Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Patent
1986-10-02
1989-05-16
Phillips, Delbert R.
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
530825, 424 88, 424 92, C07K 1504, A61K 39104
Patent
active
048311219
DESCRIPTION:
BRIEF SUMMARY
The invention relates to polydisperse native Pseudomonas flagellar (H) antigens (FAg) and a method of producing them from Pseudomonas aeruginosa bacterial cultures.
The bacterium Pseudomonas aeruginosa is an opportunistic pathogen which often occurs with hospital infections, mainly in patients having an impaired immune defense, such as patients suffering from burns, persons suffering from cystic fibrosis or having defective organic functions, and in tumor patients. Antibiotics are active against Pseudomonas infections only to a limited extent due to the occurrence of resistances, and therefore attempts have been made to fight infections caused by Pseudomonas aeruginosa by immunological methods.
Infections may be triggered by a variety of strains producing O-group antigens and H-antigens. According to the H antigen pattern according to Ansorg (Zbl. Bakt. Hyg. I. Abt. Orig. A 242, 228-238 (1978)), with Pseudomonas aeruginosa it is differentiated between a complex flagellar (H) antigen a having the partial antigens a.sub.0, a.sub.1, a.sub.2, a.sub.3, a.sub.4 and a uniform flagellar (H) antigen b, by using the indirect immunofluorescence technique. The partial factors a.sub.0 -a.sub.4 are independent determinants, so that a flagellar antigen pattern having several H-types results. O-groups and H-type show free combinations.
Strains usable for preparing the Pseudomonas aeruginosa bacterial cultures and the antigens produced are listed in the following table.
______________________________________ Strain H-type
______________________________________
1 170001 b
M-2 b
2 5142 a.sub.0
3 5940 a.sub.0, a.sub.2
4 5939 a.sub.0, a.sub.3
5 5933 a.sub.0, a.sub.1, a.sub.2
1210 a.sub.0, a.sub.1, a.sub.2
16990 a.sub.0, a.sub.1, a.sub.2
6 170018 a.sub.0, a.sub.3, a.sub.4
______________________________________
Isolated filaments of the flagellar antigens, which may be obtained by shaking, homogenization and subsequent centrifugation (R. Ansorg, W. Schmitt, Med. Microbiol. Immunol. (1980) 168: 217-226), are comprised of flagella and flagella fractions, united in a complex comprised of lipopolysaccharides (LPS) and impurities from the nutrient medium; such preparations by their nature are pyrogenic and not suited for an application on man.
It is known to produce Pseudomonas vaccines for preventing Pseudomonas infections, Pseudomonas aeruginosa bacterial masses and/or culture filtrates being used as starting materials which were obtained by growing the microorganisms on surface cultures or submersely in complex nutrient media. With these complex nutrient media, various extracts and/or hydrolisates of animal, microbial or vegetable proteins (so-called peptones) were used besides a carbon and energy source (mostly carbohydrates) and essential nutrient salts. Such nutrient solution supplements are not defined as to their precise composition and furthermore are variable from lot to lot. In addition to amino acids, they also contain incompletely decomposed protein fractions and undefined complexes thereof and substantially serve for covering the demand of amino acid and growth-promoting substances. Therefore, culture supernatants are always rich in substances of non-bacterial origin, which has the disadvantage that for preparing a flagellar (H) antigen of Pseudomonas aeruginosa, always several separating steps which have to follow the growing step are necessary in order to free the flagellar (H) antigen as far as possible from impurities stemming from the nutrient medium.
The separation of the crude flagellar material from the bacterial suspension was effected by so-called "shearing", i.e. exposure of the bacterial suspension to shearing forces, in a mixer, followed by centrifugation at 15,000.times.g-18,000.times.g. Thereupon, the pellet is discarded and the supernatant containing the crude flagella preparation, is subjected to a centrifugal acceleration of at least 40,000.times.g for one hour or 100,000.times.g for 20 minutes. Therein the crude antigen is obtained in the form of pellets. As has a
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Dorner Friedrich
McDonel James L.
Mitterer Artur
Montie Thomas C.
"Immuno" Aktiengesellschaft fur chemisch-medizinische Produkte
Chan Christina
Phillips Delbert R.
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