WO2000024406A1 - Procede d'activation des lymphocytes t et agents utiles a cet effet - Google Patents

Procede d'activation des lymphocytes t et agents utiles a cet effet Download PDF

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Publication number
WO2000024406A1
WO2000024406A1 PCT/AU1999/000929 AU9900929W WO0024406A1 WO 2000024406 A1 WO2000024406 A1 WO 2000024406A1 AU 9900929 W AU9900929 W AU 9900929W WO 0024406 A1 WO0024406 A1 WO 0024406A1
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Prior art keywords
etn
gpi
derivative
equivalent
cell
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PCT/AU1999/000929
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English (en)
Inventor
Louis Schofield
Diana Hansen
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The Walter And Eliza Hall Institute Of Medical Research
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Priority to EP99970921A priority Critical patent/EP1126857A4/fr
Priority to AU11425/00A priority patent/AU775222B2/en
Publication of WO2000024406A1 publication Critical patent/WO2000024406A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7032—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a polyol, i.e. compounds having two or more free or esterified hydroxy groups, including the hydroxy group involved in the glycosidic linkage, e.g. monoglucosyldiacylglycerides, lactobionic acid, gangliosides
    • 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
    • A61K39/002—Protozoa antigens
    • A61K39/015—Hemosporidia antigens, e.g. Plasmodium antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A61P33/06—Antimalarials
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates generally to a method of activating T cells and more particularly to a method of activating T cells using glycosylphosphatidylinositol (referred to herein as "GPI") molecules and derivatives or equivalents thereof. Even more particularly the method of the present invention contemplates a method of activating T cells, using GPI molecules, via a CD 1 -restricted pathway.
  • the method of the present invention is useful, inter alia, in a range of therapeutic and/or prophylactic applications including, but not limited to applications which require skewing of the TH1/TH2 response or which require the induction of antibody production.
  • Antibody responses to protein are understood to be MHC restricted. That is, the production of antibodies directed to a given T dependent antigen requires the production of cytokines by stimulated TH2 cells. Said TH2 cells are stimulated following their binding to a MHC II/peptide complex comprising a peptide derived from the processing of said antigen. Since MHC molecules are polymorphic, there exist genetically determined high and low responders to peptide vaccines.
  • GPI anchor surface proteins occur frequently among medically important parasitic and fungal taxa such as Plasmodium, Trypanosoma, Leishmania, Toxoplasma and Candida.
  • GPIs are ubiquitous among eukaryotes, described from T. brucei, T. cruzi, Plasmodium, Leishmania, and Toxoplasma, as well as yeast, insect, fish and numerous mammalian sources (for recent reviews see (1, 2)). GPIs consist of a conserved core glycan (Man ⁇ l- 2Man ⁇ l-6Man ⁇ l-4GlcNH 2 linked to the 6-position of the /wy ⁇ -inositol ring of PI. GPIs are built up on the cytoplasmic face of the endoplamic reticulum by the sequential addition of sugar residues to PI by the action of gly cosy ltransferases.
  • the maturing GPI is then translocated across the membrane to the luminal side of the ER, whence it may be exported to the cell surface, free or in covalent association with proteins.
  • the terasaccharide core glycan may be further substituted with sugars, phosphates and ethanolamine groups in a species and tissue-specific manner.
  • GPI fatty acid moieties can be either diacylglycerols, alky lacy lglycerols, monoalkylglycerols or ceramides, with additional palmitoylations or myristoylations to the inositol ring.
  • the overall picture is of a closely related family of glycolipids sharing certain core features but with a high level of variation in fatty acid composition and side-chain modifications to the sonserved core glycan.
  • the inventors have shown that the antibody response to several parasitic proteins is regulated predominantly through CDl -restricted recognition of the covalently associated GPI moiety by IL-4 producing CD4 + T cells with limited TCR repertoire diversity. In contrast, there is little evidence for MHC II restricted T cell responses to these antigens. GPI moieties, therefore, act as universal T cell sites through presentation by the non-polymorphic CDl restriction element.
  • One aspect of the present invention contemplates a method of activating T cells, said method comprising administering a T cell activating effective amount of a molecule or a complex comprising said molecule which molecule or molecule complex is capable of interacting with CDl on an immune cell to form an association with CDl which association activates helper T cells.
  • Another aspect of the present invention contemplates a method of activating T cells said method comprising administering a T cell activating effective amount of GPI or derivative or equivalent thereof or a complex comprising GPI or derivative or equivalent thereof which GPI or GPI-complex is capable of interacting with CDl on an immune cell to form an association with CDl which association activates helper T cells.
  • Yet another aspect of the present invention contemplates a method of activating helper T cells said method comprising administering a T cell activating effective amount of Plasmodium GPI or derivative or equivalent thereof or a complex comprising said Plasmodium GPI or derivative or equivalent thereof which Plasmodium GPI or Plasmodium GPI complex is capable of interating with CDl on an immune cell to form an association with CDl which association activates helper T cells.
  • said GPI comprises a structure selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substituent
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substituent
  • ⁇ represents ⁇ -linkages which may be substituted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substituted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a structure selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substituent
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substituent
  • ⁇ represents ⁇ -linkages which may be substituted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substituted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a structure selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substituent
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substituent
  • ⁇ represents ⁇ -linkages which may be substituted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substituted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a structure selected from:
  • said GPI comprises a structure selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substituent
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substituent
  • ⁇ represents ⁇ -linkages which may be substituted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substituted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a structure selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substituent
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substituent
  • ⁇ represents ⁇ -linkages which may be substituted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substituted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a structure selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substituent
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substituent
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substituted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a structure selected from:
  • GPI comprises a structure selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substituent
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substituent
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a structure selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substituent
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substituent
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • Another aspect of the present invention contemplates a method of activating helper T cells said method comprising administering a T cell activating effective amount of GPI or derivative or equivalent thereof or a complex comprising said GPI or derivative or equivalent thereof which GPI or GPI complex is capable of interacting with CDl on an immune cell to form an association with CDl which association activates CD4+ NK1.1 + T cells.
  • Yet another aspect of the present invention contemplates a method of activating CD4 + , NK1.1 + T cells said method comprising administering a T cell activating effective amount of a Plasmodium GPI or derivative or equivalent thereof or a complex comprising said Plasmodium GPI or derivative or equivalent thereof which Plasmodium GPI or Plasmodium GPI complex is capable of interacting with CDl on an immune cell to form an association with CDl which association activates CD4 + NK1.1 + T cells.
  • Still another aspect of the present invention contemplates a method of activating T helper cells said method comprising administering a T cell activating effective amount of a GPI or derivative or equivalent thereof or a complex comprising said GPI or derivative or equivalent thereof which GPI or GPI complex is capable of interacting with CDl on an immune cell to form an association with CDl which association activates T helper cells wherein said activated T cells provide B cell help.
  • Still yet another aspect of the present invention contemplates a method of activating CD4 + , NK1.1 + T cells said method comprising administering a T cell activating effective amount of a Plasmodium GPI or derivative or equivalent thereof or a complex comprising said Plasmodium GPI or derivative or equivalent thereof which Plasmodium GPI or Plasmodium GPI complex is capable of interacting with CDl on an immune cell to form an association with CDl which association activates CD4 + , NK1.1 + T cells wherein said activated T cells provide B cell help.
  • Yet still another aspect of the present invention contemplates a method of activating T helper cells said method comprising administering a T cell activating effective amount of a GPI or derivative or equivalent thereof or a complex comprising said GPI or derivative or equivalent thereof which GPI or GPI complex is capable of interacting with CDl on an immune cell to form an association with CDl which association activates T helper cells wherein said activated T cells induce or otherwise upregulate a THl-type response.
  • a further aspect of the present invention contemplates a method of activating T helper cells said method comprising administering a T cell activating effective amount of a GPI or derivative or equivalent thereof or a complex comprising said GPI or derivative or equivalent thereof which GPI or GPI complex is capable of interacting with CDl on an immune cell to form an association with CDl which association activates T helper cells wherein said activated T cells induce or otherwise upregulate a TH2-type response.
  • Another further aspect of the present invention provides a method of inducing, in a mammal, an immune response directed to a GPI said method comprising administering to said mammal a T cell activating effective amount of GPI or derivative or equivalent thereof which GPI is capable of interacting with CDl on an immune cell to form an association with CDl which association activates helper T cells.
  • Still another further aspect of the present invention provides a method of inducing, in a mammal, an immune response directed to an antigen, said method comprising administering to said mammal a T cell activating effective amount of GPI or derivative or equivalent thereof complexed to said antigen, which GPI-antigen complex is capable of interacting with CDl on an immune cell to form an association with CDl, which association activates helper T cells.
  • Still yet another further aspect of the present invention contemplates a method of treating a mammal said method comprising administering to said mammal an effective amount of GPI or derivative or equivalent thereof or a complex comprising said GPI or derivative or equivalent thereof which GPI or GPI complex is capable of interacting with CDl on an immune cell to form an association with CDl which association activates helper T cells.
  • GPI or derivative or equivalent thereof which GPI or GPI complex is capable of interacting with CDl on an immune cell to form an association with CDl which association activates helper T cells.
  • Another aspect of the present invention contemplates the use of GPI or derivative or equivalent thereof or a complex comprising said GPI or derivative or equivalent thereof which GPI or GPI complex is capable of interacting with CDl on an immune cell, in the manufacture of a medicament for the activation of helper T cells in a mammal.
  • Yet another aspect of the present invention provides a method for the treatment and/or prophylaxis of a mammalian disease condition characterised by a micro-organism infection, said method comprising administering to said mammal an effective amount of GPI or derivative or equivalent thereof or a complex comprising said GPI or derivative or equivalent thereof which GPI or GPI complex is capable of interacting with CDl on an immune cell to form an association with CDl which association activates helper T cells.
  • Still another aspect of the present invention provides a method for the treatment and/or prophylaxis of a mammalian disease condition characterised by the insufficiency or absence of an appropriate TH1 response said method comprising a ⁇ jriinistering to said mammal an effective amount of GPI or derivative or equivalent thereof or a complex comprising said GPI or derivative or equivalent thereof which GPI or GPI complex is capable of interacting with CDl on an immune cell to form an association with CDl which association induces or otherwise upregulates a TH1 response.
  • Still yet another aspect of the present invention provides a method for the treatment and/or prophylaxis of a mammalian disease condition characterised by the insufficiency or absence of an appropriate TH2 response said method comprising administering to said mammal an effective amount of GPI or derivative or equivalent thereof or a complex comprising said GPI or derivative or equivalent thereof which GPI or GPI complex is capable of interacting with CDl on an ⁇ nmune cell to form an association with CDl which association induces or otherwise upregulates a TH2 response.
  • Yet still another aspect of the present invention is directed to a composition which activates T cells, said composition comprising a GPI or derivative or equivalent thereof or a complex comprising GPI or derivative or equivalent thereof which GPI or GPI-complex is capable of interacting with CDl on an immune cell to form an association with CDl which association activates helper T cells.
  • a furter aspect of the present invention relates to an ⁇ nmunogenic composition
  • an ⁇ nmunogenic composition comprising as the active component GPI or derivative or equivalent thereof or GPI complex, as broadly described above.
  • Another further aspect of the present invention is directed to a pharmaceutical composition capable of activating T cells, said composition comprising a GPI or derivative or equivalent thereof or a complex comprising GPI or derivative or equivalent thereof which GPI or GPI-complex is capable of interacting with CDl on an immune cell to form an association with CDl, which association activates helper T cells, together with one or more pharmaceutically acceptable carriers and/or diluents.
  • Figure 1 is a graphical representation of the survival from PbA infection in Balb/c mice wild type (O), Balb/c mice CD Id-/- (•), and C57B/6 (A) mice. The results are from a single experiment representative of 2 independent experiments.
  • Figure 2 is a graphical representation of the percentage of CD4 + cells producing IL-4 or IFN- ⁇ during the infection with PbA.
  • Balb/c wild type ( ⁇ ) and Balb/c CDl-/- ( ⁇ ) mice were infected with 1 x 106 PbA infected red blood cells.
  • the animals were sacrificed and the spleen cells stained for CD4+ and intracellular cytokine content. The percentage of CD4+ cells producing IL-4 or IFN- ⁇ is indicated.
  • FIG. 3 Diagrammatical representation of GPI structures used in this study . Purification and compositional analyses are as described.
  • A COOH-terminal GPIs from T. brucei nd P. falciparum. Boxed areas represent modifications found in PfGPI. The cleavage site of mfVSG by phosphatidylinositol-specific phospholipase C (PI-PLC) is indicated.
  • B Free iM2 iM4 and EP-iM4 GPIs of L. mexicana. Nomenclature is as described, where all isomers contain one mannose in al-3 linkage, EP indicates ethanolamine phosphate, and M2, M4 indicate number of mannose residues, as shown.
  • C Chemically synthesized rat brain Thy-1 GPI.
  • FIG. 4 Response of peripheral NKT cells to purified GPIs in vitro.
  • A As dete ⁇ riined by forward (FSC) and side (SSC) light scatter, splenocytes from SPZ-primed class II ' donors proliferate within 48h. exposure to PfGPI (unshaded) compared with medium controls (shaded). The responding cells are NK1.1 + , CD4 + , and include a V a 14 + , CD4 + subset.
  • Splenocytes from class II " ' " donors were exposed to various antigens and [ 3 H]TdR incorporation determined after 4 days.
  • FIG. 6 CDl -restricted antibody formation to neo-GPI-proteins and malaria SPZ.
  • A Donor nu/nu mice were primed twice with P. berghei SPZ or twice with LPS FLU . Splenocytes were cultured in the presence of lOU/ml IL-2, with and without antigen (O. lmg/ml sham-OVA FLU , PfGPI-OVA FLU , or 5xl0 4 SPZ), anti-Class I, anti-Class II and anti-CD 1, with 10 4 NKT cells from SPZ-primed Class II " ' " donors.
  • Antigen-specific IgG production was quantified by ELISPOT against fluoresceinated Dog serum albumin for responses to OVA FLU , and rCS for responses to SPZ.
  • the present invention is predicated, in part, on the surprising determination that the helper T cell response to several parasitic proteins is predominantly regulated through CD1- restricted recognition of the GPI moiety by CD4+ T cells. This determination has facilitated the development of methodology for application, inter alia, in improving immune responses via regulation of helper T cell stimulation, for example, where the immune response is desired for the purpose of therapeutic or prophylactic vaccination.
  • one aspect of the present invention contemplates a method of activating T cells, said method comprising administering a T cell activating effective amount of a molecule or a complex comprising said molecule which molecule or molecule complex is capable of interacting with CDl on an immune cell to form an association with CDl which association activates helper T cells.
  • the present invention contemplates a method of activating T cells said method comprising administering a T cell activating effective amount of GPI or derivative or equivalent thereof or a complex comprising GPI or derivative or equivalent thereof which GPI or GPI-complex is capable of interacting with CDl on an immune cell to form an association with CDl which association activates helper T cells.
  • activating helper T cells is a reference to upregulating one or more of the functions which said T cells are capable of performing upon stimulation by an antigen, such as, but not limited to, one or more of cell division, differentiation, cell surface molecule expression or cytokine production.
  • association should be understood in its broadest sense to include any form of interaction between a molecule and CDl .
  • Said molecule and CDl may interact via, for example, a covalent bond, ionic bond, hydrogen bond, van Der Waals forces or other interactive bonding mechanism.
  • reference to “GPI” should be read as including reference to all forms of GPI and derivatives or equivalents thereof.
  • Reference to “derivatives” or “equivalents” should be understood to include reference to fragments, parts, portions, chemical equivalents, mutants, homologs and analogs. Chemical equivalents of GPI can act as a functional analog of GPI. Equivalents may not necessarily be derived from GPI but may share certain conformational similarities.
  • chemical equivalents may be specifically designed to mimic certain physiochemical properties of GPI.
  • Chemical equivalents may be chemically synthesised or may be detected following, for example, natural product screening.
  • Equivalents also include synthetic carbohydrates and peptide mimetics. Homologs of GPI contemplated herein include, but are not limited to, GPI from different species.
  • GPI molecules suitable for use in the present invention may be derived from any natural or synthetic source. This includes, for example, GPI moieties derived by genetic manipulation of expression systems and by manipulations of the GPI post-translational modification of proteins via recombinant DNA techniques such as glycosylation inhibitors.
  • GPI moieties suitable for use in the present invention include but are not limited to microorganism GPI moieties which cause disease conditions Such as, the parasitic, fungal and yeast taxa Plasmodium, Trypanosoma, Leishmania, Toxoplasma and Candida.
  • said GPI is a parasite GPI and even more preferably a Plasmodium GPI.
  • the present invention contemplates a method of activating helper T cells said method comprising administering a T cell activating effective amount of Plasmodium GPI or derivative or equivalent thereof or a complex comprising said Plasmodium GPI or derivative or equivalent thereof which Plasmodium GPI or Plasmodium GPI complex is capable of interating with CDl on an immune cell to form an association with CDl which association activates helper T cells.
  • said Plasmodium is V. falciparum.
  • said GPI comprises a structure selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substiment
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a structure selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substiment
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a structure selected from: EtN-P-[M ⁇ 2][G]M ⁇ 2M ⁇ 6M ⁇ 4G-Y EtN-P-[M ⁇ 2] [X]M ⁇ 2M ⁇ 6M ⁇ 4G- Y EtN-P-[M ⁇ 2][EtN-P]M ⁇ 2M ⁇ 6M ⁇ 4G-Y
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substituent
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a structure selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substiment
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a structure selected from:
  • said GPI comprises a structure selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substiment
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a structure selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substiment
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a structure selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substiment
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a structure selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substiment
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a structure selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substiment
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • lipid or “phospholipid” should be understood in its broadest sense and includes, but is not limited to, diacylglycerol, alky lacy lglycerol, monoalkylglycerol, ceramide, sphingolipids and phospholipids such as phosphatidylethanolamine, phosphayidylcholine and phosphatidylserine.
  • any of these preferred structures may be further modified by substituents of positive, negative or neutral charge such as phosphates, phosphoglycerol, hexosamines, amino acids, thiols etc in any position and with any type of linkage. This may be particularly useful in the generation of self GPI structures which are typically more highly substituted along the length of the glycan than protozoal GPIs.
  • GPI "complex” is a reference to a GPI moiety coupled to any other molecule.
  • Said molecule may be any molecule to which an immune response is sought, for example, a carbohydrate or a peptide, polypeptide or protein such as, but not limited to, peptides, polypeptides or proteins naturally anchored to GPI moieties (for example, malarial CS protein, MSP-1, MSP-2, Leishmanial PSA-2 or GP63) or any peptides, polypeptides or proteins artificially coupled to a GPI moiety (for example an influenza antigen).
  • Said molecule and said GPI moiety may be covalently linked or may be linked by ionic, hydrogen or other interactive bonding mechanisms. Coupling may be achieved by a variety of techniques including, but in no way limited to, use of a specific expression system or via chemical synthesis.
  • said molecule is a protein.
  • immunodetaenor cell should be understood as a reference to any cell of the immune system such as, but not limited to, myeloid cells, stromal cells or antigen presenting cells (for example macrophages).
  • helper T cells should be understood as a reference to any cell expressing a T cell receptor (expression of a "T cell receptor” is defined as the expression of one or more of an ⁇ , ⁇ , ⁇ and/or ⁇ T cell receptor chain in either homodimeric or heterodhneric form) which can become activated via a CDl-restricted recognition pathway instead of, or in addition to, a capacity to become activated via a MHC II restricted recognition pathway and which acts to stimulate, upregulate or otherwise modulate any aspect of the immune response via any one or more of a variety of mechanisms including, for example, cell-cell contact or production of soluble mediators.
  • Said T cells include, but are not limited to, thymically derived T cells.
  • Preferably said T cells express CD4 and even more preferably CD4 and NK1.1. This cell type is often referred to in the art as the "NKT cell” .
  • the present invention contemplates a method of activating helper T cells said method comprising administering a T cell activating effective amount of GPI or derivative or equivalent thereof or a complex comprising said GPI or derivative or equivalent thereof which GPI or GPI complex is capable of interacting with CDl on an immune cell to form an association with CDl which association activates CD4+ NK1.1 + T cells.
  • GPI GPI moiety
  • a molecule complexed to the GPI moiety by a non-polymorphic CDl restriction element.
  • CDl is expressed on a variety of cells including, for example, macrophages. Coupling of the GPI moiety (or the molecule complexed to the GPI) to the CDl may be by covalent bonding. Recognition of the CD1-GPI unit by a subclass of CD4 + T cells leads to their activation.
  • T cells do not recognise the GPI moiety or the molecule complexed to the GPI via the traditional MHC II restricted route of presentation, instead said T cells are activated through CDl- restricted recognition.
  • Said T cells represent a subset of the T cell population of an individual. For example, T cells exhibiting the phenotype CD4 + , NK1.1 + are able to become activated via the CDl-restricted recognition pathway.
  • the present invention should be understood to extend to methods of activating T cells by administration of GPI or GPI complex wherein said T cells are activated by one or both of CDl or MHC II restricted recognition.
  • the present invention contemplates a method of activating CD4 + , NK1.1 + T cells said method comprising adrninistering a T cell activating effective amount of a Plasmodium GPI or derivative or equivalent thereof or a complex comprising said Plasmodium GPI or derivative or equivalent thereof which Plasmodium GPI or Plasmodium GPI complex is capable of interacting with CDl on an immune cell to form an association with CDl which association activates CD4 + NK1.1 + T cells.
  • Plasmodium is P. falciparum.
  • the T cells of the present invention can co-operate with B cells to result in CDl-restricted antibody production, the specificity of said antibody being directed to the GPI moiety (where said GPI is administered in isolation) or to a molecule complexed to the GPI moiety (where a GPI complex is administered).
  • said complexed molecule may be a protein to which an antibody response is desired, such as an influenza antigen.
  • Activation of said B cells is supported by the production of cytokines, such as IL-4, by the CDl-restricted activated T cells.
  • GPI-anchoring therefore, permits antibody formation by a non-MHC restricted immunological process.
  • MHC which is a highly polymorphic molecule
  • CDl is non-polymorphic thereby resulting in little observed non-responsiveness in human populations.
  • the present invention contemplates a method of activating T helper cells said method comprising administering a T cell activating effective amount of a GPI or derivative or equivalent thereof or a complex comprising said GPI or derivative or equivalent thereof which GPI or GPI complex is capable of interacting with CDl on an immune cell to form an association with CDl which association activates T helper cells wherein said activated T cells provide B cell help.
  • T helper cells are CD4 + , NK1.1+ T cells.
  • said GPI molecule is a parasitic, fungal or yeast GPI moiety. Most preferably, said GPI molecule is a parasite GPI and even more preferably a Plasmodium GPI.
  • a related aspect of the present invention contemplates a method of activating CD4 + , NK1.1 + T cells said method comprising administering a T cell activating effective amount of a Plasmodium GPI or derivative or equivalent thereof or a complex comprising said Plasmodium GPI or derivative or equivalent thereof which Plasmodium GPI or Plasmodium GPI complex is capable of interacting with CDl on an immune cell to form an association with CDl which association activates CD4 + , NK1.1 + T cells wherein said activated T cells provide B cell help.
  • Plasmodium is P. falciparum.
  • said GPI comprises a structure selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substiment
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a structure selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substituent
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a structure selected from:
  • said GPI comprises a structure selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substituent
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a structure selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substiment
  • ⁇ represents ⁇ -linkages which may be substituted with ⁇ -linkages wherever required
  • - numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a structure selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substiment
  • ⁇ represents ⁇ -linkages which may be substituted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a structure selected from:
  • GPI comprises a structure selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substiment
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a structure selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substiment
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a structure selected from: EtN-P-[M ⁇ 2] [G]M ⁇ 2M-Y EtN-P-[M ⁇ 2] [X]M ⁇ 2M-Y EtN-P-[M ⁇ 2] [EtN-P]M ⁇ 2M-Y
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substiment
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • B cell help should be understood as a reference to the provision, by said activated T cells, of signals which act to stimulate, up-regulate or otherwise modulate or maintain B cell and/or plasma cell viability or functional activity.
  • Said signals may take any form including, for example, cell/cell contact or the production of soluble mediators such as cytokines.
  • the T cells activated in accordance with the method of the present invention produce a range of cytokines which can modulate aspects of the immune response other than antibody production.
  • CD-I restricted NKT cells produce very high levels of the cytokines IL-4 and IFN- ⁇ suggesting that these cells are involved in the downstream regulation of TH1/TH2 differentiation. Accordingly, these cells are thought to be involved in the aetiology of those diseases which show a pronounced TH1/TH2 dependence, including cerebral malaria, tuberculosis, leprosy, leishmaniasis , type I diabetes, autoimmune arthritis and systemic lupus erythromatosis. They are also thought to be involved in tumour rejection.
  • the up-regulation of NKT cell proliferation and differentiation therefore provides a mechanism for skewing the T helper cell response towards a TH1 or a TH2 response.
  • Skewing of the CDl restricted T helper cell response may be achieved by techniques which are known to those skilled in the art. These techniques include, for example, the route by which the GPI molecule/GPI complex is administered, co- stimulatory signals which are provided together with the GPI molecule/GPI complex and/or the choice of vehicle which is used to administer the GPI molecule/GPI complex.
  • administration of the GPI molecule/GPI complex is alum or liposomes will skew the CDl restricted T helper response towards a TH2 type response while administration of these molecules utilising ISCOM's will skew the response to either the TH1 or TH2 type response depending on the route of adniinistration which is utilised.
  • the administration of an IL-12 or IFN- ⁇ co-stimulatory agent together with the GPI molecule/GPI complex is likely to skew the response towards a TH1 type response while the administration of an IL-4 co-stimulatory agent together with the GPI molecule/GPI complex is more likely to skew the response towards a TH2 type response.
  • the method of the present invention therefore provides not only a mechanism of activating T helper cells in a non-MHC restricted fashion in response to a GPI stimulus, but the method may also be adapted to skew the nature of the immune response which is ultimately effected.
  • up-regulation of the subject T cells can be designed to provide B cell help or to otherwise effect a TH1 or TH2 type response.
  • another related aspect of the present invention contemplates a method of activating T helper cells said method comprising administering a T cell activating effective amount of a GPI or derivative or equivalent thereof or a complex comprising said GPI or derivative or equivalent thereof which GPI or GPI complex is capable of interacting with CDl on an immune cell to form an association with CDl which association activates T helper cells wherein said activated T cells induce or otherwise upregulate a THl-type response.
  • T helper cell is a CD4+ NK 1.1+ cell.
  • GPI molecule is a parasitic, fungal or yeast GPI moiety and even more preferably a Plasmodium GPI.
  • Yet another related aspect of the present invention contemplates a method of activating T helper cells said method comprising administering a T cell activating effective amount of a GPI or derivative or equivalent thereof or a complex comprising said GPI or derivative or equivalent thereof which GPI or GPI complex is capable of interacting with CDl on an immune cell to form an association with CDl which association activates T helper cells wherein said activated T cells induce or otherwise upregulate a TH2-type response.
  • T helper cell is a CD4+ NK1.1+ cell.
  • GPI molecule is a parasitic, fungal or yeast GPI moiety and even more preferably a Plasmodium GPI.
  • THl-type or TH2-type response should be understood as a reference to a T helper cell response which exhibits one or more of the functional characteristics of a TH1 or TH2 response, respectively, and which is directly or indirectly mediated by the CDl restricted T cells which are activated in accordance with the method of the present invention. These characteristics include, but are not limited to the cytokine profile which is induced in the subject. For example, a TH1 response is characterised by the up- regulation of IFN- ⁇ expression while a TH2 response is characterised by the up-regulation of IL-4 production.
  • said GPI comprises a structure selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substiment
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a structure selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substiment
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a structure selected from:
  • GPI comprises a structure selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substituent
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substiment
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a structure selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substiment
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a structure selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substiment
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a strucmre selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substiment
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a strucmre selected from:
  • said GPI comprises a strucmre selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substiment
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a strucmre selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substituent
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • a further aspect of the present invention relates to the use of the invention in relation to disease conditions.
  • the invention in relation to disease conditions. For example:
  • compositions directed to molecules naturally GPI-anchored such as, for example, the malarial CS protein, MSP-1, and
  • MSP-2 Leishmanial PSA-2 and gp63.
  • MHC II restricted T cell activation is undesirable, such as where said MHC II restricted T cell would engender an autoimmune reaction.
  • the M protein of Group A Streptococcus exhibits T cell epitopes which are cross-reactive with human heart tissue but exhibits B cell epitopes protective against the bacterium. Said B cell epitope may be coupled to GPI to facilitate non-MHC restricted antibody formation.
  • the use of the present invention to regulate a TH1/TH2 response provides a mechanism to therapeutically or prophylactically treat disease conditions which show a pronounced TH1/TH2 dependence such as, but not limited to, cerebral malaria, mberculosis, leprosy, leishmaniasis, type I diabetes, autoimmune arthritis, systemic lupus erythromatosis.
  • a pronounced TH1/TH2 dependence such as, but not limited to, cerebral malaria, mberculosis, leprosy, leishmaniasis, type I diabetes, autoimmune arthritis, systemic lupus erythromatosis.
  • skewing towards or inducing a THl response in subjects suffering from neoplasia, cancer or Leishmaniasis is desirable. Skewing towards or induction of a TH2 response is desirable where a subject is suffering from SLE, type I diabetes, autoimmune arthritis and cerebral malaria, for example.
  • the method of the present invention may also be utilised to provide B cell help, thereby leading to an antibody response which is useful, for example, in the treatment or prophylaxis of parasitic infections such as malaria and leishmaniasis.
  • the production of B cell help can be achieved, for example, by inducing a TH2 response.
  • the present invention provides a method of inducing, in a mammal, an immune response directed to a GPI said method comprising administering to said mammal a T cell activating effective amount of GPI or derivative or equivalent thereof which GPI is capable of interacting with CDl on an immune cell to form an association with CDl which association activates helper T cells.
  • said T cell is a CD4+ T cell and even more preferably a CD4+ NK1.1 + T cell.
  • said activated T cell provides B cell help.
  • GPI is Plasmodium GPI.
  • the present invention provides a method of inducing, in a mammal, an immune response directed to an antigen, said method comprising administering to said mammal a T cell activating effective amount of GPI or derivative or equivalent thereof complexed to said antigen, which GPI-antigen complex is capable of interacting with CDl on an immune cell to form an association with CDl, which association activates helper T cells.
  • said T cell is a CD4+ T cell and even more preferably a CD4+ NK1.1 + T cell.
  • said activated T cell provides B cell help.
  • said activated T cell induces or otherwise up- regulates a THl or TH2 response.
  • said GPI comprises a strucmre selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substituent
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substiment
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a strucmre selected from:
  • said GPI comprises a strucmre selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substiment
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a strucmre selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substiment
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a strucmre selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substiment
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a strucmre selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substiment
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a strucmre selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G non-N-acetylated glucosamine
  • GJ. is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substiment
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a strucmre selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substiment
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a strucmre selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substiment
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a strucmre selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substituent
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substiment
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • another aspect of the present invention contemplates a method of treating a mammal said method comprising a ⁇ mnistering to said mammal an effective amount of GPI or derivative or equivalent thereof or a complex comprising said GPI or derivative or equivalent thereof which GPI or GPI complex is capable of interacting with CDl on an immune cell to form an association with CDl which association activates helper T cells.
  • said mammal has a parasitic infection and more preferably a Plasmodium infection.
  • said activated helper T cells provide B cell help.
  • GPI is Plasmodium GPI.
  • said mammal is in need of the induction, modulation or skewing of a helper T cell response along the THl -or TH2 pathway and said activated T helper cells induce, modulate or skew the T helper response along the THl or TH2 pathway.
  • the present invention contemplates a method of treating a mammal said method comprising administering a mammalian helper T cell activation effective amount of a GPI or derivative or equivalent thereof or a complex comprising said GPI or derivative or equivalent thereof which GPI or GPI complex is capable of interacting with CDl on an immune cell to form an association with CDl which association activates helper T cells.
  • said mammal has a parasitic infection and more preferably a Plasmodium infection.
  • said activated helper T cells provide B cell help.
  • GPI is Plasmodium GPI.
  • said mammal is in need of the induction, modulation or skewing of a helper T cell response along the THl or TH2 pathway and said activated T helper cells induce, modulate or skew the T helper response along the THl or TH2 pathway.
  • Yet another aspect of the present invention contemplates the use of GPI or derivative or equivalent thereof or a complex comprising said GPI or derivative or equivalent thereof which GPI or GPI complex is capable of interacting with CDl on an immune cell, in the manufacture of a medicament for the activation of helper T cells in a mammal.
  • said mammal has a parasitic infection and more preferably a Plasmodium infection.
  • said activated helper T cells provide B cell help.
  • GPI is Plasmodium GPI.
  • said mammal is in need of the induction, modulation or skewing of a helper T cell response along the THl or TH2 pathway and said activated T helper cells induce, modulate or skew the T helper response along the THl or TH2 pathway.
  • mammal includes humans, primates, livestock animals (e.g. horses, cattle, sheep, pigs and donkeys) laboratory test animals (e.g. mice, rats, rabbits, guinea pigs) companion animals (e.g. dogs and cats) and captive wild animals (e.g. kangaroos, deer, foxes).
  • livestock animals e.g. horses, cattle, sheep, pigs and donkeys
  • laboratory test animals e.g. mice, rats, rabbits, guinea pigs
  • companion animals e.g. dogs and cats
  • captive wild animals e.g. kangaroos, deer, foxes.
  • the mammal is a human or laboratory test animal. Even more preferably the mammal is a human.
  • the mammal undergoing treatment may be human or an animal in need of therapeutic or prophylactic treatment.
  • the present invention provides a method for the treatment and/or prophylaxis of a mammalian disease condition characterised by a micro-organism infection, said method comprising administering to said mammal an effective amount of GPI or derivative or equivalent thereof or a complex comprising said GPI or derivative or equivalent thereof which GPI or GPI complex is capable of interacting with CDl on an immune cell to form an association with CDl which association activates helper T cells.
  • microorganism infection is a parasitic infection and more preferably a Plasmodium infection. Still more preferably said activated T cells provide B cell help.
  • the present invention provides a method for the treatment and/or prophylaxis of a mammalian disease condition characterised by the insufficiency or absence of an appropriate THl response said method comprising administering to said mammal an effective amount of GPI or derivative or equivalent thereof or a complex comprising said GPI or derivative or equivalent thereof which GPI or GPI complex is capable of interacting with CDl on an immune cell to form an association with CDl which association induces or otherwise upregulates a THl response.
  • the present invention provides a method for the treatment and/or prophylaxis of a mammalian disease condition characterised by the insufficiency or absence of an appropriate TH2 response said method comprising administering to said mammal an effective amount of GPI or derivative or equivalent thereof or a complex comprising said GPI or derivative or equivalent thereof which GPI or GPI complex is capable of interacting with CDl on an immune cell to form an association with CDl which association induces or otherwise upregulates a TH2 response.
  • Reference to a disease condition "characterised by the insufficiency or absence of an appropriate" THl or TH2 response should be understood as a reference to a disease condition in which the appropriate response is either not functional or else is functional at a level too low to be therapeutically or prophylactically effective. It should also be understood as a reference to a condition where one form of helper T cell response is in effect but where it is desirable to skew the response to the other form (for example, skewing a THl response to a TH2 response or vice versa). The skewing of the helper T cell response is particularly useful in disease conditions where one or more of the symptoms exhibited by a patient are due to the functional features characteristic of a THl or TH2 response, for example the cytokine profile.
  • an “effective amount” means an amount necessary at least partly to attain the desired immune response, or to prevent or to delay the onset or inhibit progression or halt altogether, the onset or progression of a particular condition being treated. This amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated, the capacity of the individual's immune system to synthesise antibodies, the degree of protection desired, the formulation of the vaccine, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
  • treatment and prophylaxis are to be considered in its broadest context.
  • treatment does not necessarily imply that a mammal is treated until total recovery.
  • prophylaxis does not necessarily mean that the subject will not eventually contract a disease condition.
  • treatment and prophylaxis include amelioration of the symptoms of a particular condition or preventing or otherwise reducing the risk of developing a particular condition.
  • the term “prophylaxis” may be considered as reducing the severity of onset of a particular condition. “Treatment” may also reduce the severity of an existing condition or the frequency of acute attacks (for example, reducing the severity of initial infection).
  • the GPI or GPI complex defined in accordance with the present invention may be coadmimstered with one or more other compounds or molecules.
  • coadmimstered is meant simultaneous admimstration in the same formulation or in two different formulations via the same or different routes or sequential administration by the same or different routes.
  • sequential administration is meant a time difference of from seconds, minutes, hours or days between the administration of the two types of molecules, These molecules may be administered in any order.
  • the present invention should also be understood to extend to immunogenic compositions for use in the methods as hereinbefore defined.
  • the present invention is directed to a composition which activates T cells, said composition comprising a GPI or derivative or equivalent thereof or a complex comprising GPI or derivative or equivalent thereof which GPI or GPI-complex is capable of interacting with CDl on an immune cell to form an association with CDl which association activates helper T cells.
  • GPI is Plasmodium GPI.
  • said GPI comprises a strucmre selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substiment
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a strucmre selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is .any other substiment
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a strucmre selected from:
  • said GPI comprises a strucmre selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substiment
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a strucmre selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substiment
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a strucmre selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substiment
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a strucmre selected from:
  • GPI is any lipid or phospholipid.
  • said GPI comprises a strucmre selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substiment
  • ⁇ represents ⁇ -linkages which may be substituted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a strucmre selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substiment
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a strucmre selected from: EtN-P-[M ⁇ 2] [G]M ⁇ 2M-Y EtN-P-[M ⁇ 2] [X]M ⁇ 2M-Y EtN-P-[M ⁇ 2] [EtN-P]M ⁇ 2M-Y
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substiment
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • the present invention relates to an immunogenic composition
  • an immunogenic composition comprising as the active component GPI or derivative or equivalent thereof or GPI complex, as broadly described above.
  • said GPI is Plasmodium GPI.
  • said GPI comprises a strucmre selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substiment
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a strucmre selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substiment
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a strucmre selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substiment
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a strucmre selected from: M ⁇ 2[M ⁇ 2][G]M ⁇ 2M ⁇ 6M ⁇ 4G-Y M ⁇ 2[M ⁇ 2] [X]M ⁇ 2M ⁇ 6M ⁇ 4G- Y M ⁇ 2[M ⁇ 2] [EtN-P]M ⁇ 6M ⁇ 4G-Y
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substiment
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a strucmre selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substiment
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a strucmre selected from:
  • ethanolamine P is phosphate
  • M mannose
  • G non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substiment
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a strucmre selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substiment
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a strucmre selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid -labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substiment
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a structure selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substiment
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a strucmre selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substiment
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • Still another aspect of the present invention is directed to a pharmaceutical composition capable of activating T cells, said composition comprising a GPI or derivative or equivalent thereof or a complex comprising GPI or derivative or equivalent thereof which GPI or GPI-complex is capable of interacting with CDl on an immune cell to form an association with CDl, which association activates helper T cells, together with one or more pharmaceutically acceptable carriers and/or diluents.
  • said GPI is Plasmodium GPI.
  • said GPI comprises a strucmre selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substiment
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a strucmre selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substiment
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a strucmre selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substituent
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a strucmre selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substiment
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a strucmre selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substiment
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a strucmre selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substiment
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a strucmre selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substiment
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a strucmre selected from:
  • said GPI comprises a strucmre selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substiment
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • said GPI comprises a strucmre selected from:
  • EtN is ethanolamine
  • P is phosphate
  • M mannose
  • G is non-N-acetylated glucosamine
  • [G] is any non-N-acetylated hexosamine including glucosamine, or any other nitrous-acid labile substiment
  • Ino is inositol or inositol-phosphoglycerol
  • [X] is any other substiment
  • ⁇ represents ⁇ -linkages which may be substimted with ⁇ -linkages wherever required
  • numeric values represent positional linkages which may be substimted with any other positional linkages as required
  • Y is any lipid or phospholipid.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion or may be in the form of a cream or other form suitable for topical application. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of superfactants.
  • the preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilisation.
  • dispersions are prepared by incorporating the various sterilised active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.
  • the active ingredients When the active ingredients are suitably protected they may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet.
  • the active compound For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • Such compositions and preparations should contain at least 1 % by weight of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit. The amount of active compound in such therapeutically useful compositions in such that a suitable dosage will be obtained.
  • Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.1 ⁇ g and 2000
  • the tablets, troches, pills, capsules and the like may also contain the components as listed hereafter: a binder such as gum, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring.
  • a binder such as gum, acacia, corn starch or gelatin
  • excipients such as dicalcium phosphate
  • a disintegrating agent such as corn starch, potato starch, alginic acid and the like
  • a lubricant such as magnesium stearate
  • a sweetening agent such as sucrose, lactose or saccharin
  • a flavouring agent such as peppermint, oil of wintergreen, or
  • tablets, pills, or capsules may be coated with shellac, sugar or both.
  • a syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour.
  • any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
  • the active compound(s) may be incorporated into sustained-release preparations and formulations.
  • Pharmaceutically acceptable carriers and/or diluents include any and all solvents, dispersion media, coatings, antibacterial and antimngal agents, isotonic and absorption delaying agents and the like.
  • the use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active material for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired as herein disclosed in detail.
  • the principal active ingredient is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in dosage unit form as hereinbefore disclosed.
  • a unit dosage form can, for example, contain the principal active compound in amounts ranging from 0.5 ⁇ g to about 2000 mg. Expressed in proportions, the active compound is generally present in from about 0.5 ⁇ g to about 2000 mg/ml of carrier.
  • the dosages are dete ⁇ riined by reference to the usual dose and manner of administration of the said ingredients.
  • the pharmaceutical composition may also comprise genetic molecules such as a vector capable of transfecting target cells where the vector carries a nucleic acid molecule capable of expressing, for example, a functional equivalent to a GPI or derivative thereof.
  • the vector may, for example, be a viral vector and it may be administered by any suitable method including, for example transfection directly into the cells of the mammal being treated or transfection into a host cell, such as a bacterium, yeast or attenuated parasite, which is then introduced into the mammal.
  • Pronase was obtained from Boehringer Mannheim. Octyl-Sepharose, Protein-G Sepharose, n-octylthiogluco-pyranoside (n-otg), phenylmethylsulfonylfluoride (PMSF), /7-tosyl-L-lysinechloromethylketone (TLCK) , N-tosyl-L-phenylalaninechloro- methylketone (TPCK), /?-chloromercuriphenylsulphonic acid (p-CMPS), aprotinin, leupeptin, pepstatin, iodoacetamide, iodoacetic acid and n-ethylmaleimide (NEM) were obtained from Sigma Chemical Co.
  • Sephadex was from Pharmacia. Analytical or HPLC grade, acetic acid, butanol, chloroform, diethyl ether, ethanol, methanol and water were obtained from BDH and Waters. Silica G60 TLC plates were from Merck Darmstadt. Tritiated mannose, glucosamine, myristic and palmitic acids were from Amersham.
  • the recombinant P. falciparum CS protein 2.3 consists of the entire gene except for the C-terminal 21 amino acids.
  • the P. berghei rCS encompasses amino acids 81-277, including the central repetitive domain.
  • mice Male female C57B1/6 wild-type mice, C57B1/6 lacking the MHC Class II gene, or the CDl.1 and CD 1.2 genes, congenic mice on the C57B1/10 or Balb background, Balb/c nu/nu mice and other mouse haplotypes used in the study were maintained in specific pathogen free animal facilities.
  • Bone marrow was derived from the femurs and tibiae of Balb/c, Balb/B, Balb/K donors. T cells and NK cells were depleted by complement-mediated lysis of Thy-1 + , CD4 + , Lyt.2 + and asialo-GM + cells. Bone marrow aspirates in RPMI 1640 + 3% BSA at 10 7 cells/ml were incubated on ice with the appropriate dilution of specific antibody, followed by centrifugation at 2°C and resuspension in low toxicity, mouse
  • Sub ⁇ i ute Sheet (Rule 26) RO/AU lymphocyte absorbed, sterile-filtered Guinea pig complement at 37 °C in RPMI 1640 + 3 % BSA for 1 hr. Syngeneic or allogeneic recipients, maintained on acidified water and tetracycline, were irradiated by cobalt source (1000 Rads) and inoculated i.v. with 10 7 T-depleted cells. Animals were maintained on acidified water and rested for 12 weeks prior to testing for chimerism and use in experiments.
  • thymic lobes were obtained by dissection from neonatal Balb/c, Balb/B, and Balb/K mice and implanted into the interscapular dorsum of adult Balb/c nulnu mice via a subcutaneous incision. Mice were maintained in sterile isolators on acidified water and rested 12 weeks prior to testing for chimerism and use in experiments.
  • the FCB-1 line of Plasmodium falciparum were grown in vitro by the method of Trager and Jensen. Synchronous development of parasites was maintained by the sorbitol method of Lambros and Vanderberg.
  • 3 H-palmitic acid conjugated to defatted bovine serum albumin in molar ratio 1: 1, 3 H-glucosamine or 3 H-mannose were added at a final specific activity of lOmCurie/ml, to RPMI 1640 cultures of 2xl0 10 parasites at the late trophozoite/early schizont stage for 2 hours (for labelling of GPI precursors) or 8 hours (for labelling of protein-bound GPI).
  • Parasites were harvested by 0.05% Saponin lysis and centrifugation in the cold at 15,000g for 20 minutes, followed by two washes in PBS and storage at -70°C.
  • the GPI-anchored MSP-1 and MSP-2 merozoite surface proteins were purified to homogeneity.
  • Biosynthetically labelled malaria parasites at the late schizont stage were lysed in 0.05% Saponin and centrifuged at 15,000g for 20 minutes, and washed as above. The pellet was extracted in 25mM n-octyl-thioglucopyranoside (n-otg), 1 %
  • Sub ⁇ itute Sheet (Rule 26) RO/AU BSA, ImM EDTA, O. lmM EGTA, ImM PMSF, ImM TPCK, O. lmM TLCK, 5mM pCMPS, lmg/ml pepstatin, lmg/ml leupeptin, ImM NEM, 5mM iodoacetamide, 150mM NaCl, 25mM Tris/HCl pH 7.4 by sonication on ice. The extact was clarified by centrifugation at 20,000g for 30 minutes in the cold, and the supernatant decanted
  • T. brucei 118 (MIT AT 1.5) was purified from the blood of infected Wistar rats by DEAE chromatography. lxlO 11 parasites were pre-incubated for 30 minutes in glucose-
  • RPMI 1640 supplemented with 40mM fructose and then labelled in the same medium for 90 minutes either with [ 3 H]-myristic acid conjugated to defatted BSA, or with [ 3 H] -glucosamine.
  • the medium contained lmg/ml tunicamycin. Parasites were washed in cold medium without label, and taken up in lOmM ZnCl 2 , followed by centrifugation at 45,000g. The pellet was put into boiling 25mM n-otg,
  • Substitute Sheet (Rule 26) RO/AU lmM CaCl 2 , ImM MgCl 2 , and ImM MnCl 2 , and passed over a Con-A sepharose column, followed by washing with 10 column volumes of extraction buffer. The column was first eluted with detergent buffer containing 0.5M a-methyl- mannopyranoside and 0.5M a-glucopyranoside, followed by 25mM n-otg in 8M urea. Aliquots were subject to SDS-PAGE and fluorography or staining with Coomassie blue.
  • GPI-anchored P. falciparum MSP-1, MSP-2 and T. brucei 118 (MITat 1.5) mfVSG were labelled with fatty acid or glucosamine as required and purified as above, to lOmg/ml. 600ml methanol was added to 150ml aliquots followed by 150ml CHCI3 and 450ml H2O. The samples were vortexed and microfuged, the supernatant taken for scintillation counting, and the interphase and lower phase mixed with 450ml methanol and re-centrifuged.
  • the pellet was repeatedly extracted with CMW 10:10:3 until partitioning of fatty-acid label into the supernatant was minimal, partitioned between water and water-saturated butanol, precipitated with acetone at -20°C, and the proteins taken up by sonication in 6M Urea, ImM DTT, ImM iodoacetic acid. After 15 minutes at room temperature, the sample was diluted 6 fold and made to 5mM CaCl 2 . 2.5% pre-digested Pronase B was added, and incubated for 72h at 37 °C with 2 additions of 0.25% pronase.
  • the sample was loaded in 5% 1-propanol, 0.1M NH 4 OAc onto pre-equilibrated Octyl-Sepharose, washed and eluted in a linear gradient of 1- propanol (5-60%) in water. GPIs eluted at 35-40% 1-propanol and were spotted onto TLC plates (Si-60) and run in the solvent system C/M/HAc/W 25:15:4:2. The origin was scraped, GPIs eluted and partitioned between water and water-saturated butanol.
  • mexicana were purified to homogeneity. Briefly, promastigotes were extracted twice in CMW (1:2:0.8), the insoluble material removed by centrifugation, and the CMW phase partitioned with 0.6 volume water. The dried upper phase was chromatographed on Octyl-Sepharose as above, and eluted GIPLs further purified by HPTLC using CM/IN NH 4 OH (10:10:3), and scrapings extracted with CMW (1:2:0.8). GIPL concentration and compositional purity was determined by GC-MS, following acid methanolysis and trimethylsilyl (TMS) derivatization.
  • TMS trimethylsilyl
  • /ny ⁇ -Inositol content was measured following acid hydrolysis (6N HCl, 110°C, 16 h) and TMS derivatization, with selected ion monitoring for m/z 305 and 318.
  • scy // ⁇ -Inositol was used as internal standard throughout.
  • GPIs were loaded onto a A DEAE column in 99% methanol, 1 % water and washed with ten column volumes of solvent. They were subsequently eluted in lOO M Ammonium Acetate in 99% methanol, 1 % water and dried under Nitrogen.
  • Base-hydrolysed GPI glycans were spiked with phenol red and blue dextran in lOOmM Ammonium Acetate and further size-fractionated by passage through a 1cm x 1.2 metre Biogel P4 column equilibrated in lOOmM Ammonium acetate in water.
  • the column had previously been exhaustively calibrated by repeated analytical runs with GPI mixed with acid hydrolysed dextran markers to yield the relative elution position of glucose units detected by staining with orcinol in concentrated sulfuric acid. The column runs proved to be highly reproducible.
  • the dextran markers were omitted.
  • the GPI peak was detected by scintillation counting of aliquots.
  • Glycan concentration and compositional purity was determined by GC-MS, following acid methanolysis and trimethylsilyl (TMS) derivatization.
  • yo-Inositol content was measured following acid hydrolysis (6N HCl, 110°C, 16 h) and TMS derivatization, with selected ion monitoring for m/z 305 and 318.
  • scy // ⁇ -Inositol was used as internal standard throughout.
  • GPIs were coupled to proteins by two methods, (i) GPIs were exposed to ImM Traut's reagent (2-iminothiolane) in 40% 1-propanol, 60mM triethanolamine, 7mM potassium phosphate, lOOmM NaCl, ImM EDTA, pH 8.0 in the cold for 90 minutes under nitrogen. The sample was then desalted by gel filtration at 4°C through a small amount of ImM Traut's reagent (2-iminothiolane) in 40% 1-propanol, 60mM triethanolamine, 7mM potassium phosphate, lOOmM NaCl, ImM EDTA, pH 8.0 in the cold for 90 minutes under nitrogen. The sample was then desalted by gel filtration at 4°C through a small
  • Substitute Sheet (Rule 26) RO/AU immobilized dextran desalting column equilibrated in 40% 1-propanol in water. The propanol was dried off under nitrogen and the sample added to maleimide-activated KLH (Pierce) in coupling buffer (7mM potassium phosphate, lOOmM NaCl, ImM EDTA, pH 7.2) overnight followed by quenching with cysteine.
  • KLH maleimide-activated KLH
  • the sample was then desalted by gel filtration at 4°C through a small Biogel P4 column equilibrated in 30mM n-otg, 7mM potassium phosphate, lOOmM NaCl, ImM EDTA, pH 7.2 and added to maleimide-activated KLH (Pierce) in coupling buffer (7mM potassium phosphate, lOOmM NaCl, ImM EDTA, pH 7.2) overnight.
  • the degree of conjugation was estimated by both by comparison of cpms before and after dialysis of the sample against PBS, or by use of Ellmans reagent to quantify sulfhydryl groups. Excess reactive groups were quenched with cysteine.
  • 2xl0 5 cells in ice cold murine tonicity RPMI 1640 with 0.05% Sodium azide and 1 % BSA were incubated with optimally titrated FITC- , biotin- or phycoerythrin-conjugated monoclonal antibodies to murine CD3, CD4, CD5, CD8, CD25, CD28, CTLA-4, CD44, CD69, Thy-1 (NIMR-1 or G7), .and NK1.1 as required.
  • Cytokine levels were determined by specific capture ELISAs (Pharmingen). Treated and control samples were incubated at 37 °C for 2 h in 96-well plates precoated with monoclonal anti-mouse IFN-g, IL-2 and IL-4, followed by washing and probing under the same experimental conditions with biotinylated second antibody anti-mouse IFN-g,
  • Sub ⁇ itute Sheet (Rule 26) RO/AU IL-2 and IL-4, followed by streptavidin-peroxidase. After addition of the substrate and color development, the plates were read by Titertek Multiscan MCC/340 automated ELISA reader. Cytokine mass was calculated by interpolating the results with the standard curve plotted with titrated recombinant mouse IFN- ⁇ , IL-2 and IL-4 of known mass.
  • Antigen (tetanus toxoid, P. falciparum rCS, P. berghei rCS, NANP 40 -BSA, FLU-BSA or BSA alone) at lOug/ml in phosphate binding buffer was incubated overnight in 50ul volumes in flat-bottomed Immunlon 96-well plates, followed by extensive washing with buffer. The plates were blocked with 1 % BSA in PBS for several hours. From a 1/32 dilution, sera were titrated two-fold in 1 % BSA in PBS, and 50ul aliquots incubated in triplicate for 2 hours at room temperature, followed by extensive washing with 1 % BSA, 0.05% Tween-20 in PBS.
  • mice were primed twice with P. berghei SPZs or twice with L P S FLU 1Q 5 S pi enoC y tes were placed in culmre for 7 days in the presence of IL-2, with and without antigen, anti-Class I, anti-Class II and anti-CD 1 as indicated, with 10 4 CD4 + , NK1.1 + cells positively selected by FACS sorting and Dynal Detachabead from
  • P. berghei SPZs or GPI-OVA FLU were added with or without antibodies to CDl, MHC Class I or MHC Class II at various concentrations and cultured in RPMI 1640 + 10% FCS, 5xl0 "5 M b-ME and left for 8 days.
  • Antigen-specific IgG production in the culmre supernatant was determined by ELISA against recCS and fiuoresceinated Dog serum albumin (Sigma) as capture antigen.
  • nude mice engrafted with irradiated neonatal allogeneic thymi produced anti-CS IgGl and IgG2 in response to sporozoites at levels similar to recipients of syngeneic thymic implants, but did not respond to recCS or other protein antigens (Table 1).
  • Class IT' 0 mice lacking both Class II and Class Il-restricted CD4 + T cells, and reportedly incapable of mounting IgG responses to T-dependent protein antigens (4-6), responded to native but not recombinant CS protein with high titres of IgGl, IgG2a and IgG2b, similar to wildtype controls (Table 2).
  • these data demonstrate that thymically-derived CD4 + cells are required for IgG formation in response to the native CS protein, but that the Class II MHC is not a restriction element in this response.
  • GPIs purified from Plasmodium falciparum, Leishmania mexicana and Trypanosoma brucei were covalently linked in a molar ratio of 1: 1 with haptenated Ovalbumin (GPI-OVA FLU ).
  • GPI-OVA FLU haptenated Ovalbumin
  • IgG responses in Class II 0/0 mice were obtained only in response to OVA F U coupled to intact GPI, and not to OVA FLU coupled to the deacylated GPI glycan, indicating the GPI lipid domain is required, and the glycan not sufficient, for the phenomenon (Table 2).
  • GPI-OVA FLU was unable to induce anti-FLU IgG responses in nude mice or euthymic animals treated with lytic anti-CD4 indicating that the GPI does not provide a sufficient signal to drive immunoglobulin class switch in the absence of CD4 + cells.
  • Substitute Sheet (Rule 26) RO/AU pro files revealed both a relative and absolute increase in ' this cell population. This was also accompanied by the production of high levels of IL-4. This unusual population of T cells was considerably expanded following exposure of mice to sporozoites. The proliferative and cytokine response of NK1 + CD4+ T cells to purified GPIs could be blocked by anti-CD 1 monoclonal antibody IB 1(7).
  • splenocytes from sporozoite primed animals were exposed to sporozoites, they produced measurable levels of anti-CS IgG in the culmre supernatant after 8 days.
  • This antibody formation in vitro could be substantially blocked by anti-CD 1 mAb 1B1, but less so by antibodies to Class II and not at all by anti-Class I.
  • the NK1.1 + cells were able to cooperate with B cells in CDl-restricted antibody formation to GPI-OVA FLU but not OVA 1 TM.
  • COOH-terminal GPI's were purified from affinity-purified GPI-anchored proteins of P. falciparum (PfGPI) and Trypanosoma brucei membrane-form variant surface glycoprotein (mfVSG), and non-protein-linked free GPIs from Leishmania mexicana.
  • PfGPI P. falciparum
  • mfVSG Trypanosoma brucei membrane-form variant surface glycoprotein
  • GC-MS gas chromatography-mass spectrometry
  • Substitute Sheet (Rule 26) RO/AU used.
  • NK1.1 + CD4 + cells from wild-type and class II " ' " mice when cultured with irradiated wild-type or class II " ' " antigen-presenting cells (APCs), responded to these purified GPIs as determined by incorporation of [ 3 H] -thymidine ([ 3 H]TdR), and the production of high levels of IL-4 and IFN- ⁇ .
  • APCs antigen-presenting cells
  • NKT cells did not respond to GPIs when cultured with irradiated APCs from ⁇ 2 M “ ' " and CD1.1/CD1.2 “ ' “ (CDl “ ' “ ) donors, or with wild-type and class II " ' " APCs in the presence of anti-CD 1.1 , but responded fully in the presence of isotype controls .
  • the proliferative and IL-4 response to GPI of NKT cells and the V ⁇ 14 + , CD4 + subset in unfractionated splenocytes could also be blocked by the anti-CD 1 mAb 1B1.
  • the recognition of GPIs by NKT cells is MHC-independent and CDl-restricted.
  • NKT cells produced IL-4 in response to CDl.1 -transfected J774 macrophages in the absence of exogenous antigen, but not to sham-transfected controls. Nonetheless, the response was enhanced when CDl.1 -transfectants were pulsed with GPI. However, in the absence of exogenous antigen no cytokine expression was detected in response to APCs expressing normal level of CD 1.1. Thus high levels of CDl.1 expression in transfected cells may alone be sufficient to drive proliferation.
  • NKT cells can respond to mammalian GPIs (for example, synthetic Thy-1 GPI)
  • CD 1.1 -transfectants may be able to present endogenous GPIs to NKT cells.
  • the P. berghei ANKA murine cerebral malaria model has several features in common with the human cerebral malaria syndrome. Specifically, it is a TNF- ⁇ and IFN- ⁇ dependent encephalitis associated with upregulation of ICAM-1 on the cerebral microvascular endothelium, an increase in both parasite and macrophage/neutrophil
  • Substitute Sheet (Rule 26) RO/AU adherence to these target cells, and attendant neurological complications. Unlike human cerebral malaria, there is a breakdown of the blood-brain barrier in the terminal stages of the murine syndrome. However, in the proximal stages the murine disease reflects more accurately the inflammatory cascade leading to cerebral involvement in humans. This syndrome manifests most clearly in C57B16 and CBA mice, and is clearly a IFN- ⁇ dependent, THl type disease. The TH2-promoting cytokine IL-4 can counter this condition and lack of IL-4 makes the condition worse.
  • Balb/c mice are usually refractory to the cerebral malaria syndrome (they preferentially manifest a TH2 response). It was observed however that Balb/c CD1.1/CD1.2 gene targeted (knockout) mice showed a high rate of P. berg&ei ' -induced cerebral malaria ( Figure 1). This suggests that a CD1- dependent mechanism protects against cerebral malaria. Furthermore, in vivo lysis of NKT cells by passive transfer of lytic monoclonal antibody causes expression of the cerebral syndrome, demonstrating a protective effect against cerebral malaria of NKT cells.
  • T cell-dependent IgG responses to protein antigens are thought to be exclusively MHC class II- restricted.
  • allogeneic bone-marrow irradiation chimeras were similar to syngeneic controls in responding to malaria sporozoites (SPZ) with IgG to the Circumsporozoite (CS) protein, despite inability to respond to the nominal protein antigens tetanus toxoid (TT) or a full-length recombinant Plasmodium falciparum CS
  • Sub ⁇ itute Sheet (Rule 26) RO/AU protein (recCS). Nude mice cannot respond to SPZ with" anti-CS IgG, and passive transfer of depleting anti-CD4 antibodies into euthymic animals abolishes the anti-CS response to SPZ. However, nude mide engrafted with irradiated neonatal allogeneic thymi mounted anti-CS IgG responses to SPZ similar to recipients of syngeneic thymi, but did not respond to recCS or TT. Mice lacking both class II and class Il-restricted CD4 + T cells, and unable to respond to T-dependent antigens, produced anti-CS IgG (mean log 2 reciprocal titer of 10) in response to SPZ. Thus CD4 + T cells are required for the IgG response to the native CS protein, but this may proceed through a MHC class II-independent route.
  • inositolphosphoglycan lacking a lipid tail
  • IPG inositolphosphoglycan
  • FLU hapten fluorescein
  • native and synthetic GPIs and IPG were exposed to 2-iminothiolane to introduce a sulfhydryl onto free amino groups, de-salted and conjugated in a molar ratio of 1:1 to fluoresceinated, maleimide-activated ovalbumin (OVA FLU ).
  • Sub ⁇ stitute Sheet (Rule 26) RO/AU responses in class II " ' " mice require linkage of antigen to GPI with an intact lipid, which may be of diacyglycerol or alkylacylglycerol composition.
  • splenocytes from wild-type and class II ' ⁇ animals primed to P. berghei SPZ were exposed to 0.5 ⁇ M of the structures shown in Fig. 3, plus dipalmitoyl-PI.
  • both glycan and fatty acids are required for reconigition, and NKT cells from SPZ-primed donors respond to a range of GPIs from diverse protozoal and mammalian taxa.
  • the results may reflect either broad recognition of diverse antigens by the general population of NKT cells or heterogeneous responses from a clonally mixed population.
  • cells were expanded in the presence of either PfGPI or Thy-1 GPI for four days, rested in IL-2, then re-stimulated with homologous or heterologous antigen. Cells expanded by either antigen responded significantly less well to the heterologous stimulus (Fig. 4B).
  • Substitute Sheet (Rule 26) RO/AU
  • NK1.1 + CD4 + cells from wild-type and class II " ' " mice primed to SPZs were cultured with irradiated wild-type or class II " ' " antigen-presenting cells (APCs), they responded to purified GPIs as determined by incorporation of [ 3 H]-thymidine ([ 3 H]TdR), and the production of high levels of IL-4 (Fig. 5A).
  • APCs antigen-presenting cells
  • NKT cells did not respond to GPIs when cultured with irradiated APCs from ⁇ 2 M “ ' “ and CD1.1/CD1.2 “ ' “ (CDl “ ' “ ) donors, or with wild- type and class II " ' " APCs in the presence of anti-CDl. l (1B1), but responded fully in the presence of isotype controls (Fig. 5A).
  • the proliferative and IL-4 response to PfGPI of NKT cells and the V ⁇ 14 + , CD4 + subset in unfractionated splenocytes could also be blocked by the anti-CD 1 mAB 1B1 (Fig. 5B).
  • NKT cells produced IL-4 in response to CD 1.1 -transfected J774 macrophages in the absence of exogenous antigen, but not to sham-transfected controls. Nonetheless, the response was enhanced when CDl.1 -transfectants were pulsed with PfGPI (Fig. 5A).
  • the response of NKT cells to CD1.1 has been adduced in support of the proposition that this cell population may play a physiological role in the absence of associative recognition of antigen.
  • no cytokine expression was detected in response to APCs expressing normal level of CDl .1.
  • high levels of CDl .1 expression in transfected cells may alone be sufficient to drive proliferation.
  • NKT cells can respond to mammalian GPIs (for example, synthetic Thy-1 GPI (Fig. 4B)
  • CDl.1 -transfectants may be able to present endogenous GPIs to NKT cells.
  • NKT cells cooperated with B cells by ELISPOT assay in CDl-restricted IgG formation to GPI-OVA FLU and native P. berghe CS protein,but not OVA FLU (Fig. 6A).
  • CDl " ' " mice and wild-type controls were exposed to mfVSG FLU , SPZ or recCS. Responses to mfVSG FL ⁇ and SPZ were significantly curtailed in CDl " ' " mice (Fig. 6B), indicating that under these conditions
  • Sub-stitute Sheet (Rule 26) RO/AU the CDl-restricted pathway of IgG formation is a significant component of responses to the native CS protein. Both groups responded equally to recCS, confirming that class Il-dependent responses are unaffected by loss of CDl.
  • Irradiated H-2 congenic mice received 10 7 T-depleted syngeneic or allogeneic bone- marrow cells.
  • Thymic lobes from neonatal Balb/c, Balb/B, and Balb/K mice were irradiated and implanted into adult Balb/c nulnu mice. Mice were rested 12 weeks prior to testing for chimerism.
  • P. falciparum and P. berghei SPZ were dissected from Anopheles freeborni and A. stephensi respectively. Some preparations were kept at - 70 °C before use. Mice were primed with TT, recCS, or SPZ, boosted after 1 week, and IgG responses analyzed by ELISA.
  • RecCS derived from the T4 strain and consisting of the entire sequence except for the C-terminal 21 amino acids (GPI-anchor signal sequence), was used as immunizing antigen and to detect responses to J°. falciparum SPZs.
  • the P. berghei rCS (detection antigen only for P. berghei SPZs) encompasses amino acids 81-277.
  • Antigen-specific end-titers were defined as the last titration giving values statistically different from binding to plates without antigen. A log 2 reciprocal titer of 4.0 was the background cutoff. To TT, recCS and P.
  • syngeneic thymic chimeras mounted mean log 2 reciprocal IgG titers of 15.14, 13.5 and 13.5, respectively.
  • the equivalent values for allogeneic chimeras were 4.5, 4.25 and 13.
  • the results were confirmed using P. falciparum SPZ.
  • IgG responses to rCS, recCS or FLU were determined as above in class II ' ' " and wild- type mice receiving P. berghei SPZ, recCS, mfVSG FLU , sVSG FLU or OVA FLU , either sham-conjugated (sham-OVA FL ⁇ ) or conjugated to P. falciparum GPI (PfGPI-OVA FLU ), Thy-1 GPI (Thy-l-OVA FLU ) or Thy-1 IPG (IPG-OVA FLU ) .
  • Class II ' ' animals failed to raise IgG to recCS, sVSG FLU , sham-OVA FLU and IPG-OVA 1 (mean log 2 reciprocal titers ⁇ 4). However, they produced IgG in response to SPZ, PfGPI-
  • Substitute Sheet (Rule 26) RO/AU OVA FLU , mfVSG FLU and Thy-l-OVA FLU (log 2 reciprocal titer of 10,8 8.25 and 9.75, respectively).
  • Ig isotypes included IgGl, IgG2a and IgG2b.
  • HPTLC high performance thin layer chromotography
  • Substitute Sheet (Rule 26) RO/AU Table 1 MHC Class II is not a restriction element in IgG formation to SPZs.
  • Antigen TT recCS nativeCS
  • Substitute Sheet (Rule 26) RO/AU Table 3.
  • the IgG response to the native CS protein is reduced in CDl. l 0 ' 0 mice

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Abstract

Cette invention se rapporte, de façon générale, à un procédé d'activation des lymphocytes T et, plus particulièrement à un procédé d'activation des lymphocytes T utilisant des molécules de glycosylphosphatidylinositol (appelé ici GPI) et des dérivés ou équivalents de ces molécules. Cette invention concerne encore plus précisément un procédé d'activation des lymphocytes T utilisant des molécules de GPI, via un canal limité par CD1. Ce procédé peut être utilisé, entre autres, dans une large gamme d'applications thérapeutiques et/ou prophylactiques, y compris notamment des applications qui nécessitent un infléchissement de la réponse TH1/TH2 ou qui nécessitent l'induction de la production d'anticorps.
PCT/AU1999/000929 1998-10-27 1999-10-27 Procede d'activation des lymphocytes t et agents utiles a cet effet WO2000024406A1 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
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US8058401B2 (en) 1998-09-14 2011-11-15 The Walter And Eliza Hall Institute Of Medical Research Immunogenic compositions and uses thereof
US8470343B2 (en) 1998-09-14 2013-06-25 The Walter And Eliza Hall Institute Of Medical Research Immunogenic compositions and uses thereof
US7575755B1 (en) * 1998-09-14 2009-08-18 The Walter And Eliza Hall Institute Of Medical Research Immunogenic compositions and uses thereof
WO2002000673A1 (fr) * 2000-06-26 2002-01-03 Rodaris Pharmaceuticals Limited Messagers phosphoglycane et leurs applications medicales
WO2002000674A1 (fr) * 2000-06-26 2002-01-03 Rodaris Pharmaceuticals Limited Messagers phosphoglycane et leurs applications medicales
US8038986B2 (en) 2002-07-26 2011-10-18 The Walter And Eliza Hall Institute Of Medical Research Immunogenic compositions and diagnostic and therapeutic uses thereof
EP1545599A4 (fr) * 2002-07-26 2006-04-05 Inst Medical W & E Hall Compositions immunogenes et utilisations diagnostiques et therapeutiques de celles-ci
EP1545599A1 (fr) * 2002-07-26 2005-06-29 The Walter And Eliza Hall Institute Of Medical Research Compositions immunogenes et utilisations diagnostiques et therapeutiques de celles-ci
US8501183B2 (en) 2002-07-26 2013-08-06 The Walter And Eliza Hall Institute Of Medical Research Immunogenic compositions and diagnostic and therapeutic uses thereof
US7521481B2 (en) 2003-02-27 2009-04-21 Mclaurin Joanne Methods of preventing, treating and diagnosing disorders of protein aggregation
US8859628B2 (en) 2003-02-27 2014-10-14 JoAnne McLaurin Method for preventing, treating and diagnosing disorders of protein aggregation
US9833420B2 (en) 2003-02-27 2017-12-05 JoAnne McLaurin Methods of preventing, treating, and diagnosing disorders of protein aggregation
WO2007060652A1 (fr) * 2005-11-24 2007-05-31 Enzo Biochem, Inc. Beta-glycolipides en tant qu'immunomodulateurs
US7897580B2 (en) 2005-11-24 2011-03-01 Yaron Ilan β glycolipids as immuno-modulators

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