2 U / H - - Ex. 2x2 4/5 - 4/5 Ex. 0 4/5 5/5 F. 4/5 G

End of description

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Claims (1)

Claims 1. Antigen preparation containing an emulsion in which Bacteroides nodosus organisms obtained or consisting thereof Antigenic material is contained, thereby characterized in that the emulsion is a 2-phase water-in-oil emulsion containing the antigenic material and an aluminum adjuvant in the aqueous phase of the emulsion contains. 2. Preparation according to claim 1, characterized in that the B.nodosus organisms, as described, are heavily piled. 3. Preparation according to one of the preceding claims, characterized in that the B.nodosus organisms contain the heavily piled variant of the strain registered with ATCC under No. 314-5. U. Preparation according to one of the preceding claims, characterized in that the aluminum adjuvant is selected from potassium alum, aluminum hydroxide or aluminum phosphate. 130008/0789 P (089) 988272 Telegrams: Bank accounts: Hype-Bank Munich 4410122850 988274 TELEX: Bayet Vereinsbank Munich 453100 (bank code 70020270) 3310 05 24 560 BERG d Postscheck Munich 653 43-808 (bank code 700 100 80) 5. Preparation according to one of the preceding claims, characterized in that the aluminum adjuvant is selected from potassium alum and thixotropic β-type aluminum hydroxide gels. 6. Preparation according to one of the preceding claims, characterized in that the emulsion contains a non-ionic emulsifier. 7. Preparation according to one of the preceding claims, characterized in that the emulsion contains a lipophilic emulsifier. 8. Preparation according to claim 7, characterized in that the lipophilic emulsifier is a Is mannitan or sorbitan fatty acid ester. 9. Preparation according to claim 7 or 8, characterized that the lipophilic emulsifier is mannide monooleate. 10. Preparation according to one of claims 7 to 9, characterized in that the lipophilic Emulsifier with 5 to 12% by volume of the oil phase of the emulsion is present. 130008/0789 11. Preparation according to one of claims 1 to 6, characterized in that the emulsion contains a hydrophilic emulsifier. 12. Preparation according to claim 11, characterized in that the hydrophilic emulsifier is a Is a polyoxyalkylene derivative of a polyhydric alcohol fatty acid ester. 13. Preparation according to claim 11 or 12, characterized in that the hydrophilic emulsifier Is polyoxyethylene (20) sorbitan monooleate. 14. Preparation according to one of claims 11 to 13, characterized in that the hydrophilic Emulsifier is present at 0.2 to 7% by volume of the aqueous phase of the emulsion. 15. Preparation according to one of claims 1 to 14, characterized in that the oil phase the emulsion contains a mineral oil. 16. Preparation according to claim 15, characterized in that the oil is a liquid paraffin oil is. 130008/0789 -H- 17. Preparation according to one of claims 1 to 16, characterized in that the aqueous Phase with 10 to 60 vol.% Of the emulsion is present. 18. Preparation according to one of claims 1 to 17, characterized in that the aqueous Phase with 20 to 50% by volume of the preparation is present. 19. Preparation according to one of claims 1 to 18, there characterized in that the anti 7 11 genetic material with 2.5x10 to 1x10 B.] per ml of the preparation is available. 7 11 genetic material with 2.5x10 to 1x10 B.nodosus organisms 20. Vaccine with a sterile preparation of an antigen preparation, which contains an emulsion in that obtained from or consisting of Bacteroides nodosus organisms Antigen material is contained, characterized in that the emulsion is a 2-phase water-in-oil emulsion which contains the antigen material and an aluminum adjuvant in the aqueous phase of the emulsion. 21. Vaccine according to claim 20, characterized in that the aqueous phase of the emulsion includes a bacteriostat. 22. Process for the production of an antigen preparation according to one of claims 1 to 19, in which the B.nodosus- 130008/0789 Antigenic material obtained from or consisting of organisms dispersed in an aqueous medium, and the aqueous medium is emulsified with an oil, characterized that an aluminum adjuvant is present in this aqueous medium. 23. Vaccine according to claim 20 or 21 for the prophylaxis or treatment of a dead leg infection in a sheep. - / 6 130008/0789