Chemistry: molecular biology and microbiology – Plant cell or cell line – per se ; composition thereof;...
Patent
1995-08-07
1998-02-10
Rollins, John W.
Chemistry: molecular biology and microbiology
Plant cell or cell line, per se ; composition thereof;...
435420, 435421, 4354301, 4241951, 4241931, C12N 5100
Patent
active
057168482
DESCRIPTION:
BRIEF SUMMARY
This application is the national phase of PCT NL93/00220, filed Oct. 29, 1993, and claims priority under 35 U.S.C. .sctn.119, based on EP 92203365.9, filed on Oct. 30, 1992.
The present invention relates to cultured cells of Quillaja sp., a method for preparing active substances from Quillaja sp., various products comprising the active substances, a method for preparing Immune-Stimulating COMplexes (ISCOM's) from the active substances and vaccins and adjuvants comprising the ISCOM's, the adjuvant ISCOM-matrix (Lovgren, K. thesis, ISBN 91-576-3202-2), the adjuvant Quil A (Dalsgaard, K., Arch. ges. Virusforsch. 44, 243-254 (1974), the adjuvant QS 21 (Jia-Yan Wu, J. Immunology 148, 1519-1525 (1992), and other saponin adjuvants.
Immune-stimulating complexes (so-called ISCOM's) are negatively charged pentagonal dodecahedra that form spontaneously on mixing cholesterol and the saponins of Quillaja sp. During their formation proteins and other lipids can be incorporated. ISCOM's have been found to strongly enhance immune responses and are therefore used as an immunological adjuvant and carrier/delivery system in e.g. vaccins. Quil A, QS 21, and other saponin adjuvants are products derived from the natural bark having immunological adjuvant activity in a variety of vaccines.
For the production of ISCOM's there is a growing need for inter alia Quil A. Quil A is a mixture of the active substances (saponins) originating from the bark of the Quillaja sp. tree growing mainly in Chile. The natural sources of Quillaja bark are limited. In fact, old trees are already rare today and yet about 1000 tons of bark per year are exported from Chile. Because the increasing demand for active substances (sapanins) for various purposes a shortage of material is to be expected in the future.
It is therefore highly desirable to find other ways of isolating Quil A and other active substances of Quillaja (saponins) for the preparation of ISCOM's, as adjuvants, and for various other applications.
It has now been found that cells, tissues or organs of the Quillaja sp. plant body can be cultured in vitro in a liquid or on a solid medium.
The invention thus provides cultured cells of Quillaja sp. for the preparation of active substances from Quillaja sp. The cultured cells may originate from a callus cell culture, wherein tissues or organs are grown on a solid medium. The term "callus" refers to an amorphous lump of cells having lost their organ-forming capacity, and which lump is formed when a fragment of the plant body is tissue cultured on a solid medium. The so obtained callus shows an external form resembling the agglutination tissue of the plant body. The cultured cells may also originate from a suspension cell culture. The term "suspension" cell culture refers herein to a fine flocky dispersion of the cells formed when pieces of callus are further inoculated and cultured in a liquid medium under aerobic conditions.
According to the invention various kinds of Quillaja plants can be used for tissue or suspension culture, for example Quillaja saponaria Molina, Quillaja smegmadermos, Quillaja brasiliensis and the like.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a densitometric scan of a HPTLC plate developed as described in Example 3, to which was applied an extract from a callus cell culture. In FIG. 1, "A" represents the point of application of the extract to the plate, "1" and "2" represent the two major bands, and "F" indicates solvent front.
FIG. 2 shows a densitometric scan of a HPTLC plate developed as described in EXAMPLE 3, to which was applied the non-dialysable fraction of a callus cell culture extract. In FIG. 2, "3" and "4" indicate the two major bands.
FIG. 3 shows an electron microscope photograph of the ISCOM matrices described in EXAMPLE 5.
FIG. 4 graphically illustrates the hemolysis of sheep red blood cells by extracts of the various cell cultures described in EXAMPLE 4.
FIG. 5 illustrates the results of testing extracts of a cell suspension culture and a callus culture for immunological adjuvant activity, as de
REFERENCES:
patent: 5057540 (1991-10-01), Kensil et al.
Henry, M et al., Quillaic acid production in Saponaria officinalis cell suspension culture, Chem Abs 97(25):688, abstract 97:214231c 1982.
Neumuller, O.-A., Rompps Chemie-Lexikon, 8ste Ed., 1987, Franckh'sche Verlagshabdlung, Stuttgart, Germany, p. 3452 (with Translation).
Chemical Abstracts, White, et al., A purified saponin acts as an adjuvant for a T-independent antigen, 117:5649n Jul. 6, 1992.
Chemical Abstraacts, Gaunt, et al., Short term toxicity of quillaia extract in rats, 82:56161p Mar. 3, 1975.
E.F. Steinmetx, Codex Vegetablis, No. 941 1957.
Dalsgard Kristian
Henry Max
O'Connor Christensen
Rollins John W.
Seed Capital Investment (SCI) B.V.
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