WO1995009179A1 - Composes presentant une activite d'adjuvant - Google Patents

Composes presentant une activite d'adjuvant Download PDF

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Publication number
WO1995009179A1
WO1995009179A1 PCT/NL1994/000238 NL9400238W WO9509179A1 WO 1995009179 A1 WO1995009179 A1 WO 1995009179A1 NL 9400238 W NL9400238 W NL 9400238W WO 9509179 A1 WO9509179 A1 WO 9509179A1
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WO
WIPO (PCT)
Prior art keywords
quadri
plant cell
compounds
cell extract
adjuvant
Prior art date
Application number
PCT/NL1994/000238
Other languages
English (en)
Inventor
Kristian Dalsgaard
Max Henry
Ricardo Manuel Guillermo San Martin Gamboa
Henk Johan Grande
Søren KAMSTRUP
Original Assignee
Seed Capital Investments (Sci) B.V.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from NL9301690A external-priority patent/NL9301690A/nl
Application filed by Seed Capital Investments (Sci) B.V. filed Critical Seed Capital Investments (Sci) B.V.
Priority to AU10777/95A priority Critical patent/AU1077795A/en
Publication of WO1995009179A1 publication Critical patent/WO1995009179A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/20—Carbocyclic rings
    • C07H15/24—Condensed ring systems having three or more rings
    • C07H15/256—Polyterpene radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J63/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton has been modified by expansion of only one ring by one or two atoms
    • C07J63/008—Expansion of ring D by one atom, e.g. D homo steroids

Definitions

  • the present invention relates to compounds with adjuvant activity, in particular saponins.
  • the invention relates to compounds in very pure form, hydrolyzed deriv ⁇ atives of these pure compounds and modified derivatives thereof.
  • the invention further relates to methods for manufacturing the compounds, the use of the compounds in ISCOM matrices, ISCOMS, adjuvants and vaccines, and the ISCOM matrices, ISCOMS, adjuvants and vaccines as such which contain the compounds, and to a method for controlling the immune response in addition to the targeted release of medicines.
  • the compounds can also be used for precise and selective hemolysis of erythrocytes.
  • Saponins are substances which occur inter alia in plants of for instance the genera Saponaria. Gypsophilia and in particular Ouillaia. Although the chemical structure of these saponins has not been fully clarified, they contain in any case gypsogenin or the gypsogenenic acid respectively Ouilla ic acid derived therefrom, all triterpenoids and a number of sugar chains. In many cases there also occurs a fatty acid residue which is linked to a sugar residue on the triterpenoid structure.
  • This fresh material obtained in a similar manner as known QUIL A thus consists of less components and has a lower toxicity and a similar or higher adjuvant activity than QUIL A. Furthermore, from this material relatively high amounts of the two single main components may be purified by means of purification techniques known per se. such as reversed phase HPLC, because of its simple composition as compared to QUIL A.
  • the invention therefore provides a plant cell extract, which is a more pure starting material, and in itself already suitable as an adjuvant.
  • the invention further relates to compounds obtainable by separating into fractions an extract of fresh plant material consisting of substantially living cells which originates from saponin- containing plants.
  • plant cell extract is an extract obtainable by extracting plant material, such as single cells but also complete small trees or parts thereof, such as roots, cambium, phloem etc., with water.
  • plant cell extract will be used in addition to "New Quil A" in order to differentiate the new extract according to the invention from known heterogenous QUIL A.
  • saponins are those compounds which can be obtained directly from a plant extract and do not undergo any subsequent hydrolysis or modification treatment.
  • Seraponin-like compounds are compounds which do undergo such a hydrolysis or modification.
  • the general term “compounds” includes both de saponins and the saponin-like compounds.
  • fresh plant material When “fresh plant material” is mentioned in the text, this means plant material extracted essentially “directly after harvesting” without subjecting the material to weather conditions, such that conditions stimulating oxidation and decomposition can occur as less as possible. Furthermore it appeared that plant material isolated from parts of a plant which are not differentiated or have been differentiated a short time before, such as the cambium, young wood and phloem as well as the roots, comprise less decomposition products derived from saponin.
  • ISCOM matrix represents a matrix for immuno- stimulating complexes as described in WO 90/03184.
  • ISCOMS is meant an ISCOM matrix having enclosed therein at least one antigen, as described in US patent 4,578,269.
  • the sources from which the saponin-like compounds according to the present invention can be recovered are very diverse. An important condition however is that use is made of fresh plant material consisting of substantially living cells. It is also important that the material is processed immediately after harvesting thereof. Suitable sources are for instance the trunks of trees up to 15 years old of the type Ouillaia saponaria and the roots of such trees. In principle the layer of living cells of older trees consisting of growing wood, cambium and phloem and the roots could also be used, but this is less desirable with regard to the scarcity of older trees.
  • tissue cultures and suspension cell cultures of cells originating particularly from the cambium are also very suitable sources, as described in the Netherlands patent application 93.01404, the content of which is incorporated herein by way of reference, as well as root cultures and the like.
  • QUADRI-1 and QUADRI-2 Two different compounds are found, which are called QUADRI-1 and QUADRI-2.
  • QUADRI-1 and QUADRI-2 From analytical RP-HPLC it is found that very pure substances of both QUADRI-1 and QUADRI-2 99.99% pure material can be obtained in this way.
  • the invention further relates to the hydrolysis products derived from the saponins. It has been found that by selective hydrolysis of the pure saponins the adjuvant activity can be varied. Different compounds have been found by means of different methods of alkaline hydrolysis.
  • QUADRI-2B which are found to be identical, both with respect to their NMR spectra and with respect to the point of time they leave the column at about 13.1 (figure 8 and figure 10) (see also table 2) .
  • the compounds according to the invention, and then particularly the hydrolyzed derivatives thereof, can also serve as basis for modified compounds. It is thus for instance possible to link a peptide to the acid group in position 23 of product B, respectively the fucose of product A.
  • the compound modified with the peptide can itself or in combination with other normal saponins or saponin-like compounds be used as adjuvant and also in the formation of ISCOMs.
  • Another possible application of the compound according to the invention is the targeted release of medicines.
  • hydrolyzed compounds could be useful for this purpose.
  • the saponins according to the present invention can be prepared by means of a per se known method. Such a method is for instance described by Dalsgaard (supra) . Separation into fractions of the product ("New Quil A") obtained by means of this method can take place using preparative HPLC, as for instance RP-HPLC, e.g. on a Vydac C4-column with a gradient of 30-45% acetonitril with 0.15 % TFA. Any separation of an extract into its separate components can however be used in the preparation of the compounds according to the invention.
  • the compounds according to the invention exhibit adjuvant activity to a lesser or greater extent. They can be used alone or in combination, for instance in vaccines. It is possible to manufacture an ISCOM matrix from compounds according to the invention or combinations thereof together with for example cholesterol and phosphatidylcholin. The inclusion of one or more antigens in the matrix provides ISCOMs.
  • the present invention is illustrated in this de ⁇ scription with reference to saponins and saponin-like compounds of Ouillaia saponaria. but is not limited thereto.
  • ISCOMS ISCOMS it has been demonstrated that, in addition to the MHC-class 2 response usual for normal adjuvants which results in increased T-helper cell con ⁇ centration, they can also induce a heretofore unique MHC- class 1 response. It has been further demonstrated that the induced T-helper cells are mostly of the TH-1 type and thus give protection and are to a lesser extent or not at all of the TH-2 type which result in an over-sensitivity reaction.
  • QUADRI-1 and QUADRI-2 per se and as part of ISCOMS possess such an adjuvant function and can thus also induce cytolytic T-lymphocytes (CTLs) .
  • CTLs cytolytic T-lymphocytes
  • the hydrolyzed compounds also exhibit these properties but not to the same extent. They possibly help to dissolve the antigens and appear to induce a lower MHC-class 2 immune response.
  • QUADRI-1 and QUADRI-2 are surrounded by many more sugar residues than the derived products QUADRI-1A, -IB, -ID and QUADRI-2A, -2B, -2D generated by selective hydrolysis, which will influence among other things the hydrophobicity and the three- dimensional structure.
  • the presentation of antigens can hereby be selectively influenced.
  • Saponins can also be used for the hemolysis of erythrocytes.
  • a known method wherein use is made of hemolysis is the counting of white blood cells using a Coulter counter. Normally speaking, the accuracy of the count is influenced highly negatively by the presence of the red blood cells. By lysing the red blood cells the disruptive background is removed. Saponins can be used to lyse erythrocytes. It has been found that the existing saponin preparations are not reliable enough to standardize this method of counting white blood cells. There is a demand from both industry and the legislator for a pure preparation with which this white blood cell determination can be performed in reproducible manner.
  • the saponins, and optionally saponin-like compounds, according to the invention meet this requirement.
  • Another field of application of the compounds according to the invention is the targeted release of medicines.
  • a quantity of medicine is included in an ISCOM matrix.
  • molecules with a targeted function such as for instance antibodies, which are for example included in the matrix, the ISCOMS can be transported to the desired location in the body.
  • Figure 2 shows the RP-HPLC elution pattern of QUIL A that is isolated from a suspension cell culture. In addition to the two peaks characteristic of QUADRI-1 and
  • QUADRI-2 a number of peaks can also be distinguished which were later shown to be deacylated versions of QUADRI-2.
  • HPLC pattern of QUIL A from old bark is shown in figure 3.
  • the arrows in figure 3 indicate the positions of QUADRI-1 and QUADRI-2 and of deacylated QUADRI-2. It can be seen from this figure that the material from the old bark is not just much more heterogenous than the material recovered according to the invention from young trees, but also shows double peaks where in fig. 1 single peaks may be seen.
  • FIG 11 By way of example in figure 11 is shown the HPLC elution pattern of a component known under the name B4b. It is known of B4b that it has a very high ISCOM forming capability but a low adjuvant activity (WO 90/03184) .
  • the QUADRI-1 and QUADRI-2 peaks are absent from the HPLC pattern and it is precisely the deacylated QUADRI-1A and QUADRI-2A which are present. From this it appears that fraction B4b comprises various components upon analysis on RP-HPLC. The absence of QUADRI-1 and QUADRI-2 can now also explain why B4b does provide ISCOMS but has no adjuvant activity normal for ISCOMS.
  • Figures 4 and 5 show RP-HPLC elution patterns for the compounds QUADRI-1 and QUADRI-2 respectively. It can be seen from the figures that both fractions are more than 99.99% pure.
  • QUADRI-1 contains 415.9 ⁇ g sugars per mg. This corresponds with 41.6% carbohydrates.
  • QUADRI-2 is less glycosylated and contains 359.6 ⁇ g per mg (35.9% carbohydrates).
  • QUADRI-2 in particular was characterized still further making use of GC-MS, NMR (500 MHz) , UV/vis-spectra, Fluorescence and Circular Dichroism.
  • GC-MS sugar analysis in addition to the sugars, Quillajic acid was also found to be indicated at high column temperature as the trimethylsilylated methylester (fig. 13) . Noticeably, the Quillajic acid did not appear to be homogeneous but displayed at least two bulges. It is not clear whether this was possibly induced by the isolation and analysis method itself.
  • Peak 3 showed 1.9 xylose in comparison to rhamnose and therefore corresponds with the residue associated with QA- 21-V2 consisting successively of xylose, xylose, rhamnose and fucose.
  • peak 4 also corresponds in sugar analysis with the structure proposed by Higuschi of successively apiose (0.8), xylose (1.3), rhamnose (1.0) and fucose (0.9). The three peaks correspond in area with a presence of 22% trisaccharide, 17% tetrasaccharide xylose, xylose, rhamnose, and fucose, 61% tetrasaccharide apiose, xylose, rhamnose and fucose.
  • a NOE 2D-NMR of the fragment of QUADRI-2A showed in respect of sugar residues no other adjacent group than the glucuronic acid. What was noticeable here however was that in water/methanol both aldehyde resonances displayed NOE in conjunction with two ⁇ -resonances of the glucuronic acid. This shows that two conformers are present which, once formed, are reasonably stable.
  • the emission spectrum has a maximum at about 350 nm but is quite wide. Excitation spectra recorded at two emission wavelengths (394 and 350 nm) therefore show differences. When the intensity is recorded at 394 nm (in the edge of the emission) the excitation spectrum displayed a clear shift to higher wavelength. Once again this can be explained by the presence of an internal complex.
  • QUADRI-2 is characterized by a constant part formed by QUADRI-2B, to which via the fucose is bonded the double fatty acid residue with the arabinose thereon. This part possibly forms the essential component of the molecule resulting in the adjuvant activity.
  • the remaining part (C) of the molecule is not homo ⁇ geneous either in composition or in its spatial structure, but in terms of the internal sugar chain C consists of respectively apiose, xylose and rhamnose, respectively xylose, xylose, rhamnose, fucose, respectively xylose, rhamnose, fucose, together referred to as QUADRI-2.
  • the individual substances are not separated with the RP- HPLC purification method known for this type of substances, whereby internal complex formation can result in at least two peaks in RP-HPLC and whereby this technique is already per se unable to unambiguously define QUADRI-1 and/or QUADRI-2.
  • QUADRI-1 is possibly also an adjuvant because of the identical nature of the fragments QUADRI-IB and QUADRI-2B and the fatty acid part.
  • glucose also occurs in this molecule. The substance is however, when isolated, much more toxic and therefore less suitable. Possible changes resulting from the presence of the glucose on the spatial structure could result in this increased toxicity in water compared to QUADRI-2, but this phenomenon is not yet fully understood.
  • ovalbumin is a soluble protein which occurs in the cytosol, of which it is known that saponins in that case offer fewer advantages than other adjuvants than in the case of glycosylated proteins which occur in the membrane.
  • QUADRI-2 50, 100, 200, 400 or 800 ⁇ g QUADRI-l respectively QUADRI-2 were administered subcutaneously to groups of 6 mice in each case. Even the maximum dose used of 800 ⁇ g QUADRI-2 per mouse of 20 g was found not to have lethal consequences for any of the 6 test animals. This means that the LD 50 of QUADRI-2 is greater than 40 mg/kg test animal. In the case of QUADRI-l an LD 50 of approximately 7.5 mg/kg was found. QUIL A® from Superfos was used as reference material. The LD 50 of this preparation amounted to approxi- mately 25 mg/kg.
  • Adjuvant activity was compared for QUADRI-l and 2 by administering 100 ⁇ g ovalbumin in the presence of differing amounts of adjuvant to different groups of guinea-pigs (fig. 22).
  • 125 ⁇ g QUADRI-2 gives approximately a hundred-fold increase in the titre of antibodies against ovalbumin. 25 ⁇ g has roughly the same effect.
  • QUADRI-2 is used as component of the ISCOM matrix there is then found to be a maximum (again about a hundred-fold) with the use of 25 ⁇ g QUADRI-2.
  • the avidity of the generated antibodies against ovalbumin is also found to differ when QUADRI-2 is added not separately but in the ISCOM matrix.
  • mice could not stand 25 ⁇ g pure QUADRI-l and died. If a glycosylated antigen is used as glycoprotein D2 (gD) instead of ovalbumin, the effectiveness of QUADRI-2 is then found to be very great compared to Al(OH) 3 .
  • gD glycoprotein D2
  • the mice were injected again with the same mixture ("booster") . Titres were measured after 14 days and 21 days. After the first injection a quantity of 5 ⁇ g QUADRI-2 was found to be necessary to obtain an adjuvant activity of about a factor 5.
  • QUADRI-l is converted for the most part into a new compound which is character- ized by a retention time of about 8 minutes (see figure 7) .
  • This compound is the hydrolysis product QUADRI-IA. 30% acetonitrile is then added to an alkaline solution (pH 12) of QUADRI-l, which solution is heated for 16 hours at 100°C (so-called "strong hydrolysis”) .
  • the elution pattern then shows, in addition to the peak of the void volume, two other peaks (see figure 9) , the first of which, with a retention time of about 8.7 minutes, corresponds with the peak occurring after the mild hydrolysis (compare figure 7) , and the second of which corresponds with QUADRI-IB.
  • a solution of one or more of the compounds according to the present invention is prepared by dissolving a number of mg of the compound in a corresponding number of ml of water (this is solution A) .
  • Phosphatidylcholin is dissolved in water with 20% of the detergent Mega-10® (Sigma) to a concentration of 10 mg/ml. 10 mg cholesterol per ml is dissolved therein by stirring at 50°C. This is solution B.
  • An antigen solution is obtained by dissolving a deter ⁇ mined number of mg of antigen in a corresponding number of ml of water. This is solution C.
  • 20 ⁇ l B is added per ml A.
  • ISCOMS are prepared from 1 ml A, 1 ml C and 20 ⁇ l B. The mixtures are shaken on a tumbler for 3 to 4 hours. If necessary the Mega-10® is removed by dialyzing against physiological salt solution (0.85% NaCl, 0.01 M phosphate buffer, pH 6.5-7.2).
  • Figure 24 is an electron microscopic photo illustrat ⁇ ing the ISCOM-forming properties of QUADRI-2.
  • Figure 25 shows the ISCOM matrices formed with one of the two hydro- lysis products obtained after mild hydrolysis of QUADRI-l (the product which showed a value of 2.405 in the sedimen ⁇ tation experiments of figure 28) . The hydrolysis products are found to form smaller ISCOM matrices than the starting products.
  • the Zeta potential shows the stability of a particle in a solution.
  • a physiological salt solution (0.85% NaCl, 0.01 M phosphate buffer, pH 7.2)
  • a value of -22 mV was obtained. This means that a very stable micelle (ISCOM) is obtained.
  • ISCOM matrices prepared with hydrolyzed saponins also have the same high stability, as characterized by their Zeta potential of -22 mV.
  • New Quil A itself and the compounds according to the invention, whether or not they are hydrolyzed and/or modified, can be used independently as adjuvant or be involved in the forming of the ISCOM matrix or ISCOMS.
  • the compounds are suitable for use in hemolysis and they can be used for the targeted release of medicines.
  • the substances have the advantage that they can be identified as pure components and as a limited group of 3 almost homologous compounds having strongly related properties with respect to adjuvant activity, and in the case of QUADRI-2 show together a low toxicity, because of which QUADRI-2 in particular and/or its components can therefore probably be registered as adjuvant acceptable for use in for instance human vaccines.

Abstract

L'invention concerne un extrait cellulaire végétal présentant une activité d'adjuvant que l'on peut obtenir en extrayant une matière végétale fraîche constituée de cellules vivantes provenant de végétaux produisant de la saponine du genre Quillaja, Saponaria ou Gypsophilia. L'invention porte également sur des composés présentant une activité d'adjuvant que l'on peut obtenir en isolant chaque composant de l'extrait cellulaire végétal. L'extrait et les composés ainsi obtenus peuvent être utilisés dans des adjuvants et des vaccins.
PCT/NL1994/000238 1993-09-30 1994-09-30 Composes presentant une activite d'adjuvant WO1995009179A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU10777/95A AU1077795A (en) 1993-09-30 1994-09-30 Compounds with adjuvant activity

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
NL9301690 1993-09-30
NL9301690A NL9301690A (nl) 1993-08-12 1993-09-30 Verbindingen met adjuvans-activiteit.

Publications (1)

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WO1995009179A1 true WO1995009179A1 (fr) 1995-04-06

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WO (1) WO1995009179A1 (fr)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996011711A1 (fr) * 1994-10-12 1996-04-25 Iscotec Ab Preparations a base de saponine et leur utilisation dans des complexes immunostimulants
AU686891B2 (en) * 1994-10-12 1998-02-12 Iscotec A.B. Saponin preparations and use thereof in iscoms
WO2000009075A2 (fr) * 1998-08-14 2000-02-24 Galenica Pharmaceuticals, Inc. Saponines modifiees chimiquement et leur utilisation comme adjuvants
WO2008030154A1 (fr) * 2006-09-08 2008-03-13 Hemocue Ab Agent d'hémolyse
EP2011517A1 (fr) 2001-04-04 2009-01-07 Nordic Vaccine Technology A/S Complexes liant les polynucléotiques comprenant des stérols et de la saponine
US7776343B1 (en) 1999-02-17 2010-08-17 Csl Limited Immunogenic complexes and methods relating thereto
US8808692B2 (en) 2001-12-21 2014-08-19 Csl Limited Compositions comprising immunoreactive reagents and saponins, and methods of use thereof
US20150313991A1 (en) * 2012-12-10 2015-11-05 Universidad De La Republica De Uruguay Vaccination adjuvant, preparation and vaccines containing the same
WO2019191317A1 (fr) 2018-03-28 2019-10-03 Obi Pharma Inc. Nouvel adjuvant de saponine et son procédé d'évaluation

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988009336A1 (fr) * 1987-05-29 1988-12-01 Cambridge Bioscience Corporation Adjuvant a base de saponine
WO1990003184A1 (fr) * 1988-09-30 1990-04-05 Bror Morein Matrice a activite immunomodulatrice
WO1992006710A1 (fr) * 1990-10-23 1992-04-30 De Staat Der Nederlanden Vertegenwoordigd Door De Minister Van Welzijn Volksgezondheid En Cultuur Complexes immunogenes, notamment des iscomes
WO1993005789A1 (fr) * 1991-09-18 1993-04-01 Cambridge Biotech Corporation Conjugues de saponine/antigene et leur utilisation
WO1994010291A1 (fr) * 1992-10-30 1994-05-11 Seed Capital Investment (Sci) B.V. CELLULES DE CULTURE DE $i(QUILLAJA SP.)

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988009336A1 (fr) * 1987-05-29 1988-12-01 Cambridge Bioscience Corporation Adjuvant a base de saponine
WO1990003184A1 (fr) * 1988-09-30 1990-04-05 Bror Morein Matrice a activite immunomodulatrice
WO1992006710A1 (fr) * 1990-10-23 1992-04-30 De Staat Der Nederlanden Vertegenwoordigd Door De Minister Van Welzijn Volksgezondheid En Cultuur Complexes immunogenes, notamment des iscomes
WO1993005789A1 (fr) * 1991-09-18 1993-04-01 Cambridge Biotech Corporation Conjugues de saponine/antigene et leur utilisation
WO1994010291A1 (fr) * 1992-10-30 1994-05-11 Seed Capital Investment (Sci) B.V. CELLULES DE CULTURE DE $i(QUILLAJA SP.)

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Title
CHEMICAL ABSTRACTS, vol. 119, no. 13, 27 September 1993, Columbus, Ohio, US; abstract no. 136941, G. KERSTEN: "New Inactivated Vaccines: Immune-Stimulating Complexes" page 657; column 1; *
K. DALSGAARD ET AL: "A Study of the Isolation and Characterisation of the Saponin Quil-A. Evaluation of its Adjuvant Activity, with a Special Reference to the Application in the Vaccination of Cattle against Foot and Mouth Disease", ACTA VETERINARIA SCANDINAVICA, SUPPLEMENTUM, no. 69, 1978, COPENHAGEN, DK, pages 1 - 40 *
PHARM. WEEKBL., vol. 128, no. 29, July 1993 (1993-07-01), pages 834 - 838 *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996011711A1 (fr) * 1994-10-12 1996-04-25 Iscotec Ab Preparations a base de saponine et leur utilisation dans des complexes immunostimulants
AU686891B2 (en) * 1994-10-12 1998-02-12 Iscotec A.B. Saponin preparations and use thereof in iscoms
US6352697B1 (en) 1994-10-12 2002-03-05 Iscotec A.B. Saponin preparations and use thereof in iscoms
WO2000009075A2 (fr) * 1998-08-14 2000-02-24 Galenica Pharmaceuticals, Inc. Saponines modifiees chimiquement et leur utilisation comme adjuvants
WO2000009075A3 (fr) * 1998-08-14 2000-06-22 Galenica Pharmaceuticals Inc Saponines modifiees chimiquement et leur utilisation comme adjuvants
US6262029B1 (en) 1998-08-14 2001-07-17 Galenica Pharmaceuticals, Inc. Chemically modified saponins and the use thereof as adjuvants
US8173141B2 (en) 1999-02-17 2012-05-08 Csl Limited Immunogenic complexes and methods relating thereto
US7776343B1 (en) 1999-02-17 2010-08-17 Csl Limited Immunogenic complexes and methods relating thereto
EP2011517A1 (fr) 2001-04-04 2009-01-07 Nordic Vaccine Technology A/S Complexes liant les polynucléotiques comprenant des stérols et de la saponine
US8808692B2 (en) 2001-12-21 2014-08-19 Csl Limited Compositions comprising immunoreactive reagents and saponins, and methods of use thereof
WO2008030154A1 (fr) * 2006-09-08 2008-03-13 Hemocue Ab Agent d'hémolyse
US20150313991A1 (en) * 2012-12-10 2015-11-05 Universidad De La Republica De Uruguay Vaccination adjuvant, preparation and vaccines containing the same
WO2019191317A1 (fr) 2018-03-28 2019-10-03 Obi Pharma Inc. Nouvel adjuvant de saponine et son procédé d'évaluation

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