DE2134930A1 - Antigenic extracts - Google Patents

Description

DR. BERG DIPL.-ING. STAPF

PATENTANWÄLTE 8 MÜNCHEN 8O, MAUERKIRCHERSTR. 45

Dr. Berg Dipl.-Ing. Stopf, 8 Munich 80, Mauerkircherstraße 45

Your mark

Your writing Our sign

date

July 13, 1971

Attorney file 21 248
Be / Ro

l'he Wellcome Foundation Limited London (Great Britain)

"Antigenic Extracts"

The present invention relates to immunogenic products and yakzins for protection against infections with Bruceila Abortus, and methods of making such products and vaccines.

A354

-2-

1 09884/1903

213A930

So far vMe attempts have been made, satisfactory To provide vaccines to protect humans and animals, especially cattle, against brucellosis »In practice cattle are usually immunized with a vaccine prepared from a suspension of a live attenuated B. Abortus originates, preferably of the well-known strain S.19. The live vaccine provokes the formation of serum antibodies in vaccinated animals. This is a serious disadvantage when vaccination is combined with elimination of sick! animals should be carried out because the animals infected with the carrier are not easily affected by those with the attenuated strain distinguished 19 vaccinated animals can be.

A vaccine can also be obtained from a suspension of killed whole cells, for example of the Brucella abortus strain 45/20, emulsified in a mineral oil. This type of vaccine has been approved by a number of governments. It is less prone to undesirable formation than the S.19 strain To evoke serum antibodies, however, the administration of the killed vaccine in an oily emulsion the undesirable side effect of causing very permanent local damage. This can be considerable cause physical discomfort and pain. In addition to these consequences, the affected parts must be discussed, or removed when the animals are slaughtered

~ 3—

109884/1903

resulting in a loss of meat. Bs can do this be a serious disadvantage if the animals are used for meat

be held.

Ii -tGX'üon and other (British Journal of Experimental Pathology, 1947, 28, 223-236) have antigenic substance isolated by Zentrifug'ieren of phenol-saline suspensions of B. abortus strain 544, grown on Leberagar, Typically the product was found to be high in lipids, carbohydrates and formyl. It also has very high toxicity and causes protracted local edema at the vaccination site even when given in small doses. About 20 years later, Ellwood and others (British Journal of Experimental Pathology, 1967, 48, 28-39) examined extracts of B. Abortus obtained with sodium dodecyl sulfate (NDS) and isolated immunogenic fractions having a lipoid content of 35-42 $, 10-12 $ carbohydrate (with an additional 12-15% amino sugar), about 5% formic acid and only 10 fo protein. Lysine was predominant in the amino acid portion of these products. These products caused (similar to the earlier Paterson preparations) hypersensitivity in animals and led to the formation of serum antibodies with high titer. These properties cannot be accepted in the case of substances intended for vaccines in domestic animals, regardless of their ability to protect against infection.

-4-

109884/1903

213A93Q

It has now been found that a material is obtained from strains of Bo Abortus (and from other Bruceila sp ")
that provides sufficient protection against such forms of brucellosis. The material has a high protein content, but is essentially free of lipopolysaccharides and formyl groups. If it is administered subcutaneously in suitable formulations, it does not cause any local reactions and it does not have to be stored in highly irritating or pain-producing oily adjuvants in order to form sufficient protection, although less irritating aluminum adjuvants can of course be used without such disadvantages » It is also non-agglutinogenic and does not provoke the formation of serum antibodies that make the plague
interfere with examinations provided by infection.

It accordingly provides the present invention in one respect a process for the production of an immunogenic, nonagglutinogenic, protein-containing material or fraction of a species of the Bruceila group, in particular of B. Abortus,
available, for which the living bacterial cells are extracted in aqueous suspension with a surface-active agent (surfactant), a water-soluble organic solvent, preferably ethanol, is added as a protein precipitant, and the protein-containing agent obtained in this way. en precipitate of others initially present in the suspension

Materials. Advantageously, together the

bacterial

-5-109384 / 1903

213493Q

rush fragments and the precipitated proteinaceous Fraction first, for example by means of centrifugation, isolated from the suspension and then by redissolving the protein-containing fraction in a suitable solvent, like a buffered saline solution, are separated »Me bacterial fragments and the others remaining as residue Insoluble materials can then be removed in a suitable manner such as by centrifugation.

Any suitable strain of Bruceila species can be used in the manufacture of the immunogenic material in accordance with the present invention can be used, especially the non-agglutinating strains. In the case of B. Abortus, the strain is 45/20 in rough form a suitable source; however, other strains with higher agglutination properties can also be used because obviously the agglutinogen would not appear in the extracts, but such sources are unmistakable are less preferred "

The "surfactants" useful for the purposes of the present invention can be any of a wide variety of known agents of the non-ionic or ionic type containing a hydrocarbon chain of, for example, 4-20, preferably 8-16 carbon atoms can be selected. Anionic surfactants such as long chain alkyl benzene sulfonate salts proved to be suitable, but the

-6-

109884/1903

- ^β - 213493Q

Alkali metal salts of dodecyl sulfate more readily available stand and be preferred.

The cultivation and isolation of the living cells can be carried out in the usual manner. For best results, adjust the concentration of the suspension to approximately 8-10 cells / ml water or exhausted medium and add a suitable surface-active agent, suitably sodium dodecyl sulfate (HDS), to the extraction of the immunogenic materials from the bacteria to effect. The concentration of the agent is preferably below 10% w / v, suitably in the case of HDS at about 2.5% w / v, but with smaller amounts in the range of about or below 1 fo w / v . up to $ 0.5> w / v or even up to 0.1 ^a / o w / v. can work satisfactorily. The suspension is left to stand until the extraction is complete. It has been found that approximately 17 to 24 hours is sufficient to achieve reasonable results at tree heat (20 C.), although the optimal time may vary with the type and concentration of surfactant and temperature. Satisfactory extraction at 52 ° C using a concentration of 4% w / v can therefore be obtained. HDS and at 37 ⁰ C at a concentration of 2.5 i ° wt / vol. HDS can be achieved.

According to the present method, the mixture of the immunogenic fractions and contaminating other materials

-7-

108884/1903

21 3A930

by. Treatment with a water-miscible, organic
Solvent precipitated. These include, for example, the lower alkanols to propanol, preferably ethanol
or acetone or similar solvents, such as dioxane, Celloso lve and carbidols, which contain a fraction of enriched,
protein-containing, non-agglutinogenic, immunogenic material. For example, it was found that the addition
of approximately the same volume of ethanol to the extract is sufficient to obtain such a fraction. Precipitation can be followed by centrifugation to facilitate precipitation and aid in the separation of the solid material from the aqueous phase.

If the precipitate immediately follows the extraction, other components of the suspension are also removed by the subsequent centrifugation. The solid phase can then
further, for example by redissolving the protein-containing, non-agglutinating component in an aqueous buffer and by removing the insoluble, bacterial debris by means of centrifugation at low speed. To well-known ones suitable for such purposes
Buffers include, for example, phosphate saline and succinate buffer solution

Chemical analysis of the typical, purified, non-agglutinogenic Fractions obtained according to the present invention and made available suggests that

-8th-

109884/1903

that about half of the components are carbohydrates, since they only provide glucose residues on hydrolysis without amino sugars by normal methods. A little more than half; desKohlehydrätanteils "was not dialyzable The protein content bet-rug at least about 15» usually about 20, a maximum to about 25 parts by weight S ^;.. based on the dry weight Approximately 80 fo the dry weight of Haterials' sedimented as the sole component (sedimentation constant Spo ' ^w = 1 »55) in the ultracentrifuge, from which it can be concluded that the carbohydrate and protein are linked in some way to form at least one loose complex.The non-dialyzable lipoid content was below 5, usually around 1.7 G-ew., -%.

The extent or absence of agglutinogenic activity can suitably be checked by the method described in the "WH ₀ O. Technical Report Series" 86 (1954), page IJ or in the addition to the "Veterinary Record", 1967, (" Interpretation of the B-. Abortus- serum application test ") is described. The level of such effectiveness can be expressed as serum agglutination titer (SAT) in international units. Although it was found that the SAT value in animal experiments after vaccination with a preparation containing immunogenic material produced according to the invention is almost 0, would any value for the SAT below, for example, 10 or preferably b, still ala essentially not ag ^ 'lutinogon and im

109884/1903 ~ ⁹ "

213493Q

- Q -

To be considered sufficient within the scope of the present invention because values above 10 or even around 20 or 30, normally in the case of killed vaccines that are officially approved for this purpose will be accepted as relatively satisfactory · The favorable properties of the vaccine according to the present invention Invention were also due to the lack of complement binding antibodies and the actual or almost complete absence confirmed by incomplete antibodies.

The immunogenicity of this preparation can be tested in mice or guinea pigs, but these experiments may be deceptive and uncharacteristic or unrelated to the immunogenicity in animals to be immunized with the preparation. For example, more reliable results for a preparation obtained from a B _e Abortus strain can be obtained by experiments on pars, for which purpose this artificial vaccination was inseminated and provoked in a suitable manner with a virulent strain of B. Abortus. The number of abortions in the group can be recorded and the presence or absence of infection determined by isolating Brucellae from any tissue or secretion or determined according to the level of agglutinins in the serum after calving «,

-10-

109884/1903

- ίο -

The present invention accordingly provides in one aspect a proteinaceous, non-agglutinogenic material produced by a species of the Bruceila group, which material is immunogenic with respect to infections of virulent Bruceila species and is essentially free of iipopolysaccharides and formyl groups. In a special way this material has additional chemical and biological properties, which are described below.

In another aspect, the invention provides a yakzin that a proteinaceous, non-agglutinogenic material or fraction, as previously defined, contains Advantageously Contains such a vaccine for binders from 2 to 20 mg, preferably about 5 to 20 mg, based on the dry weight, of the material previously described, along with a pharmaceutically acceptable solid or liquid carrier. The solid preparation can be stored in a freeze-dried Form, such as a freeze-dry excipient or a stabilizer, for example dextran or SaooharoBe contains, are presented. A liquid preparation can preferably the immunogenic material in a concentration of 1 τ 5 mg dry weight / ml in a suitable pharmaceutically acceptable buffer, such as a phosphate-buffered saline solution with a pH value in the neutral Area, e.g. pH 7 ^ 5 included. An aluminum adjuvant, for example potash alum, can also be used in the sterile vaccine

-11-

-109804/190 3

- li -

preparation be included _o

It was found that the dosages given above are sufficient to immunize cattle, but that others Suckers, humans or 'animals,' a proportionally corresponding one Need length for protection,

In a further aspect, therefore, the invention provides a method of immunizing mammals against brucellosis, including liian an effective dose of a valczin, as above defined, administered, this protein-containing, non-agglutinating Material contains that the counter-infection is with a corresponding Brueellastamni imniünogeri, the plug in the receiver 'can »- · - ■ - ■ · ..-... ·

Example 1 " ^: '

1 ", The preparation of the immunogenic material " - ''. 'It became the Bruceila Abortus tribe. 45/20, the rough variant, isolated from McEwen, A ₀ D ₀ and Roberts, RST Oomp. Path. Ther. 1936, 49, 97, used. "

2 Behavior and Reconstitution of the Tribes "

The above mentioned strain and the virulent, smooth B ₀ Abortus strain 544, which is used extensively for provocation, were kept in the freeze-dried state or in liquid nitrogen

■■ - ■■■ ■ ".--: ■ ■ · -, .- · -v, _.. ■ '-12-:

109884/1903

BAD ORlGiMAt

they were surface-seeded on solid media and the obtained growth was checked for homogeneity checked on the morphology of the colonies

3. Culture medium

It contained a Papainnährlösung of horse muscle with a G-esamtstickstoffkonzentration from 0.45 fo, yeast extract ( "mannitols") 1 fo, fructose 4 f> $ anhydrous disodium hydrogen phosphate 0.16 fo Gew./Vol. The pH was adjusted to 6.5 and the medium was sterilized by filtering through a Seitz filter pad.

4. Cultivation and collection (isolation)

The 45/20 strain was deep-cultured in the above medium with aeration for 3 days, then isolated and let settle in the cold to a final concentration of approximately 8-10 cells / ml exhausted medium «,

5 » Production of the extracts

Hatriumdodecylsulfat (HDS), the suspension of the Iebenden cells of strain 45/20 was added (10 cells / ml), the concentration of HDS to 2.5 fo Gew./YoI. was discontinued. The suspension was allowed at 20 ° 0 stand for 24 hours after an equal volume had been stirred in 95 $ sodium ethanol in the gelatinous mass, which was then the centrifuged at 2O ⁰ G with a Gravitätsbeschleunigung 1800 2 Scales, wherein the supernatant layer

109884/1903 "" ¹ ^

was discarded. The fiber deposit was in 0.02M Disodium hydrogen phosphate buffered saline solution at pH 7 »5 added to the initial volume, as before centrifuged gently and the residue discarded.

The dry matter content of the solution contained the active ingredient, but no lipopolysaccharide complexes were present because the non-dialysable lipoid content was only 1.7 <? <> . The overall analysis is given in Table 1 »

6. The preparation of a vaccine

The preparation prepared above, which after removal of the residue to a content of at least 5 mg / ml immunogenes Protein was discontinued as sterile yakzin to protect cattle against experimental provocation used with a virulent B. abortion strain. A substantial part of the dry residue consisted of this solution of inorganic materials such as sodium chloride and buffers.

7. Analysis of the extract

Table 1 see page -14-

109884/1903

Table 1

213493Q

percentage Dry weight Existing substances Before dialysis Free amino nitrogen 2.4 Total amino nitrogen 3.9 Amino acids after hydrolysis 54.0 Protein (acc. To Lowry) 15.2 Total lipoid content 11.5 Non-dialyzable lipoid 1.7 Carbohydrate 48.1 Formic acid «· O Aminopimelic acid n.f ο Amino sugar n.f. Pentose n.f. Sedimentation-Sp ₀ .W 1.35 After dialysis • «« 2.3 16.4 24.4 3.2 3.2 49.6 . Sp • · ο • β • · · O O O

Sp = tracks} n “f. = nothing found

57 wt -io carbohydrate which was not dialyzable., Was 48 fo, based on the dry weight. The hydrolysis yielded only glucose; no amino sugars were found. It
therefore seemed unlikely that the carbohydrate was derived from a capsular mucopolysaccharide. But it can be the polysaccharide part of a glucoprotein. The fact that 80 % of the sample precipitated as the only component in the ultracentrifuge suggests that a large part of the carbohydrate and protein detected could be associated in such a complex.

-15-

109884/1903

Only $ 1.7> lipoid left after dialysis. It was therefore unlikely that any major component of the extract was either a lipopolysaccharide or a lipopolysaccharide-protein complex, an assumption already being doubted by the absence of any other than glucose residues after hydrolysis and by the result of ultracentrifugation, which has a sedimentation constant of Sp ₀ » W = 1.35 making up for a 80 ^o ß> of the extract component resulted.

8. Experimental vaccination of cattle

Uncovered heifers, free of brucellosis, were vaccinated subcutaneously (day 0) in the middle of the neck and again on the opposite side 84 days later with doses of 2 ml of the vaccine described in Part 6. The animals were then artificially inseminated in the 3 week period, beginning 14 days after the second vaccination, and on the 197th lay with approx McEwen is isolated and is used extensively to provoke immunity to B. Abortus ^. At this point, heifers were in foal for 124 to 149 days. Agglutination and complement fixation antibodies were determined prior to vaccination and at appropriate times thereafter. Appropriate specimens for Bruoella were obtained when discarded or calved

109884/1902

213A930

by. Cultures and while these turned out to be negative, checked by animal vaccination. A cow was considered infected if the remaining level of agglutinins was higher, than 25 international units longer than 21 days after stopped calving, or if Brucellae were isolated from any tissue or secretion "

9. Controls

Ten uncovered heifers were vaccinated with a tested vaccine, this being an inactivated strain of B. Abortus 45/20 in an oily adjuvant.

The vaccinated animals were then artificially fertilized and tested as in Part 8, another 5 animals received none Yaking, however, were similarly fertilized and tested.

The results are shown in Table 2.

Table 2 see page -17-

109884/1903

Table Immunity achieved in pregnant cows by vaccination with various preparations

! O OO DO

Every
vaccinated
with 2 ml Extent of maximum
local reac
actions (damage)
ο
(cm / animal) Jach de:
2nd vaccination U] agglutination
Maximum titer in I.U. After the 1st
Vaccination After the 2nd
Vaccination Result de: r
Verification number
infi
adorns number
Abortions preparation
according to part 6 After
1st vaccination O Before the vac
financing SAT KP SAT Number in
d.group 2 O Officially ge
approved
killed ad
3 uvant s vaccine
(Part 9) O 60 + 37 ^? SAT
G O O O 5 3 "X
• «4
ι
O Not vaci
ned con
trolls X
60 + 42 - 6 "8 + 8 19 + 12 26 + 77 32 + 15 10 4th 4th - 8 ο4 + 11 O O O 5 I, O

χ Persistent response

SAT = serum agglutination titer Ki ¹ = complement binding titer

ro

CJ j> CO CJ O

It can be seen that none of the animals vaccinated with the vaccines of the present invention developed damage or showed agglutination. There were no abortions or calving. Although part of the animals
infected, there was no evidence of any
serious symptoms.

The killed vaccine provided a similar protection,
but showed the usual extensive local reaction and noticeable agglutination titers. The non-vaccinated controls were almost all infected and showed a very high level
Occurrence of abortions.

It can be concluded from this that the preparation according to the invention, which contains the protein-containing immunogenic fraction of the extract of
B. abortion, actually prevents local reactions and agglutination without any significant loss of immunogenicity and is superior to the best currently available. available vaccine of the killed type proven.

-19- 109884/1903

Claims (1)

Patent claims: 1. A process for the preparation of an immunogenic, non-agglutinogenic, protein-containing material from a species of the Bruceila group, characterized in that the living bacterial cells are extracted in aqueous suspension with a surface-active agent, a water-miscible organic solvent is added to precipitate the extract and the separates protein-containing precipitate from the ί other materials in the suspension « 2. The method according to claim 1, characterized that the species is Brucella Abortus. 3 ο method according to claim 1 characterized that the species is the rough variant of Brucella Abortus strain 45/20 « 4β method according to any one of claims 1 to 3 thereby characterized that the surface-active coat is anionic " 5. The method according to claim 4, characterized in that the anionic surface-active Agent used an alkali metal salt of dodecyl sulfate. -21-109884 / 1903 6. The method according to claim. 5 characterized that the salt is the sodium salt of dodecyl sulfate is ο 7. The method according to any one of claims 1-6, characterized in that the living cells with between 10 and 2.5 i ° Gew./YoI. Surface active agent to be treated. 8 ο method according to claim 7, characterized in that the living cells with 0.1 - 2.5 '/ <> weight weight / volume. Surface active agent treated _a 9 ο method according to any one of claims 4-8 thereby marked that the living cells with treated with an anionic surfactant at room temperature for 17-24 hours » 10. The method according to any one of claims 1-9, characterized in that the water-miscible solvent is a limed alkanol _o Ho method according to claim 10, characterized in that the lower alkanol is ethanol " -22- 109884/1903 213A930 12o method according to any one of the preceding claims, characterized in that the protein-containing Separate precipitate from the rest of the suspension by centrifugation. 13 β method according to any one of claims 1-12 thereby characterized in that the material is filled into a container, whereby a sterile, liquid Receives vaccine preparation with a content of 2 - 20 mg dry weight of the material in the preparation, 14 ο method according to claim 13 characterized that the preparation is an aluminum adjuvant contains. 15. The method according to claim 14, characterized in that the aluminum adjuvant is potassium space _e 16. The method according to claim 14, characterized that the preparation is freeze-dried. -23- 109884/1903 / 17 / Protein-containing, non-agglutinogenic material characterized by a species of the Bruceila group that the material is immunogenic . with regard to infections of virulent Brucella spp., and is essentially free of lipopolysaccharides and formyl groups o 18. Material according to claim 17, characterized in that it is obtained from a strain of Brucella Abortus _o 19. Material according to claim 17, characterized that it is obtained from the rough variant of the Brucella Abortus strain 45/20 » 20. Material in accordance with any one of claims 17 to 19 characterized in that it has a protein content of between 15 and 25f ° and a lipoid • between 1 and 5%, based on the dry weight of the material. 21. Material according to one of claims 17 to 20, characterized in that it has a sedimentation constant S ₂₀ .w = approximately 1.35 in the ultracentrifuge. -24- copy 10388W1903 22. Material according to one of claims 17 "to 21 d a lurch marked that it is in shape a sterile, injectable vaccine preparation is available. 23. Material according to claim 22, characterized that it is in the form of a liquid preparation β 24-0 material according to claim 22, characterized that it is in the form of a freeze-dried preparation " 25. Material according to claim 22, characterized in that there is an aluminum adjuvant in the preparation having" 26 ο Material according to claim 25, characterized in that the ¹ adjuvant is potassium alum. 27. Material according to one of claims 22 to 26, characterized in that it is in a Amount from 2 to 20 mg, based on the dry weight of the material in the preparation. GOPY