DE1154234B - Method of making a staphylococcal vaccine - Google Patents

Description

Method of making a staphylococcal vaccine In recent For 60 years many attempts have been made to produce organic products induce immunization against infection by staphylococci. These products, some of them are technically available and have daunting results in people result. Today the problem of staph infections is even more serious than it is has been for the past 25 years. This is particularly surprising since it has been successful satisfactory immunizers against other bacterial infections such as typhoid diseases, diphtheria, whooping cough, tatanus and pneumonia, protect, were manufactured.

It has now been found that by extracting the intact cells certain strains of Staphylococcus aureus with phenol and an aqueous medium and Separating the phenol and the insoluble solid material from the aqueous solution can obtain an excellent staphylococcal vaccine.

The two extractions can either be carried out at the same time, or, preferably, the phenol extraction is carried out prior to the extraction with an aqueous Medium carried out. The particular strains of Staphylococcus aureus that are used can be used to produce the vaccines according to the invention are UC76, Smith, S193N4, H&D, Castellani, MCD138, SK4473, K6, K93 and K93M.

Of these strains, K93M, MCD138, S193N4 and UC76 are preferred. All of these strains of Staphylococcus aureus are in the culture collection of Parke, Davis & Company of Detroit, Michigan and in the culture collection of the Fermentation Laboratory, Northern Utilization Research and Development Division, US Department of Agriculture at Peoria, Illinois, under the following Numbers given in the table. Parke, Davis Northern Utilization Stamm & Company Research Laboratory Collection collection number number UC76. 05068 NRRL B-2467 Smith ... 05067 NRRL B-2468 S193N4 .... 05089 NRRL B-2469 H&D .......... 05087 NRRL B-2471 Castellani 05088 NRRL B-2472 MCD138 ...... 05085 NRRL B-2473 SK4473 ........ 05086 NRRL B-2474 K6 ........ 05090 NRRL B-2475 K93 ........... 05091 NRRL B-2476 K93M ....... 05092 NRRL B-2477 The intact cells of the aforementioned strains of Staphylococcus aureus used as starting material can either be alive, ie able to multiply, or dead, ie incapable of multiplying, or represent a mixture of both, ie partially killed. The exact process used to produce the killed or partially killed staphylococci is not critical. Some inactivation methods that can be used to produce satisfactory killed or partially killed starting materials are heating to about 600 ° C. for 11 / s, treatment with ß-propiolactone (0.1-0.2%) at 37 ° C. for 2 hours, and with phenol (1 %) at 25 ° C for about 18 hours.

The aqueous solvent used for the extraction is preferable Water; Physiologically acceptable aqueous salt solutions, such as physiological, can also be used Saline solutions, Hanks' saline solution and the like can be used. The amount of aqueous Solution is not critical, but it is preferred not to apply more than necessary to effectively pull out the desired antigen. If excessively much aqueous solvent is used, the potency per cubic centimeter of the finished vaccine is undesirable low and requires the use of an inconveniently large volume of vaccine. The amount of phenol for the extraction is also not critical.

If extractions are carried out at the same time, one must be sufficient Amount of phenol are used to produce a phenolic phase - but that is insufficient to dissolve any aqueous solvent present. If the Phenol extraction is carried out prior to extraction with the aqueous solution the phenolic phase contain sufficient water to make the phenol liquid, but not enough water to separate a separate aqueous phase, d. That is, the phenol must contain between 5 and 30% water.

As the intact cells of the staphylococci used as starting material are moist and contain water, it is usually not necessary to have very much water add to phenol to liquefy it.

The normal preferred method is to use a phenol which is am End contains no more than about 15% water. Although the phenol extraction is an essential one Step of the process is, you have not been able to determine exactly what happens with it or how it fulfills its essential task.

However, it appears that phenol is being used in a new way and Acts wisely and not just as a solvent for contaminating proteins works.

An examination of the phenolic phase shows that it is only a very small one Amount of protein and contains practically no antigenic substances. This leads to the view that the phenol causes the cell walls to break down in order to provide the antigen for extraction to be released with the aqueous solvent and at the same time the bulk of the Denature egg white so that it is neither in the phenol nor in aqueous solvents is soluble.

The finished aqueous vaccine should not be significantly more than about 0.2 01o contain phenol. Any excess phenol that makes its way into the final product finds can by dialysis or by extraction with a volatile, with water immiscible organic solvents, e.g. B. with ether or chloroform, with subsequent removal of any dissolved organic solvents from the aqueous Solution can be removed in vacuo.

In another embodiment, the phenol can be used when both extractions of the procedure are not carried out at the same time, eliminated during the procedure will. It does this as follows: The cells of the staphylococci are made with phenol extracted, and the phenol is removed from the insoluble solids by centrifugation, Filtration, decanting, etc. separated. The insoluble solids then become with a volatile organic solvent for phenol, e.g. B. with ether, chloroform, absolute ethanol etc. and any liquid organic solvents, which remain in phenol and insoluble solids after washing, allowed to evaporate before the insoluble solids with the aqueous solvent extracted.

If necessary, the p-value of the finished vaccine is increased to about 6 to 8, preferably set to about 7.2. If the vaccine is not isotonic it is made isotonic, preferably by adding sodium chloride. The finished vaccine can be made sterile by various methods; z. B. they can be exposed to heat, ultraviolet radiation or by adding chemical Sterilize preservatives. It is preferred to use the finished vaccine one Incorporate preservatives.

Some suitable preservatives are Thimerosal 1: 10000 (cf. "The Merck Index of Chemicals and Drugs ((, 1960, p. 1034), benzethonium chloride 1-20000 (cf. loc. cit., p. 130) or phenol 0.20 / o.

The aqueous staphylococcal vaccine obtained by the method of the invention can be generated is free of undissolved solids and components of the culture medium used to produce the staphylococci. It is not viable and contains very small amounts of nitrogen compared to the starting material cells used. It produces high immunity when administered parenterally not only against infection by strains of Staphylococcus aureus leading to its Manufacture were used, but also against infection by other strains of Staphylococcus aureus. The vaccine is unique in that the amount of it produced Protective antibodies in test animals can be determined and their effectiveness to protect against strong harmful doses of different strains of Staphylococcus aureus is clearly proven. The vaccine does not produce any localized when administered Reactions or system reactions.

The invention is illustrated by the following examples: Example 1 250 cc of sterile culture broth with the following composition: peptone, from casein produced by pancreas digestion (trypticase) ..... 17.0 g papain digestion product from soy flour ............. (Pythone) ..... 3.0 g sodium chloride .......... .......... 5.0 g dipotassium phosphate ................ 2.5 g dextrose ................... ..... . Put 2.5 g of water ................... ....... 1000.0 g in a 500 cc flask one teaspoon of stock culture of UC76 Staphylococcus aureus is used as the germ mass; the culture is developed at 37 ° C under aseptic conditions for 6 hours. Ever 30 cc of this culture of Staphylococcus aureus UC76 are placed in three 6-1 bottles given containing 3 l of the sterile culture broth described above; the inoculated Cultures are developed at 37 "C under aseptic conditions for 24 hours. 30 cc of 30% phenol are added to each culture for viability degrade the staphylococcus; the cultures are then left for 16 to 18 hours Stand room temperature. The cultures were then pooled. A sample of 10 cc was removed and found to contain still living staphylococci. The pooled cultures were cooled to +4 to + 100 ° C. and placed in a Sharples centrifuge at a flow rate of 225 ccm per minute and 25,000 rev / min. centrifuged. The wet packed cells were removed from the centrifuge and weighed; it was 8.2 grams of mass, or approximately 3.43. 1012 organisms. A small Sample, d. H. 0.2753 g, the mass was overnight in an oven at 105 "C dried, to determine the water in the wet cell mass. The dry cell weight was determined to be 27.8 01o of the wet cell weight.

The 8.2 g wet cells of Staphylococcus aureus UC76 were in placed in a Waring blender, and 50 g of crystalline phenol and 10 com of water were added. The phenol liquefied.

The mixture was mixed at room temperature for 10 minutes. the The final temperature of the dirty gray mixture was 41 "C. It separated on standing the mixture does not differ from each other. The mixture (49 ccm) was cooled to + 10 ° C and 60 minutes at 4000 rev / min. centrifuged. The centrifuged mixture passed of two layers, namely a dark brown, supernatant liquid and a gray precipitate packed with you.

The supernatant liquid (51 cc) was separated and discarded. 60 cc of the 830/0 pure phenol (50 g phenol and 16 com water) became the gray Precipitate was added and the mixture was thorough in a Waring blender for 1 minute mixed thoroughly, creating a light gray suspension with a volume of 61 ccm received. The suspension was at '+10 "C for 60 minutes at 4000 rev / min. centrifuged, and the supernatant 54 cc was separated from the precipitate and discarded. The precipitate was suspended in 25 cc of distilled water; the Suspension was dialyzed at +4 "C against running tap water.

The dialysis bag was kept in motion during dialysis. Dialysis against running tap water was continued for about 3 days. The dialysis bag was placed in standing distilled water at t4 ° C and dialysis for continued for about 24 hours without changing the distilled water. The suspension was transferred from the dialysis bag to a measuring cup; the bag was with Rinsed with 1 ccm of distilled water.

The caster was added to the suspension; the total volume was 31 cc and the pH value 5.9.

The suspension was transferred to a 50 cc bottle, and the The measuring beaker was rinsed with 1 cc of distilled water. The wash water was added to the suspension and the mixture frozen out.

The mixture was then thawed (volume: 28 ccm) and the pn value by adding three drops of 1N sodium hydroxide solution and three drops of 0.1N sodium hydroxide solution adjusted from 6.0 to 7.5, the mixture was shaken vigorously by hand, 2 hours Centrifuged at 4 to 10 "C and 7710XG and an additional 11/2 hours at 12100XG. Es very little turbidity remained in the supernatant. The watery one supernatant liquid consisting of the desired vaccine product separated from the precipitate (volume: 21 ccm, prr value: 7.7). An investigation this vaccine for immunogenic activity against UC76 Staphylococcus aureus in Carworth Farms no. 1Mice showed that the product had a potency PD50 of 15,000 per cubic centimeter against had an LD20 of about 10,000. This experiment was described as follows carried out.

One hundred and five Carworth Farms No. 1 albino mice (18 to 22 g) were made divided into seven groups of fifteen mice each for inoculation. The mice in each group were simultaneously subcutaneously with 0.2 cc of the alternating sterile inoculated physiological saline solution of the vaccine. The one for each group applied Dilutions of the vaccines are as follows: Group 1: 0.2 cc of a dilution 1: 100 Group 2: 0.2 cc of a dilution 1: 300 Group 3: 0.2 cc of a dilution 1: 1000 Group 4: 0.2 cc of a dilution 1: 3000 Group 5: 0.2 cc of a dilution 1: 10000 Group 6: 0.2 ccm of a dilution 1: 30000 Group 7: 0.2 ccm of a dilution 1: 100,000 Seven days later, the mice in each group and thirty mice in one became Control group by intraperitoneal injection of 10,000 LD50 Staphylococcus aureus UC76 treated (0.5 cc of a 1: 100,000 dilution in a 6 hour culture of Staphylococcus aureus UC76 in the broth described above).

The mice were observed for 5 days and deaths were noted. The percentage of survivors in each group was calculated and from this Survivors count the protective dilution for 500 in the PD50 mice after the procedure calculated by Miller and Tainter (cf.

Proceedings Society Experimental Biological Medicine, Vol. 57, 1944, pp. 264ff.). The survivor data for this experiment are presented in the table below. dilution of 0.2 cc deaths on days after O / o Group of the inoculation though used living Vaccine 0 j 1 j 2 21314 control untreated 0 1 30 0 0 0 0 1 100 0 0 0 0 0 100 2 1: 300 0 0 0 0 0 100 3 1: 1000 0 1 0 1 0 0 93.3 4 1: 3000 0! 4 3 0 0 53.3 5 1: 10000 0 15 0 0 0 0 6 1: 30000 0 1 15 0 0 0 0 7 1: 100000 0! 15 0 0 1 0 0 It can be seen from the table above that all control mice died within 24 hours and that with 0.2 cc vaccine at a dilution of 1: 3000 only a little more than 50% of the mice were protected. The actual PD50 of the vaccine was calculated from this data to be 3100 per 0.2 ccm or 15 500 per cubic centimeter.

The dialysis in the above procedure can be eliminated by washing out the precipitate, which is insoluble in phenol, with ether and by the ether present in the solid mass is allowed to evaporate before the extraction with water.

Example 2 Ten test tubes with 10 cc of sterile culture broth the same composition as in Example 1 were each inoculated with a teaspoon full of a stock culture UC76 Staphylococcus aureus and at 37 "C under aseptic Conditions developed 6 hours. 2 cc of these live cultures were used, about forty-three Roux flasks each, each containing 200 ccm of sterile solid culture medium of the following composition contained to inoculate.

Peptones from casein by pancreas digestion (trypticase) ............ 15.0 g papain digestion product made from soy flour (Phytone) ................... 5.0 g Sodium chloride ................... 5.0 g agar ................... ...... ... 15.0 g Distilled water ............... 1000.0 g The inoculated cultures were at 37 "C developed under aseptic conditions for 18 hours. The living staphylococci aureus UC76 were obtained from the surface of forty-three cultures with a minimum amount sterile physiological saline solution washed off, and the suspension obtained was 1 hour at 4000 rev / min. centrifuged. The supernatant liquid was discarded, and the packed cells (40.1 g wet weight; about 1.8 1013 organisms) were suspended in 219 cc of distilled water; the volume of the suspension was 245 cc. A 9 cc sample was taken and the remaining 236 cc of suspension were divided into two equal parts. 118 ccm of aqueous suspension of living Staphylococcus each aureus UC76 cells were mixed with 256.3 g phenol and 156 cc water in a Waring blender mixed for 8 minutes. The temperature rose from 15 "C to 360 C. The resulting Mixtures were combined and cooled to +10 "C (volume: 800 ccm). It formed two layers. The mixture was at +10 "C for 60 minutes at 4000 rev / min. centrifuged, causing a disintegration into four phases; an aqueous phase on the top layer, a middle precipitation phase below the aqueous phase, one Phenolic phase under the intermediate precipitate and a precipitate phase under the Phenolic phase. The aqueous phase was siphoned off and stored at +4 ° C. The phenol phase was siphoned off and discarded. The interphase precipitation was with the other Precipitation phase combined and diluted with 10 com of distilled water that is used had been used to rinse the vessels during the transfer. The combined precipitations and wash water were mixed with the aqueous phase and the resulting suspension was at 4000 rev / min. Centrifuged for 2 hours. The aqueous phase (volume: 400 ccm) was separated and dialyzed against running tap water at + 4 ° C.

The dialysis bag was kept in motion during dialysis. Dialysis against running tap water was continued for about 2 days. The dialysis bag was then placed in standing distilled water transferred at +4 "C, and dialysis continued for about 2 days without changing the distilled water. The dialysis material had a volume of 450 ccm, was clear and colorless. 3.825 grams of sodium chloride were added added to the aqueous vaccine to make it physiological, and the Pn value was adjusted from 6.9 to 7.2 by adding 0.15 ccm of 0.1 N sodium hydroxide solution. the Vaccine was transferred to a sterile 500 cc bottle and thimerosal was placed in a concentration of 1: 10000 was added, and the vaccine was overnight at Stored at room temperature. A 30 cc sample was used for sterilization trials taken out, and an assay for immunogenic activity according to the method, as described in Example 1 was carried out.

The rest of the vaccine was stored at +4 "C, and after the final Sterilization as well as safety and effectiveness tests in a sealed with rubber Bottle transferred, each containing 10.5 ccm. The sterilization attempts in liquid thioglycolic acid medium indicated that the vaccine was sterile.

Immunogenic tests according to the method as described in Example 1 showed that the vaccine had an effectiveness of 975 PD50 per cubic centimeter against exhibited 10,000 LDs0 inoculation with the UC76 strain of Staphylococcus aureus.

If desired, dialysis can be eliminated in the above procedure by extracting the aqueous (400 ccm) phase with several parts of ether and then frees the aqueous solution from the ether by evaporation in vacuo.

Vaccine solutions similar to those described in Examples 1 and 2 similar can be made by using any of the following strains Staphylococcus aureus used in place of the UC76 strain of Staphylococcus aureus, to inoculate the nutrient media as described in the previous examples: Smith, S193N4, H&D, Castellani, MCD138, SK4473, K6, K93 and K93M.

Claims (1)

PATENT CLAIM: Process for the production of a staphylococcal vaccine, characterized in that the intact cells from UC76, Smith, S139N4, H&D, Castellani, MCD138, Sk4473, K6, K93 or K93M of Staphylococcus aureus with phenol, which contains 5 to 300 / o water and then with water or an aqueous salt solution or extracted simultaneously with phenol and an aqueous medium and the phenol and separating the insoluble solids from the resulting aqueous solution.