Saponin preparations and use thereof in iscoms

Details Saponin preparations and use thereof in iscoms Saponin preparations and use thereof in iscoms

1999-02-22

2002-03-05

Nolan, Patrick J. (Department: 1644)

Drug, bio-affecting and body treating compositions

Nonspecific immunoeffector, per se ; or nonspecific...

C424S278100, C424S283100, C424S184100, C514S885000

Reexamination Certificate

active

06352697

ABSTRACT:

This application is a 371 national stage application of PCT/AU95/00670, filed Oct. 12, 1995.
FIELD OF THE INVENTION
This invention relates to saponin preparations, particularly to saponin preparations based on defined compositions of purified saponin fractions derived from the bark of
Quillaja saponaria
Molina. The invention also extends to immunostimulating complex (iscom) matrices prepared using these saponin preparations, as well as to immunogenic iscoms in which immunogens are incorporated into or associated with an iscom matrix. Such immunogens will usually be proteins or peptides derived from bacteria, viruses or other microorganisms, but they may, in addition, be any other protein, peptide or other chemical entity which can induce an immune response.
The saponin preparations of this invention, and iscom matrices prepared using them, have particular activity as adjuvants, that is as products which result in a specific increase in the immunogenicity of a vaccine component
BACKGROUND OF THE INVENTION
The adjuvant properties of saponin have been long known as has its ability to increase antibody titres to immunogens. As used herein the term “saponin” refers to a group of surface-active glycosides of plant origin composed of a hydrophilic region (usually several sugar chains) in association with a hydrophobic region of either steroid or triterpenoid structure. Although saponin is available from a number of diverse sources, saponins with useful adjuvant activity have been derived from the South American tree
Quillaja saponaria
Molina. Saponin from this source was used to isolate a “homogeneous” fraction denoted “Quil A” (Dalsgaard, 1974).
Acute toxicity is a major concern for both the veterinary and human use of Quil A in vaccine preparations. One way to avoid the acute toxicity of Quil A is the use of iscoms, an abbreviation for Immuno Stimulating COMplexes. This is primarily because Quil A is less toxic when incorporated into iscoms, because its association with cholesterol in the iscom reduces its affinity for cholesterol in cell membranes and hence its cell lytic effects. In addition, a lesser amount of Quil A is required to generate a similar level of adjuvant effect. Iscoms are small, cage-like structures generally 30 to 40 nm in diameter which retain this structure on freeze drying. The final formulation of a typical immunogenic iscom with an optimal amount of immunogenic protein is a weight ratio of Quil A, cholesterol, phosphatidyl choline, and protein (1:1:1:5). Such a typical iscom is estimated to contain 5 to 10% by weight Quil A, 1 to 5% cholesterol and phospholipids, and the remainder protein. Peptides can be incorporated into iscoms either directly or by chemical coupling to a carrier protein (e.g. influenza envelope protein) after incorporation of the carrier protein into iscoms.
As an adjuvant, the iscom confers many advantages including powerful immunostimulatory effects, low toxicity, ability to induce both cellular (including CTL) and humoral responses, and it is inexpensive in both reagent and manufacturing cost. However, in the past, iscoms have had two major disadvantages; firstly, the Quil A used in their preparation was a complex and ill defined mixture of a biologically-derived product, and batch-to-batch variation was therefore to be expected; and secondly, iscoms still showed injection-site reactivity and low but detectable in vivo toxicity.
Since the recognition of the adjuvant activity of Quil A (Dalsgaard, 1974) several groups have further fractionated this material into a number of “purified”components (Morein et al., Australian Patent Specification No. 632067; Kersten, 1990; Kensil, 1988; Kensil 1991). These components were subsequently shown to have variable properties especially in regards to adjuvant activity, haemolytic activity and ability to form iscoms. The use of purified Quil A components conferred two potential advantages for their use in a human vaccine. Firstly, the purified component could be characterized and therefore made reproducibly; and secondly, the components could be selected for optimal usefulness.
The immunomodulatory properties of the Quil A saponins and the additional benefits to be derived from these saponins when they are incorporated into an iscom have been described in various publications, e.g. Cox and Coulter, 1992; Dalsgaard, 1974; Morein et al., Australian Patent Specifications Nos. 558258, 589915, 590904 and 632067. In Australian Patent Specification No. 632067, the separation of a preparation of Quil A into three distinct fractions called B4B, B3 and B2 is described, along with HPLC chromatographic procedures for this fractionation. More carefully defined and controllable procedures for the fractionation of Quil A have now been devised which result in three major fractions with increasing degrees of hydrophobicity in the purification system used.
In work leading to the present invention, it has now been shown that saponins derived from
Quillaja saponaria
can be separated into fractions with differing chemical and biological properties, including the important biological properties of adjuvant activity, haemolytic activity, ability to form iscoms and in vivo toxicity, and that particular compositions of these fractions can be prepared to form novel saponin preparations which are capable of forming good iscoms, having optimal adjuvant activity but minimal haemolytic and toxic activity.
SUMMARY OF THE INVENTION
According to the present invention, there is provided a saponin preparation comprising saponins of
Quillaja saponaria
, said preparation comprising from 50 to 90% by weight of Fraction A of Quil A (as herein defined) and from 50% to 10% by weight of Fraction C of Quil A (as herein defined).
Preferably, the saponin preparation comprises from 50% to 70% by weight of Fraction A and from 50% to 30% by weight of Fraction C. A particularly preferred preparation comprises about 70% by weight of Fraction A and about 30% by weight of Fraction C.
The term “Quil A” is used throughout this specification and in the claims as a generic description of a semi-purified saponin fraction of
Quillaja saponaria.
The saponin preparation may, if desired, include minor amounts (for example up to 40% by weight) of other adjuvant materials with desired immunomodulatory properties, including minor amounts of Fraction B of Quil A or of other saponins. Examples of other saponins or other adjuvant materials which are suitable for inclusion in this preparation are described in Australian Patent Specification No. 632067, incorporated herein by reference.
As described above, it is known that in order to prepare an immunostimulating complex (iscom) matrix, Quil A, a sterol such as cholesterol and optionally a lipid such as phosphatidyl choline, must be included in the reaction mixture.
In accordance with another aspect of the present invention there is provided an immunostimulating complex (iscom) matrix comprising a saponin preparation, a sterol and optionally a lipid, wherein the saponin preparation comprises from 50 to 90% by weight of Fraction A of Quil A (as herein defined) and from 50% to 10% by weight of Fraction C of Quil A (as herein defined).
Preferably, in such an iscom matrix the sterol is cholesterol, and the lipid (which is optionally present) is a phospholipid such as phosphatidyl choline.
In yet another aspect, this invention provides an immunogenic iscom which comprises an iscom matrix as described above having at least one immunogen incorporated into or associated with the iscom matrix.
An iscom matrix or an immunogenic iscom in accordance with the present invention may be prepared by techniques which are well known to persons skilled in the art, and which are described in detail in the publications Cox and Coulter, 1992 and Morein et al., Australian Patent Specifications Nos. 558258, 589915, 590904 and 632067, the disclosures of which are incorporated herein by reference.
The immunogen which is incorporated into or associated with the iscom matrix in accordance with this invention may be any chemical entity w

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