WO2001015727A2 - Compositions de polymeres et d'adjuvants a base de saponine immunostimulants et leurs utilisations - Google Patents

Compositions de polymeres et d'adjuvants a base de saponine immunostimulants et leurs utilisations Download PDF

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Publication number
WO2001015727A2
WO2001015727A2 PCT/US2000/023688 US0023688W WO0115727A2 WO 2001015727 A2 WO2001015727 A2 WO 2001015727A2 US 0023688 W US0023688 W US 0023688W WO 0115727 A2 WO0115727 A2 WO 0115727A2
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adjuvant
saponin
phosphazene
composition
immunostimulatory
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PCT/US2000/023688
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English (en)
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WO2001015727A3 (fr
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Charlotte A. Kensil
Sharon A. Jenkins
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Antigenics Inc.
Avant Immunotherapeutics, Inc.
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Priority to AU70854/00A priority Critical patent/AU7085400A/en
Publication of WO2001015727A2 publication Critical patent/WO2001015727A2/fr
Publication of WO2001015727A3 publication Critical patent/WO2001015727A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants

Definitions

  • the present invention is in the field of immune adjuvants and vaccines.
  • the compositions of the invention enhance the induction of a cell-mediated immune response and enhance antibody production.
  • antigens stimulate the production of antibodies in animals and confer protection against subsequent infection.
  • some antigens are unable to stimulate an effective immune response.
  • the immunogenicity of such a relatively weak antigen is often enhanced by the simultaneous administration of the antigen with an adjuvant.
  • Adjuvants are substances that are not immunogenic when administered alone, but will induce a state of mucosal and/or systemic immunity when combined with the antigen.
  • the development of improved vaccine adjuvants for use in human biomedical applications has, therefore, become a priority of research.
  • Saponin adjuvants have been identified and purified from an aqueous extract of the bark of the South American tree, Qiullaja saponaria Molina.
  • a commercial source of heterogeneous Quillaja saponins is "Quil A” (Dalsgaard, Acta Veterinaria Scandinavica, 69:1 (1978)).
  • Quil A may be further fractionated by HPLC.
  • the more predominant purified saponins have been identified as QS-7, QS-17, QS-18, and QS-21, also known as QA-7, QA-17, QA-18, and QA-21, respectively.
  • saponins have been substantially purified by various methods including high pressure liquid chromatography (HPLC), low pressure liquid silica chromatography, and hydrophilic interactive chromatography (HILIC).
  • HPLC high pressure liquid chromatography
  • HILIC hydrophilic interactive chromatography
  • the substantially purified saponins have been found to be useful as immune adjuvants for enhancing immune responses in individuals.
  • PAA polyacrylic acid
  • CP-AAVPD poly-2- methyl-5-vinyl pyridine
  • PMVP poly-4-vinyl-N-ethylpyridinium bromide
  • PVP-R 2 poly-4-vinyl-N-ethylpyridinium bromide
  • polyphosphazene One polyelectrolyte, polyphosphazene, has been shown to be useful both as a soluble adjuvant and as an encapsulation vehicle for antigen. Antigens may be mixed with the soluble polyphosphazene and injected directly into an animal for parenteral immunization. (Payne, et al., Pharm. Biotechnol, 6:473-493 (1995).)
  • polymers can entrap antigens, microencapsulation has been applied to the injection of pharmaceuticals to give a controlled release, i.e., poly-D,L- lactidecoglycolide (PLGA).
  • PLGA poly-D,L- lactidecoglycolide
  • polyphosphazene microspheres because of their ability to cross-link and their water-solublity characteristics, have become an interesting potential vaccine delivery vehicle.
  • a water-soluble polyphosphazene and antigen solution may be formulated into hydrogel microspheres by ionically cross-linking the carboxyl groups with divalent cations, and then used for parenteral or mucosal administration.
  • Saponin and polymeric adjuvants have each proved to be effective immune stimulators in eliciting certain types of immune responses.
  • a typical maximum level of stimulation from a single adjuvant with a given antigen It would be useful to further optimize the maximum level of immune stimulation.
  • Optimization of an adjuvant effect with a single adjuvant and a single antigen is usually defined by a dose response curve of adjuvant; a maximum or "plateau" level of immune response is achieved at an optimum dose of adjuvant. Further increases in adjuvant effect are not expected from these adjuvants alone by going to higher doses.
  • optimized adjuvant formulations that may drive the immune stimulation to a higher maximum level.
  • the present invention provides an effective immune adjuvant composition of one or more saponin adjuvant and an immunostimulatory polymeric adjuvant that provides for a higher maximum level of immune stimulation.
  • An adjuvant may provide immune stimulation that is delayed (i.e., appearing after multiple immunizations).
  • the present invention provides an immune adjuvant composition that also raises an enhanced immune response at an earlier time in an immunization schedule.
  • An adjuvant may be required in large quantities for optimal immune response.
  • optimized adjuvant formulations that allow the use of less adjuvant, but with equivalent immune stimulation to higher adjuvant doses, used alone.
  • the present invention provides an immune adjuvant composition that provides enhanced immune response (antibody and CTL) compared to low doses of the adjuvants alone.
  • Figure 1 shows the chemical structure of the saponin adjuvant QS-21.
  • Figure 2 depicts a graph showing the enhanced induction of an antibody response of mice having received immunizations with ovalbumin and various adjuvant formulations.
  • C57BL/6 mice were immunized subcutaneously with ovalbumin (25 ⁇ g) and various adjuvant formulations consisting either of QS-21 at suboptimal (1.25 ⁇ g) or optimal (10 ⁇ g) doses, polyphosphazene at suboptimal (10 ⁇ g)
  • mice received immunizations at days 0, 14, and 28. Sera were collected for analysis of anti-ovalbumin IgG at day 42.
  • Figure 3 provides a graph showing the enhanced induction of a cell-mediated immune response by the QS-21 and polyphosphazene combination, as evidenced by the CTL induction.
  • C57BL/6 mice were immunized subcutaneously with ovalbumin
  • mice received immunizations at days 0, 14, and 28.
  • Spleens were collected for analysis of antigen- specific cytotoxic T lymphocyte response at day 42.
  • Splenocytes were stimulated in vitro for 6 days with mitomycin C-treated E.G7-OVA cells. The stimulated splenocytes were used in a standard CTL assay using 51 Cr-loaded E.G7-OVA cells as targets.
  • Figure 4 provides a graph of the induced IgG antibody production.
  • the data show the influenza specific serum IgG response as measured by ELISA.
  • Balb/C mice were subcutaneously immunized with split inactivated influenza virus strain X-31 vaccine (5 ⁇ g) and various adjuvant formulations consisting either of QS-21 at suboptimal (2 ⁇ g) or optimal (20 ⁇ g) doses, polyphosphazene at suboptimal (10 ⁇ g) or optimal (100 ⁇ g) doses, as well as combinations of the two adjuvants. Mice received only a single immunization on day 0. Sera were collected for analysis of anti-influenza IgG at days 21 and 42.
  • Figure 5 shows a graph of enhanced IgG2a antibody production, a response that is influenced by Th 1 cytokines.
  • These data show the influenza specific serum IgG2a response, as measured by ELISA, from mice having received immunizations with influenza vaccine and various adjuvant formulations.
  • Balb/C mice were subcutaneously immunized with split inactivated influenza virus strain X-31 vaccine (5 ⁇ g) and various adjuvant formulations consisting either of QS-21 at suboptimal (2 ⁇ g) or optimal (20 ⁇ g) doses, polyphosphazene at suboptimal (10 ⁇ g) or optimal (100 ⁇ g) doses, as well as combinations of the two adjuvants.
  • Sera were collected for analysis of anti-influenza IgG2a at days 21 and 42.
  • Figure 6 shows a graph of the enhanced IgGl antibody production.
  • mice were subcutaneously immunized with split inactivated influenza virus strain X- 31 vaccine (5 ⁇ g) and various adjuvant formulations consisting either of QS-21 at suboptimal (2 ⁇ g) or optimal (20 ⁇ g) doses, polyphosphazene at suboptimal (10 ⁇ g) or optimal (100 ⁇ g) doses, as well as combinations of the two adjuvants. Mice received only a single immunization on day 0. Sera were collected for analysis of
  • Figure 7 depicts the functional antibody responses as measured by hemagglutination inhibition assay.
  • Balb/C mice were subcutaneously immunized
  • influenza virus (5 ⁇ g) and various adjuvant formulations consisting either of QS-21 at suboptimal (2 ⁇ g) or optimal (20 ⁇ g) doses, polyphosphazene at suboptimal (10 ⁇ g) or optimal (100 ⁇ g) doses, as well as combinations of the two adjuvants.
  • Mice received only a single immunization on day 0.
  • Sera were collected for analysis of hemagglutination inhibition titers at days 21 and 42.
  • Figure 8 provides a graph of induced IgGl antibody production for mice having received immunizations with polysaccharide and various adjuvant formulations.
  • Balb/C mice were immunized subcutaneously with 0.5 ⁇ g Type 14 S. pneumonia capsular polysaccharide (antigen) alone or with adjuvant formulations consisting of QS-21 at suboptimal (1.25 ⁇ g) or optimal (10 ⁇ g) doses, polyphosphazene (100 ⁇ g), and combinations of the two adjuvants.
  • Sera were collected and serum titers to polysaccharide were determined by ELISA on day 21 (mice immunized once, on day 0) or on day 42 (mice immunized twice, on days 0
  • FIG. 9 depicts the enhanced IgG2a response for mice having received
  • mice were immunized subcutaneously with 0.5 ⁇ g Type 14 S. pneumonia capsular polysaccharide (antigen) alone or with adjuvant formulations consisting of QS-21 at suboptimal (1.25 ⁇ g) or optimal (10 ⁇ g) doses, polyphosphazene (100 ⁇ g), and combinations of the two adjuvants.
  • Sera were collected and serum titers to polysaccharide were determined by ELISA on day 21 (mice immunized once, on day 0) or on day 42 (mice immunized twice, on days 0 and 28).
  • Figure 10 shows a graph of the induced IgG3 antibody production for mice having received immunizations with polysaccharide and various adjuvant formulations.
  • Balb/C mice were immunized subcutaneously with 0.5 ⁇ g Type 14 S. pneumonia capsular polysaccharide (antigen) alone or with adjuvant formulations consisting of QS-21 at suboptimal (1.25 ⁇ g) or optimal (10 ⁇ g) doses,
  • polyphosphazene 100 ⁇ g
  • Sera were collected and serum titers to polysaccharide were determined by ELISA on day 21 (mice immunized once, on day 0) or on day 42 (mice immunized twice, on days 0 and 28).
  • Th 1 and Th 2 immunity plays an important role in the protective response to infection with certain microbial agents, a need exists to characterize other novel adjuvant compositions that may safely induce these responses and may potentially be incorporated in future human vaccines.
  • a combination of a synthetic, water-soluble, immunostimulatory phosphazene and a saponin adjuvant was found to be a powerful stimulator of immunity compared to either adjuvant alone.
  • saponin adjuvant is a candidate adjuvant for vaccines to induce immunity.
  • an immune adjuvant composition comprising one or more saponin adjuvants and an immunostimulatory polymeric adjuvant may be administered.
  • the immunostimulatory polymeric adjuvant is a synthetic, water-soluble, immunostimulatory polyelectrolyte.
  • the synthetic, water-soluble, immunostimulatory polyelectrolyte is a polyphosphazene.
  • both the phosphazene and the saponin are biodegradable and exhibit minimal toxicity when administered to individuals, which may be humans or other animals.
  • the immunostimulatory polymeric adjuvant in the immune adjuvant composition is a polyorganophosphazene with an ionized or ionizable pendant group that contains, for example, carboxylic acid or hydroxyl moieties.
  • the preferred immunostimulatory polymeric adjuvant is poly[di(carboxylatophenoxy)phosphazene] or "PCPP.”
  • another embodiment is directed towards the immunostimulatory polymeric adjuvant polyorganophosphazene with (i) an ionized or ionizable pendant group that contains carboxylic acid or hydroxyl moieties and (ii) a pendant group that is susceptible to hydrolysis under the conditions of use, to impart biodegradability to the polymer and wherein the pendant group that is susceptible to hydrolysis is selected from chlorine, amino acid, amino acid ester, imidazole, glycerol, and glucosyl.
  • polyphosphazenes that are useful as immune adjuvants within this genus are poly[di(carboxylatophenoxy)phosphazene-co-di(glycinato)phosphazene- co(carboxylatophenoxy)(glycinato)phosphazene] and poly
  • the saponin adjuvant in the immune adjuvant composition may comprise an unpurified, partially purified, or substantially purified saponin.
  • the saponin adjuvant in the immune adjuvant composition is from Quillaja saponaria Molina. More preferably, the saponin adjuvant may encompass a partially purified saponin-enriched extract "Quil-A.” (Dalsgaard, supra.)
  • the saponin adjuvant is a substantially purified saponin, QS-7, QS-17, QS-18, or QS-21, also
  • the saponin adjuvant may cover a semisynthetic saponin adjuvant.
  • the saponin adjuvant may cover a chemically modified saponin adjuvant or a fraction thereof obtainable from an unpurified Quillaja saponaria extract, wherein the chemical modification of the saponin or fraction thereof consists of conjugation of the saponin glucuronic acid carboxyl to an antigen.
  • a second aspect of the invention provides novel vaccine compositions comprising an immunostimulatory polymeric adjuvant, one or more saponin adjuvants, and an antigen.
  • the vaccine composition stimulates Th 1 and Th 2 immunity. More preferably, the vaccine composition enhances antibody production to an antigen and enhances induction of a cell-mediated immune response to an antigen. In another embodiment, the vaccine composition, most preferably, will synergistically enhance a cell-mediated immune response to an antigen.
  • the saponin adjuvant in the vaccine composition may be an unpurified, partially purified, or substantially purified saponin.
  • the saponin adjuvant in the vaccine composition is from the tree Quillaja saponaria Molina. More preferably, the saponin adjuvant may encompass a partially purified saponin- enriched extract "Quil-A.” (Dalsgaard, supra.)
  • the saponin adjuvant is a substantially purified saponin, QS-7, QS-17, QS-18, or QS-21, also known as QA- 7, QA-17, QA-18, or QA-21, respectively.
  • QS-21 is the most preferred saponin adjuvant in the immune adjuvant composition.
  • the saponin adjuvant may cover a semisynthetic saponin adjuvant.
  • the saponin adjuvant may cover a chemically modified saponin adjuvant or a fraction thereof obtainable from an unpurified Quillaja saponaria extract, wherein the chemical modification of the saponin or fraction thereof consists of conjugation of the saponin glucuronic acid carboxyl to an antigen.
  • the immunostimulatory polymeric adjuvant is a synthetic, water-soluble, immunostimulatory polyelectrolyte.
  • the synthetic, water-soluble, immunostimulatory polyelectrolyte is a polyphosphazene.
  • both the phosphazene and the saponin are biodegradable and exhibit minimal toxicity when administered to individuals, which may be humans or other animals. More preferably, such a vaccine composition may increase an immune response to an antigen in an individual to
  • the immunostimulatory polymer in the vaccine composition is a polyorganophosphazene with an ionized or ionizable pendant group
  • the preferred immunostimulatory polymeric adjuvant is poly[di(carboxylatophenoxy)phosphazene] or "PCPP.”
  • another embodiment is directed towards the immunostimulatory polymeric adjuvant polyorganophosphazene with (i) an ionized or ionizable pendant group that contains carboxylic acid or hydroxyl moieties and (ii) a pendant group that is susceptible to hydrolysis under the conditions of use, to impart biodegradability to the polymer and
  • pendant group that is susceptible to hydrolysis is selected from chlorine, amino acid, amino acid ester, imidazole, glycerol, and glucosyl.
  • polyphosphazenes that are useful as immune adjuvants within this genus are poly[di(carboxylatophenoxy)phosphazene-co-di(glycinato)phosphazene- co(carboxylatophenoxy)(gl cinato)phosphazene] and poly [di(carboxylatophenoxy)phosphazene-co-di(chloro)phosphazene-co- (carboxylatophenoxy)-(chloro)phosphazene].
  • a third aspect of the invention is a method for stimulating the immune response to an antigen in an individual to which the antigen is administered comprising administering an effective amount of a vaccine composition comprising one or more saponin adjuvants, an immunostimulatory polymeric adjuvant, and an
  • a fourth aspect of the invention is a method for enhancing antibody production to an antigen in an individual comprising administering an effective amount of an immune adjuvant composition comprising one or more saponin adjuvant and an immunostimulatory polymeric adjuvant.
  • a fifth aspect encompasses a method for enhancing the induction of a cell-mediated immune response to an antigen in an individual comprising administering an effective amount of an immune adjuvant composition comparing one or more saponin adjuvants and an immunostimulatory polymeric adjuvant.
  • the saponin adjuvant in the immune adjuvant composition according to these method may be an unpurified, partially purified, or substantially purified saponin.
  • the saponin adjuvant in the immune adjuvant composition is from Quillaja saponaria Molina. More preferably, the saponin adjuvant may encompass a partially purified saponin-enriched extract "Quil-A.” (Dalsgaard, supra.)
  • the saponin adjuvant is a substantially purified saponin, QS-7, QS-17, QS-18, or QS-21, also known as QA-7, QA-17, QA-18, or QA-21, respectively.
  • QS-21 is the most preferred saponin adjuvant in the method using the immune adjuvant composition.
  • the saponin adjuvant may cover a semisynthetic saponin adjuvant.
  • the saponin adjuvant may cover
  • a chemically modified saponin adjuvant or a fraction thereof obtainable from an unpurified Quillaja saponaria extract wherein the chemical modification of the saponin or fraction thereof consists of conjugation of the saponin glucuronic acid carboxyl to an antigen.
  • the immunostimulatory polymeric adjuvant is a synthetic, water-soluble, immunostimulatory polyelectrolyte.
  • the synthetic, water-soluble, immunostimulatory polyelectrolyte is a polyphosphazene.
  • both the phosphazene and the saponin are biodegradable and exhibit minimal toxicity when administered to individuals, which may be humans or other animals.
  • the immunostimulatory polymeric adjuvant in the immune adjuvant composition administered in such method is a phosphazene, preferably a polyorganophosphazene with (i) ionized or ionizable pendant groups that contain, for example, carboxylic acid or hydroxyl moieties, and (ii) pendant groups that are susceptible to hydrolysis under the conditions of use, to impart biodegradability to the polymer.
  • Suitable hydrolyzable groups include, for example, chlorine, amino acid, amino acid ester, imidazole, glycerol, and glucosyl.
  • Two examples of polyphosphazenes that are useful as immunoadjuvants are poly[di(carboxylato ⁇ henoxy)phosphazene-co-di(glycinato)phos ⁇ hazene-
  • the saponins of the present invention may be unpurified, partially purified, or substantially purified saponins from any source.
  • the saponins may obtained from the tree Quillaja saponaria Molina.
  • a commercial source of partially purified heterogeneous Quillaja saponins is "Quil A". (Dalsgaard, supra.) "Quil A” may be further fractionated by HPLC.
  • HPLC high pressure liquid chromatography
  • HILIC hydrophilic interactive chromatography
  • glycosidic triterpenoid compounds which produce foam in aqueous solution, have hemolytic activity in most cases, and possess immune adjuvant activity.
  • the term encompasses the saponin per se, as well as natural and pharmaceutically acceptable salts thereof and pharmaceutically
  • spontaneous neuropeptide also encompasses biologically active fragments thereof, as well as semisynthetic saponins.
  • the adjuvant activity of such saponins may be determined by any of a number of methods known to those of ordinary skill in the art.
  • the increase in antibody titer of antibody against specific antigen upon administration of an adjuvant may be used as a criteria for adjuvant activity and determined by any of several methods.
  • one such test involves injecting CD-I mice intradermally with an antigen (for instance, i.e., bovine serum albumin, BSA) mixed with varying amounts of the potential adjuvant. Sera are harvested from the mice two weeks later and tested by ELISA for anti-BSA antibody.
  • Another such test involves injecting inbred mice such as C57BL/6 or Balb/c by subcutaneous route with a protein antigen such as ovalbumin ("OVA"), an inactivated viral vaccine such as a flu vaccine, or a polysaccharide antigen such as pneumococcal polysaccharide, mixed with the potential adjuvant.
  • an antigen for instance, i.e., bovine serum albumin, BSA
  • Sera are harvested from the mice two weeks later and tested by ELISA for anti-BSA antibody.
  • Another such test involves injecting inbred mice such as C57BL/6 or Balb/c by subcutaneous route with a protein antigen such as ovalbumin (“OVA"), an inactivated viral
  • Sera harvested from the mice after one, wo or three immunizations could be harvested and tested by ELISA for antigen-specific antibody (total immunoglobulin) or for specific mouse IgG subclasses such as IgGl or IgG2a.
  • Another such test involves injecting C57BL/6 mice with OVA, harvesting spleens after one, two, or three immunizations, stimulating splenocytes with antigen, and then assaying for cytolytic T lymphocyte activity or killing of OVA-peptide-expressing target cells.
  • a proliferative response involves injecting C57BL/6 mice with OVA, harvesting spleens after one, two, or three immunizations, stimulating splenocytes with antigen, and then assaying for cytolytic T lymphocyte activity or killing of OVA-peptide-expressing target cells.
  • QS-21 designates the mixture of components QS-21 VI and QS- 21V2 which appear as a single peak on reverse phase HPLC on Vydac C4 (5 ⁇ m particle size, 300A pore, 4.6 mm ID x 25 cm length) in 40 mM acetic acid in methanol /water (58/42, v/v).
  • the component fractions are referred to specifically as QS-21V1 and QS-21 V2 when describing experiments performed on the further purified components.
  • the chemical structure of QS-21 is shown in Figure 1. According to Kensil, et al., U.S. Patent No.
  • the carboxyl group on the glucuronic acid of Quillaja saponaria Molina can be conjugated to a protein, a peptide, or a small molecule containing a primary amine.
  • the present invention relates to a chemically modified saponin adjuvant or a fraction thereof obtainable from a crude Quillaja saponaria Molina extract, wherein the chemically modified saponin or fraction thereof comprises at least one of QS-17, QS-18, QS-21, QS-21V1, and QS-21V2, and wherein the modified saponin retains adjuvant activity.
  • spontaneous means a chemical modification of a naturally occurring compound where the compound has been modified by one or more synthetic or degradative reactions to form a compound that does not normally occur in nature.
  • the starting materials for this compound may be purified or unpurified naturally occurring compound.
  • the present invention may also employ modified or semisynthetic saponins that retain adjuvant activity when administered to an individual, which may be a human or another animal. It may employ unpurified, partially purified, or substantially purified saponins.
  • partially purified means saponins partially separated from compounds normally associated with the saponins in its natural state.
  • substantially purified means substantially free from compounds normally associated with the saponin in its natural state and exhibiting constant and reproducible chromatographic response, elution profiles, and biologic activity.
  • substantially purified is not meant to exclude artificial or synthetic mixtures of the saponin with other compounds.
  • the present invention may also employ immunostimulatory saponins isolated from other plant species.
  • a saponin from Doliches lablab has been shown to be useful as an adjuvant [Katayan, et al, Vaccine 17:2733 (1999)].
  • polymer as used herein means long chained polymers that act as immune adjuvants by increasing the immune response to an antigen in an individual to which the polymeric adjuvant and the antigen are administered.
  • polyelectrolyte as used herein means polymers that contain ionized or ionizable pendant groups that render the polymer anionic, cationic, or amphophilic.
  • the ionic groups can be in the form of a salt, or alternatively, an acid or base that is or can be at least partially dissociated.
  • the polyelectrolyte can also contain non-ionic side groups.
  • polyphosphazene as used herein means long chained polymers of phosphazenes with backbones consisting of repeating units that have immune
  • amino acid refers to both natural and synthetic amino acids
  • amino acids includes, but is not limited to alanyl, valinyl, leucinyl, isoleucinyl, prolinyl, phenylalaninyl, tryptophanyl, methioninyl, glycinyl, serinyl, threoninyl, cysteinyl, tyrosinyl, asparaginyl, glutaminyl, asparatoyl, glutaoyl, lysinyl, argininyl, and histidinyl.
  • amino acid ester refers to the aliphatic, aryl, or heteroaromatic carboxylic acid ester of a natural or synthetic amino acid.
  • alkyl as used herein, means a saturated straight, branched, or
  • cyclic hydrocarbon typically of C- to C 20 , and specifically includes methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, cyclopentyl, isopentyl, neopentyl, hexyl, isohexyl, cyclohexyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, heptyl, octyl, nonyl, and decyl.
  • (alkyl or dialkyl)amino means an amino group that has one or two alkyl substituents, respectively.
  • alkenyl and alkynyl means a C 2 to C 20 straight or branched hydrocarbon with at least one double or triple bond, respectively.
  • aryl refers to phenyl or substituted phenyl, wherein the substituent is halo, alkyl, alkoxy, alkylthio, haloalkyl, hydroxyalkyl, alkoxyalkyl, methylenedioxy, cyano, C(0)(lower alkyl), — C0 2 H, — S0 3 H, — P0 3 H, — C0 2 alkyl, amide, amino, alkylamino and dialkylamino, and wherein the aryl group may have up to 3
  • aliphatic refers to hydrocarbon, typically of C- to C 20 , that may contain one or a combination of alkyl, alkenyl, or alkynyl moieties, and which may be straight, branched, or cyclic, or a combination thereof.
  • halo includes fluoro, chloro, bromo, and iodo.
  • aralkyl refers to an aryl group with an alkyl substituent.
  • alkaryl refers to an alkyl group that has an aryl substituent, including benzyl, substituted benzyl, phenethyl or substituted phenethyl, wherein the substituents are as defined above for aryl groups.
  • heteroaryl or “heteroaromatic,” as used herein, refers to an aromatic moiety that includes at least one sulfur, oxygen, or nitrogen in the aromatic ring, and that may be optionally substituted as described above for aryl groups.
  • Nonlimiting examples are furyl, pyridyl, pyrimidyl, thienyl, isothiazolyl, imidazolyl, tetrazolyl, pyrazinyl, benzofuranyl, benzothiophenyl, quinolyl, isoquinolyl, benzothienyl, isobenzofuryl, phyrazolyl, indolyl, isoindolyl, benzimidazolyl, purinyl, carbozolyl, oxaxolyl, thiazolyl, isothiazolyl, l,2,4thiadiazolyl, isooxazolyl, pyrrolyl, pyrazolyl, quinazolinyl, phridazinyl, pyrazinyl, cinnolinly, phthalzinyl, quinoxalinyl, xanthinyl, hypoxanthinyl, pteridinyl, 5-a
  • pharmaceutically acceptable cation refers to an organic or inorganic moiety that carries a positive charge and that may be administered as a counteraction in a phosphazene polyelectrolyte.
  • heteroalkyl refers to an alkyl group that includes a heteroatom such as oxygen, sulfur, or nitrogen (with valence completed by hydrogen
  • the immunostimulatory polymer is a preferably a polyphosphazene that is at least partially soluble in water (typically to an extent of at least 0.001% by weight), an aqueous buffered salt solution, or aqueous alcohol solution.
  • the polyphosphazene preferably contains charged side groups either in the form of an acid or base that is in equilibrium with its counter ion, or in the form of an ionic salt thereof.
  • the polymer is preferably biodegradable and exhibits minimal toxicity when administered to individuals, which may be humans or other animals.
  • Polyphosphazenes are polymers with backbones consisting of alternating phosphorus and nitrogen, separated by alternating single and double bonds. Each phosphorous atom is covalently bonded to two pendant groups ("R").
  • R pendant groups
  • n is an integer.
  • R may be any of a wide variety of moieties that may vary within the polymer, including but not limited to aliphatic, aryl, aralkyl, alkaryl,
  • carboxylic acid heteroaromatic, carbohydrates, including glucose, heteroalkyl, halogen, (aliphatic)amino including alkylaminoheteroaralkyl, di(aliphatic)amino-
  • dialkylamino-, arylamino-, diarylamino-, alkylarylamino-, -oxyraryl including but not limited to -oxyphenylC0 2 H, -oxyphenylS0 3 H, -oxyphenylhydroxyl and -oxyphenylP0 3 H; -oxyaliphatic including -oxyalkyl, -oxy (aliphatic) C0 2 H
  • the groups may be bonded to the phosphorous atom through, for example, an oxygen, sulfur, nitrogen, or carbon atom.
  • the groups will vary randomly throughout the polymer, and the polyphosphazene is thus a random copolymer.
  • Phosphorous may be bound to two like groups, or two different groups.
  • Polyphosphazenes with two or more types of pendant groups may be produced by reacting poly(dichlorophosphazene) with the desired nucleophile or nucleophiles in a desired ratio.
  • the resulting ratio of pendant groups in the polyphosphazene will be determined by a number of factors, including the ratio of starting materials used to produce the polymer, the temperature at which the nucleophilic substitution reaction is carried out, and the solvent system used. While it is very difficult to determine the exact substitution pattern of the groups in the resulting polymer, the ratio of groups in the polymer may be easily determined by one skilled in the art.
  • the immune adjuvant composition comprises a biodegradable polyphosphazene of the formula:
  • a and B may vary independently in the polymer, and may be:
  • a group that is not susceptible to hydrolysis under the conditions of use including, but not limited to an aliphatic, aryl, aralkyl, alkaryl, carboxylic acid, heteroaromatic, heteroalkyl, (aliphatic(amino- including alkylamino-, heteroaralkyl, di(aliphatic)amino including dialkylamino-, arylamino-, diarylamino-, alkylarylamino-, oxyaryl including but not limited to -oxyphenylC0 2 H, -oxyphenyl S0 3 H, -oxyphenylhydroxyl and oxyphenylP0 3 H; -oxyaliphatic including -oxyalkly, -oxy(aliphatic C0 2 H, -oxy(aliphatic) S0 3 H, -oxy(aliphatic P0 3 H, and -oxy(aliphatic
  • the rate of hydrolysis of normally nonhydrolyzable groups may increase to the point that the biodegradation properties of the polymer may be affected.
  • normally nonhydrolyzable groups such as heteroaromatics other than imidazole
  • One of ordinary skill in the art using well known techniques may easily determine whether pendant groups hydrolyze at a significant rate under the conditions of use.
  • One of ordinary skill in the art may also determine the rate of hydrolysis of the polyphosphazenes of diverse structures as described herein, and will be able to select that polyphosphazene that provides the desired biodegradation profile for the targeted use.
  • the degree of hydrolytic degradability of the polymer will be a function of the percentage of pendant groups susceptible to hydrolysis and the rate of hydrolysis of the hydrolyzable groups.
  • the hydrolyzable groups are replaced by hydroxyl groups in aqueous environments to provide P — OH bonds that impart hydrolytic instability to the polymer.
  • the immune adjuvant comprises: (i) a nonbiodegradable polyphosphazene wherein none, or virtually none, of the pendant groups in the polymer are susceptible to hydrolysis under the conditions of use, or (ii) a completely biodegradable polyphosphazene wherein all of the groups are susceptible to hydrolysis under the conditions of use (for example, poly[di(glycinato)phos ⁇ hazene]).
  • Phosphazene polyelectrolytes are defined herein as polyphosphazenes that
  • the ionic groups may be in the form of a salt, or, alternatively, an acid or base that is or may be at least partially dissociated. Any pharmaceutically acceptable monovalent cation may be used as counterion of the salt, including but not limited to sodium, potassium, and ammonium.
  • the phosphazene polyelectrolytes may also be biodegradable or nonbiodegradable under the conditions of use.
  • the ionized or ionizable pendant groups are preferably not susceptible to hydrolysis under the conditions of use.
  • a preferred polyorganophosphazene immune adjuvant contains pendant groups that include carboxylic acid, sulfonic acid, or hydroxyl moieties. While the
  • acidic groups are usually on nonhydrolyzable pendant groups, they may alternatively, or in combination, also be positioned on hydrolyzable groups.
  • An example of a phosphazene polyelectrolyte having carboxylic acid groups as side chains is shown in the following formula:
  • n is an integer, preferably an integer between 10 and 10,000 to 300,000.
  • This polymer has the chemical name poly[di(carboxylatophenoxy)phosphazene] or, alternatively, poly[bis(carboxylatophenoxy)phosphazene] (PCPP).
  • the polyorganophosphazene is preferably biodegradable to prevent eventual deposition and accumulation of polymer molecules at distant sites in the body, such as the spleen.
  • biodegradable means a polymer that degrades within a period that is acceptable in the desired application, typically less than about five years and most preferably less than about one year, once exposed to a physiological solution of pH 6-8 at a temperature of approximately 25°C-37°C.
  • the immunostimulatory polymeric is a polyorganophosphazene that includes pendant groups that include carboxylic acid moieties that do not hydrolyze under the conditions of use and pendant groups that are susceptible to hydrolysis under the conditions of use.
  • preferred phosphazene polyelectrolytes with hydrolysis-sensitive groups are poly[- di(carboxylatophenoxy)phosphazene-co-di(aminoacid)phosphazene- co)carboxylatophenoxy)(amino acid)phosphazene], specifically including poly[di(carboxylatophenoxy)phosphazene-co-di(glycinato)phosphazene-
  • the toxicity of the polyphosphazene may be determined using cell culture experiments well known to those skilled in the art.
  • toxicity of PCPP may be determined in cell culture by coating cell culture dishes with the PCPP. Chicken embryo fibroblasts are then seeded onto the coated petri dishes. Three days after seeding the chicken embryo fibroblasts, the cells are inspected and cell morphology observed. Flattened cells and spindle formulation indicates non-toxicity.
  • Crosslinked polyphosphazenes for use as immunoadjuvants may be prepared by combining a phosphazene polyelectrolyte with a metal multivalent cation such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, or cadmium.
  • a metal multivalent cation such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, or cadmium.
  • immune adjuvant refers to compounds which, when administered to an individual, increase the immune response to an antigen in the individual to which the antigen is administered.
  • such individuals are mammals, and more preferably, the mammals are humans.
  • the invention is not intended to be so limiting. Any animals that may experience the beneficial effects of the vaccines of the invention are within the scope of animals which may be treated according to the claimed invention.
  • Some antigens are weakly immunogenic when administered alone or are toxic to the individual at concentrations which evoke immune responses in said individual.
  • An immune adjuvant may enhance the immune response of the individual to an antigen. The adjuvant effect may also lower the dose of said antigen effective to achieve an immune response in said individual.
  • the term "vaccine composition” herein refers to a composition capable of producing an immune response in an individual administered the composition.
  • a vaccine composition may produce immunity against disease in individuals or may provide therapy in individuals with established disease.
  • the vaccine composition may modulate immune response and be a treatment of autoimmune diseases.
  • the combination of saponin and immunostimulatory polymer of the present invention may be administered to enhance the immune response to any antigen.
  • activation of CTL activity may produce immunity against disease not only prophylactically but also therapeutically (after development of disease).
  • the vaccine composition stimulates Th 1 and Th 2 immunity. More preferably, the vaccine composition enhances antibody production to an antigen and enhances a cell- mediated immune response to an antigen.
  • the immune adjuvant composition of the invention may enhance antibody production to an antigen and enhance the cell-mediated immune response to an antigen in a positive synergistic manner.
  • the synergistic adjuvant effect of the immunostimulatory polymeric adjuvant and the saponin adjuvant described herein may be shown in a number of ways. For example, a synergistic adjuvant effect may be demonstrated as an increase in the maximum expected immune response. One may expect an additive effect of combining two adjuvants. Specifically, if one adjuvant, used at optimum doses, produces "X" and the other adjuvant, also used at optimum doses, produces "Y" antibody, then the combination may be expected to produce "X+Y” if the result is additive and not synergistic. A maximum level of response that is considerably higher than "X+Y" would be considered a synergistic effect and would be unexpected. A second indication of synergism would be the
  • Polyorganophosphazenes including phosphazene polyelectrolytes, may be synthesized by a macromolecular nucleophilic substitution reaction of poly(dichloro phosphazene) with a wide range of chemical reagents or mixture or reagents in accordance with methods known to those skilled in the art.
  • the phosphazene polyelectrolytes are made by reacting the poly(dichloro phosphazene) with an appropriate nucleophile or nucleophiles that displace chlorine.
  • Desired proportions of hydrolyzable to non-hydrolyzable side chains in the polymer may be obtained by adjusting the quantity of the corresponding nucleophiles that are reacted with poly(dichlorophosphazene) and the reaction conditions as necessary.
  • polyphosphazenes for immune adjuvant activity have a molecular weight of over
  • poly[(carboxylatophenoxy)(glycinato)phosphazene] (PC-G1PP) is prepared by the nucleophilic substitution reaction of the chlorine atoms of the ⁇ oly(dichlorophosphazene) with propyl phydroxybenzoate and ethyl glycinate hydrochloride (PC-GIPP synthesis).
  • the poly[(aryloxy)(glycinato)phosphazene] ester thus obtained is then hydrolyzed to the corresponding poly (carboxy lie acid).
  • Other polyphosphazenes may be prepared as described by Allcock, et al., Inorg, Chem.
  • Typical antigens suitable for the enhanced immune response include antigens derived from, but not limited to, any of the following: viruses, such as feline leukemia virus, feline immunodeficiency virus, influenza, HIV-1, HIV-2, rabies, measles, herpes virus, hepatitis B virus, or hoof and mouth disease; bacteria, such as anthrax, chlamydia, diphtheria, Lyme disease, Clostridium tetani, Neisseria gonorrhea, Streptococcus pneumoniae, Haemophilus influenza, tetanus toxoid, or tuberculosis; protozoans, such as Babeosis bovis or Plasmodium; or cells.
  • viruses such as feline leukemia virus, feline immunodeficiency virus, influenza, HIV-1, HIV-2, rabies, measles, herpes virus, hepatitis B virus, or hoof and mouth disease
  • bacteria such as an
  • the antigen may be a protein, a glycoprotein, a peptide, a polysaccharide, a lipid, a glycolipid, a phospholipid, a ceramide, or a nucleic acid encoding an antigenic protein or peptide of interest.
  • the antigens may be purified from a natural source, synthesized by means of solid phase synthesis, or may be obtained by means of genetic engineering.
  • the antigens may be inactivated whole or fractionated viruses or bacteria.
  • the antigens may also be semisynthetic products of an antigen purified from a natural source.
  • the antigen as defined herein elicits an immunogenic response in an individual. Preferably, such individuals are humans, however, the invention is not
  • the immunogenic response may be humoral or cell mediated.
  • the material to which the immunogenic response is to be directed is poorly antigenic, it may be conjugated to a carrier such as albumin or to a hapten, using standard covalent binding techniques, for example, with one of the several commercially available reagent kits.
  • the immunostimulatory polymeric adjuvant of the immune adjuvant composition is used to deliver a nucleic acid which encodes an antigen to a mucosal surface where the nucleic acid is expressed.
  • polyphosphazene microspheres containing the antigen, polyphosphazene and saponin adjuvant may be prepared by simply mixing the components in an aqueous solution, and then coagulating the polymer together with the substance by mechanical forces to form a microparticle.
  • the microparticle/ saponin may be stabilized, if necessary or desired, using electrolytes, pH changes, organic solvents, heat or frost to form polymer matrices encapsulating biological material (antigen, saponin).
  • the polymer may also be used to encapsulate the antigen/saponin, for example, using the method of Cohen, et al, U.S. Patent No. 5,149,543, the teachings of which are incorporated herein, or by spray drying a solution of polymer, antigen, and saponin.
  • the polymer may also be covalently conjugated with the antigen to create a
  • the saponin may be covalently conjugated to the antigen by covalent linkage between an amino or carboxyl group on the antigen and the glucuronic acid carboxyl on the saponin.
  • the polymer is cross-linked with a multivalent ion, preferably using an aqueous solution containing multivalent ions of the opposite charge to those of the charged side groups of the polyphosphazene, such as multivalent cations if the polymer has acidic side groups or multivalent anions if the polymer has basic side groups.
  • the polymers are cross-linked by di- and trivalent metal ions such as calcium., copper, aluminum, magnesium, strontium, barium, tin, zinc, and iron,
  • organic cations such as poly(aminoacid)s, or other polymers such as poly(ethyleneimine), poly(vinylamine) and polysaccharides.
  • the immunogenic vaccine composition may contain other physiologically acceptable ingredients such as water, saline, polysorbate 80, cyclodextrins, or a mineral oil such as DrakeolTM, MarkolTM, and squalene, to form an emulsion.
  • the effective compositions of the present invention may be employed in such forms as capsules, liquid solutions, suspensions or elixirs for oral administration, or sterile liquid forms such as solutions or suspensions.
  • Any inert acceptable carrier may preferably be used, such as saline, PBS, polysorbate 80 in saline or PBS, cyclodextrins, or any such acceptable carrier in
  • compositions of the present invention have suitable solubility properties for use of the present invention.
  • the immunogenic composition may be administered as a vaccine by any method known to those skilled in the art that elicits an immune response, including parenterally, orally, or by transmembrane or transmucosal administration, or other suitable means.
  • the vaccine is administered parenterally (intravenously,
  • Nonlimiting examples of routes of delivery to mucosal surfaces are intranasal (or generally, the nasal associated lymphoid tissue), oral, respiratory,
  • vaginal vaginal, and rectal.
  • the dosage is determined by the antigen loading and by standard techniques for determining dosage and schedules for administration for each antigen, based on titer of antibody elicited by the polymer-antigen administration, as demonstrated by the following examples.
  • the dosage administered may be dependent upon the age, weight, kind of concurrent treatment, if any, and nature of the antigen administered.
  • the initial dose may be followed up with a booster dosage after a period of about two to eight weeks, preferably about four weeks, to enhance the immunogenic response. Further booster dosages may also be administered.
  • the vaccine composition is administered simultaneously
  • the polymer, saponin, and antigen components of the vaccine composition are administered separately to the same or nearby site.
  • the polymer serves to attract cells of the immune system to the site, where they process the antigen.
  • the vaccine compositions, immune adjuvant compositions, and methods of use will be further understood by reference to the following non-limiting examples.
  • PCPP in combination with QS-21 may serve as an adjuvant for a subunit vaccine, ovalbumin (OVA), in mice in inducing CTL responses and /or antibody responses.
  • OVA ovalbumin
  • an immune adjuvant consisting of PCPP with suboptimal doses of QS-21 was evaluated to assess whether PCPP may affect the adjuvant effect of QS-21 at these low doses.
  • mice C57BL/6 mice (5 per group, female, 8-10 weeks of age) were immunized by subcutaneous route at days 0, 14, and 28.
  • the vaccines were 25 ⁇ g ovalbumin (OVA) subcutaneous route at days 0, 14, and 28.
  • the vaccines were 25 ⁇ g ovalbumin (OVA) (antigen) plus the indicated doses of adjuvant in a total volume of 0.2 ml phosphate- buffered saline.
  • Serum titers to OVA were determined by ELISA on sera collected on day 42 from the mice immunized as described in above.
  • IgG antibody titers were determined for individual mice (5 mice per group) and are plotted as a geometric mean titer (GMT). The IgG titers were highest in groups receiving QS-21 alone (at the 10 ⁇ g dose) or PCPP alone (at 100 ⁇ g dose) or 10 ⁇ g QS-21 in combination with either 10 or 100 ⁇ g PCPP as seen in Figure 2.
  • Splenocytes from mice immunized as described in Example 1 were removed at day 42 for use as effector cells in the cytotoxic T lymphocyte (CTL) assay.
  • CTL cytotoxic T lymphocyte
  • Splenocytes were stimulated in vitro for 6 days with mitomycin C-treated E.G7-OVA cells and then used in a standard 51 Cr release CTL assay.
  • E.G7-OVA cells loaded with 51 Cr
  • the background lysis of EL4 cells (not transfected by OVA) was subtracted from the lysis of E.G7-OVA cells to obtain a
  • mice (5 per group, female, 6-8 weeks of age) were immunized by subcutaneous route at day 0.
  • the vaccines were 5 ⁇ g influenza (antigen) plus the indicated doses of adjuvant in a total volume of 0.1 ml phosphate buffered saline.
  • Serum titers to influenza were determined by ELISA on sera collected on day 21 and day 42 from the mice.
  • IgG antibody titers as well as subclass IgGl and IgG2a antibody titers, were determined for individual mice and are plotted as a geometric mean titer. Hemagglutination-inhibition titers were also measured.
  • FIG 4 shows the influenza specific serum IgG response.
  • IgG serum antibody titers were not observed for the suboptimal doses of QS-21 (2 ⁇ g) or PCPP (10 ⁇ g) or in the unadjuvanted group. IgG responses were detectable, however, with
  • Figure 4 shows that at day 42, the GMT of the group receiving 20
  • ⁇ g QS-21 and 100 ⁇ g PCPP was 524,288, which was 3-fold higher than 100 ⁇ g PCPP and 8-fold higher than 20 ⁇ g QS-21.
  • the GMT of the group receiving 100 ⁇ g PCPP and 2 ⁇ g QS-21 was 301,124, which was 1.7-fold higher than 100 ⁇ g PCPP and 56-fold higher than 2 ⁇ g QS-21.
  • the GMT of the group receiving 10 ⁇ g PCPP and 20 ⁇ g QS- 21 was 99,334, which was 21-fold higher than 10 ⁇ g PCPP and 1.5-fold higher than 20 ⁇ g QS-21.
  • the GMT of the group receiving 10 ⁇ g PCPP and 2 ⁇ g QS-21 was 28,526, which was 6-fold higher than 10 ⁇ g PCPP and 5-fold higher than 2 ⁇ g QS-21. These titers are higher than the simple addition of titer increases.
  • the IgG2a response was not detectable in any PCPP dose used alone, with the suboptimal QS-21 dose of 2 ⁇ g, or with the unadjuvanted group.
  • the IgG2a response was minimally detected in the optimal QS-21 dose of 20 ⁇ g and in several of the combinations of PCPP and QS-21.
  • the IgG2a antibody response was significantly enhanced with the adjuvant combination of 20 ⁇ g QS-21 and 100 ⁇ g PCPP. This result was observed both at day 21 and day 42.
  • Serum titers to influenza antigen were determined by hemagglutination inhibition assay (HIA) on sera collected on day 21 and day 42 from the mice immunized in Example 3.
  • the HIA assay is one measure of functional antibody responses against the influenza virus.
  • the hemagglutination inhibition antibody response is shown in Figure 7. The results show that the hemagglutination inhibition titers were highest in groups receiving the adjuvant combination of 100 ⁇ g PCPP and
  • mice (10 per group, female, 8-10 weeks of age) were immunized by subcutaneous route at day 0.
  • the vaccines were 0.5 ⁇ g Type 14 S. pneumonia capsular polysaccharide (antigen) plus the indicated doses of adjuvant in a total volume of 0.2 ml phosphate-buffered saline.
  • Sera were collected from 5 mice per group on day 21. The remaining 5 mice per group were immunized by subcutaneous route on day 28. Sera were collected from these mice on day 42.
  • Serum titers to polysaccharide were determined by ELISA on sera collected on day 21 (mice immunized once) and on sera collected on day 42 (mice immunized twice).
  • IgG subclasses IgGl, IgG2a, and IgG3 titers were determined for an equivolume serum pool.
  • This dose of PCPP was expected to be optimal, based on results in mice with other antigens.
  • the combination of these two doses together yields an IgGl titer of 342 (8- fold higher than with QS-21, 68-fold higher than PCPP).
  • the combination of these two doses together after two immunizations (day 42 sera) yields an IgGl titer that is 19-fold higher than for QS-21 and that is 165-fold higher than for PCPP.
  • Figure 9 shows the results for the antigen-specific serum IgG2a response.
  • a single dose of polysaccharide yields an IgG2a titer of 5.
  • the addition of 10 ⁇ g of QS- 21 yields a titer of 93.
  • the addition of PCPP to the polysaccharide yields a titer of 35.
  • the combination of these doses of QS-21 and PCPP yields a titer that is still 3-fold increased over QS-21 and is 9-fold increased over PCPP.
  • Two immunizations with the combination yield a titer that is 8-fold higher than QS-21 and is 19-fold higher that PCPP.
  • Figure 10 shows the IgG3 data for these same sera.
  • a single immunization with polysaccharide yields a titer of 5.
  • the addition of QS-21 yields a titer of 80.
  • the addition of 100 ⁇ g PCPP yields a titer of 59.
  • the combination of these two doses yields a titer that is 6-fold increased over QS-21 and is 8-fold increased over PCPP.
  • Two immunizations with the combination yield a titer that is 5-fold higher than QS- 21 and is 24-fold higher than for PCPP. These titers are higher than expected from

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Abstract

L'invention concerne des compositions de vaccin comprenant des phosphazènes, des adjuvants à base de saponine, et des antigènes immunostimulants, et leur utilisation pour stimuler une immunité. L'invention concerne également des compositions immunes d'adjuvant à base de phosphazènes et d'adjuvants à base de saponine immunostimulants. Les compositions immunes d'adjuvant peuvent s'utiliser pour améliorer une réponse immunitaire induite par une cellule, et la production d'anticorps. L'invention concerne enfin une technique de stimulation immunitaire par rapport à un antigène.
PCT/US2000/023688 1999-09-02 2000-08-29 Compositions de polymeres et d'adjuvants a base de saponine immunostimulants et leurs utilisations WO2001015727A2 (fr)

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Cited By (7)

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WO2009008988A1 (fr) * 2007-07-09 2009-01-15 Apogee Technology, Inc. Composés de polyphosphazène immunostimulants pour une immunisation intradermique
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
US9061001B2 (en) 2008-10-16 2015-06-23 University Of Saskatchewan Combination adjuvant formulation
WO2018191598A1 (fr) * 2017-04-13 2018-10-18 Adjuvance Technologies, Inc. Synthèse de saponine triterpénique, intermédiaires et combinaisons d'adjuvants
US10906926B2 (en) 2015-11-06 2021-02-02 Adjuvance Technologies, Inc. Triterpene saponin analogues
US11324821B2 (en) 2017-10-16 2022-05-10 Adjuvance Technologies, Inc. Triterpene saponin analogues

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WO1998052573A1 (fr) * 1997-05-20 1998-11-26 Galenica Pharmaceuticals, Inc. Analogues de la saponine triterpenique a activite d'adjuvant et immunostimulatoire
WO1999027959A1 (fr) * 1997-11-28 1999-06-10 Commonwealth Scientific And Industrial Research Organisation Composition a base de saponine, tenant lieu d'adjuvant

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US5494673A (en) * 1993-07-12 1996-02-27 Virus Research Institute Phosphazene polyelectrolytes as immunoadjuvants
WO1998052573A1 (fr) * 1997-05-20 1998-11-26 Galenica Pharmaceuticals, Inc. Analogues de la saponine triterpenique a activite d'adjuvant et immunostimulatoire
WO1999027959A1 (fr) * 1997-11-28 1999-06-10 Commonwealth Scientific And Industrial Research Organisation Composition a base de saponine, tenant lieu d'adjuvant

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7776343B1 (en) 1999-02-17 2010-08-17 Csl Limited Immunogenic complexes and methods relating thereto
US8173141B2 (en) 1999-02-17 2012-05-08 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
WO2009008988A1 (fr) * 2007-07-09 2009-01-15 Apogee Technology, Inc. Composés de polyphosphazène immunostimulants pour une immunisation intradermique
US9061001B2 (en) 2008-10-16 2015-06-23 University Of Saskatchewan Combination adjuvant formulation
US9408908B2 (en) 2008-10-16 2016-08-09 University Of Saskatchewan Combination adjuvant formulation
US10906926B2 (en) 2015-11-06 2021-02-02 Adjuvance Technologies, Inc. Triterpene saponin analogues
WO2018191598A1 (fr) * 2017-04-13 2018-10-18 Adjuvance Technologies, Inc. Synthèse de saponine triterpénique, intermédiaires et combinaisons d'adjuvants
IL269963B1 (en) * 2017-04-13 2024-05-01 Adjuvance Tech Inc Synthesis of triterpene saponins, intermediates and adjuvant combinations
US11324821B2 (en) 2017-10-16 2022-05-10 Adjuvance Technologies, Inc. Triterpene saponin analogues

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