WO1998004272A1 - Oligosaccharides de chlamydiae - Google Patents

Oligosaccharides de chlamydiae Download PDF

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Publication number
WO1998004272A1
WO1998004272A1 PCT/US1997/013037 US9713037W WO9804272A1 WO 1998004272 A1 WO1998004272 A1 WO 1998004272A1 US 9713037 W US9713037 W US 9713037W WO 9804272 A1 WO9804272 A1 WO 9804272A1
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carbohydrate
chlamydia
mannose
type
oligosaccharides
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PCT/US1997/013037
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English (en)
Inventor
Cho-Chou Kuo
Albertina F. Swanson
Sen-Itiroh Hakomori
Noriko Takahashi
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The University Of Washington
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Publication of WO1998004272A1 publication Critical patent/WO1998004272A1/fr
Priority to US10/732,281 priority Critical patent/US7053067B2/en
Priority to US11/376,337 priority patent/US7517851B2/en

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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
    • A61K31/702—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • 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
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
    • C07H3/06—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages

Definitions

  • Chlamydia trachomatis is one of the most common causes of blindness and sexually transmitted diseases in humans.
  • C. trachomatis is an obligate intracellular bacterium which is biphasic.
  • the intracellular form is the metabolically active reticulate body and the extracellular form is the infectious elementary body (EB) (Moulder et al. (1984) in Berqey s Manual of Systemic Bacteriology (Krieg, ed.) 1:729-735, Williams & Wilkins, Baltimore).
  • EB infectious elementary body
  • MOMP major outer membrane protein
  • the MOMP is the principal structural protein of the EB and individual MOMP proteins are cross-linked by disulfide bonds to provide rigidity to the cell wall (Newhall & Jones (1983) J. Bacteriol. 154:998-1001).
  • the serologic specificity of the organism resides in the MOMP and antibodies raised to MOMP can neutralize infectivity of chlamydia (Caldwell & Perry (1982) Infect. Immun. 38:745-754; Lucero & Kuo (1985) Infect. Immun. 50:595-597).
  • MOMP and two other chlamydial proteins were identified as glycoproteins when the organisms were probed with various plant lectins (Swanson & Kuo (1990) Infect. Immun. 58:502-507). Further characterization showed the three proteins to be glycosylated by way of N-linkage, a structure means rarely found in bacteria (Wieland (1988) Biochimie. 70:1493-1504).
  • the carbohydrate moieties of the MOMP which are involved in the attachment of C. trachomatis and other chlamydiae to host mammalian cells can be used to block attachment and infectivity of chlamydiae.
  • compositions comprising same and methods for using same to block binding of and infectivity of chlamydiae in a host.
  • the instant invention includes compositions and methods for precluding attachment of chlamydiae to host cells.
  • Figure 1 depicts a chromatographic separation of pyridylaminated oligosaccharides of the glycan from the 40-kDa MOMP glycoprotein.
  • the structure of the oligosaccharide in each peak is provided in Table 3.
  • Figures 2A and 2B depict inhibition of infectivity of Chlamydia species in HeLa cells with glycopeptides from hen ovalbumin. Ovomucoid was fractionated into "high mannose-type” ( Figure 2A) and "complex-type” ( Figure 2B) glycopeptides using a ConA column. A four-fold dilution of glycopeptides was mixed with an organism suspension, incubated at 35°C for 30 min. and inoculated onto HeLa cell monolayers, in duplicate.
  • Figures 3A and 3B depict inhibition of attachment of Chlamydia trachomatis l ⁇ /434/ to HeLa cells with glycopeptides from hen ovalbumin. Experiments using "high mannose-type" glycopeptides are shown in Figure 3A and with complex glycopeptides in Figure 3B. Tritium-labeled organisms, either live or formalin-fixed, were mixed with 4-fold dilutions of glycopeptides, incubated at room temperature for
  • the carbohydrate moiety of the 40 kDa MOMP and other glycoproteins expressed on chlamydia are involved in attachment of the organism to host cells and the infectivity thereof, that is, the MOMP glycoprotein plays an essential role in the infectivity of the organism in mammalian cells.
  • the carbohydrate moiety of the MOMP glycoprotein is recognized by host mammalian cells in the process of attachment and entry of the organism into host cells.
  • Mammalian cell proteins particularly those at the cell surface membrane, often are N-glycosylated, that is, the amino group of asparagine (AsN) in the sequence, ...AsN-X-Ser/Thr... , wherein X is an amino acid, is glycosylated through a stable N-glycoside linkage, as demonstrated in Table 1.
  • GlcNAc.1 ⁇ 3 Man.1- GlcNAc.1- GIcNAc- ⁇ Asn Mancl GlcNAc/-.” ⁇
  • MOMP The carbohydrate moiety of MOMP binds ConA, wheat germ agglutinin (WGA) and Dolichos biflorus agglutinin; but does not bind to lectins from Ulex europaeus agglutinin, soybean agglutinin or Ricinus communis agglutinin.
  • WGA wheat germ agglutinin
  • Dolichos biflorus agglutinin but does not bind to lectins from Ulex europaeus agglutinin, soybean agglutinin or Ricinus communis agglutinin.
  • GalNAc N-acetyl galactosamine
  • Gal galactose
  • Fuc fucose
  • Binding to WGA is not affected by sialic acid in a hapten inhibition assay or by treatment with sialidase indicating in the chlamydia carbohydrate sialic acid is not present or not exposed on MOMP, but rather that WGA binding likely is attributable to N-acetyl glucosamine (GlcNAc) .
  • the "high mannose-type" structure may contribute to or mediate the attachment of chlamydia to a host cell, and could define infectivity, regardless of species (C. trachomatis , C . pneumoniae , or C. psittaci ) . That revelation is contrary to the generally accepted belief that N-linked structures essentially are absent in microorganisms. However, application of ultramicroanalysis techniques able to analyze a sample of less than 100 ⁇ g of glycan contributed, in part, to discarding that notion.
  • glycopeptides containing "high mannose-type", “complex-type” or “hybrid-type” structures from novel, analogous sources can be used to determine the inhibitory effects of glycopeptides on the infectivity of chlamydia.
  • Glycopeptides of the "high mannose-type” showed greater inhibition of infectivity of chlamydia species than those of the "complex-type” or "hybrid-type". The same trend was observed for inhibition of attachment of live and formalin-fixed organisms to mammalian cells.
  • those having about 8 mannose (Man) residues showed stronger inhibitory effect than those having other numbers of residues.
  • the presence of N-linked structures in MOMP glycoprotein of chlamydia was indicated by the susceptibility thereof to N-glycanase, which causes release of all oligosaccharides.
  • the released oligosaccharides were fluorescence-labeled by pyridylamination followed by two-dimensional HPLC with ion-exchange and hydrophobic chromatography to distinguish the molecules.
  • That structure is defined by the presence of: (i) a trimannosyl core; (ii) mannosyl substitution or branching at an ⁇ xl ⁇ 6Man residue of the trimannosyl core; and (iii) mannosyl substitution or branching at an i ⁇ 3Man residue of the trimannosyl core.
  • trimannosyl core consisting of two mannosyl branches linked ocl ⁇ 6 and «1 ⁇ 3 , respectively, to a mannose residue
  • a ⁇ -GlcNAc residue of a chitobiosyl structure (GlcNAc ⁇ l ⁇ 4GlcNAc) linked to AsN
  • GlcNAc ⁇ l ⁇ 4GlcNAc chitobiosyl structure
  • AsN AsN
  • a "high mannose-type" structure of interest is one with about 8 or more mannose residues.
  • structures wherein the mannose residues linked to the «l ⁇ 6Man and «l ⁇ 3Man branches of the trimannosyl core are of the i ⁇ 6Man, ⁇ l ⁇ 2 and ocl ⁇ 3Man linkage forms in combination with optional ocl ⁇ 2Man substitution at nonreducing Man residues, such as those containing about 10 or 11 mannose residues (identified as ManlO in Table 2, class 1) are of interest.
  • Man6b or Man7b depicted in Table 2 , class 4.
  • Man ⁇ showed the strongest inhibition, followed by Man9 , Man7 and Man6.
  • a mixture of Man7b and Man ⁇ b prepared from ovalbumin glycopeptide had an inhibitory effect comparable to that of Man9, Man7 and Man6.
  • a carbohydrate of interest is one containing plural mannose residues at the terminus of a linear carbohydrate backbone wherein the terminal residue of the backbone to which a mannose is bound is an N-acetyl glucosamine (GlcNAc) residue.
  • GlcNAc N-acetyl glucosamine
  • a plurality of mannose residues in a linear and branch form are present.
  • about five mannose resides are present, see for example Man6b and Man 7b.
  • Optional ⁇ l- ⁇ 2 substation can be present at the reducing ends increasing the mannose count .
  • a terminal mannose can serve as a branch point to which two mannose residues bind resulting in a bifurcated or bi-antennary molecule. Mannose residues then can form two linear chains from that branch point. Either branch itself can bifurcate resulting in a trifurcated or tri-antenarry molecule, or a tetra-antenarry molecule.
  • Preferred structures are those of the "high mannose-type" having at least five mannose residues and up through 12 or more mannose residues. Those containing 6, 7, 8, 9, 10, 11 or 12 residues are of particular interest.
  • a preferred structure also is tri-antennary, that is, contains three branches. Also, a preferred structure has a mannose residue at the reducing ends.
  • the carbohydrates of the instant invention can be made using known techniques or from commercially available starting materials.
  • the carbohydrates can be isolated from appropriate sources using known extraction techniques. Some of those methods are exemplified herein.
  • the carbohydrates of interest can be synthesized chemically or enzymatically and reference to some of those techniques is made herein.
  • the carbohydrates of interest are found on a plurality of chlamydial species and thus may typify the genus, that is, chlamydia may share a common mechanism and means for binding to and infecting mammalian cells and the instant carbohydrates are useable on any of a variety of chlamydia.
  • the carbohydrates of interest can be used to intervene in the attachment and infectivity processes of chlamydial interaction with host mammalian cells and thus can serve a prophylactic or treatment role for combatting chlamydial infection.
  • Chlamydia are responsible for a variety of maladies. In human, C. psittacosis can cause fever and pneumonia.
  • C. pneumonia is responsible for respiratory disorders, such as, pneumonia, bronchitis and sinusitis, and more recently has been correlated with the development of atherosclerotic plaques. Thus, the presence of chlamydia has been correlated with coronary heart disease, myocarditis and endocarditis.
  • C . trachomatis is associated with vision disturbances and blindness as well as with sexually transmitted disease.
  • the organism can affect a variety of reproductive organs. Chlamydial infections can be more severe in the female resulting ultimately in infertility or ectopic pregnancy. Current therapy of chlamydial infection relies primarily on known antibiotic treatment means. However, antibiotic therapy often fails to cure the infection because the organism resides and proliferates intracellularly. Thus, antibiotic therapy may be only partially effective, and particularly only in the early stages of infection.
  • the carbohydrates of the instant invention block attachment of chlamydia to mammalian cells
  • the instant carbohydrates can be used to retard the transmission and dissemination of disease as well as prohibit progression of an infection.
  • the instant carbohydrates can be used in conjunction with standard antibiotic therapy, by blocking attachment and rendering the organisms more susceptible to the known actions of the antibiotics.
  • the instant carbohydrates can be modified to enhance desirable characteristics thereof.
  • various residues can be substituted by analogs to obtain desirable characteristics, such as to prolong the half-life of the molecule thereby enhancing the stability of the molecule in circulation.
  • artificial carbohydrates containing greater than three branches can be configured, using known biosynthetic or chemical synthetic methods, for example, see Merritt et al. (1994) J. Or ⁇ . Chem. 59:4443-4449.
  • branched structures also can contain mannose residues or structurally similar replacements therefor which-provide the same function as a mannose residue but which have ancillary beneficial properties, such as resistance to certain enzymes which, for example, might catalyze the removal of terminal mannose residues from a carbohydrate.
  • ancillary beneficial properties such as resistance to certain enzymes which, for example, might catalyze the removal of terminal mannose residues from a carbohydrate.
  • the specific choice of starting materials to construct an analog molecule which can substitute for a naturally occurring "high mannose-type" molecule of interest but which contains one or more replacements of residues to obtain a molecule with enhanced binding or other beneficial features, such as prolonged half-life, can be made in view of known molecules and mimics which can substitute, for example, for mannose.
  • a means for determining whether an analog or derivative is useable is whether the resulting analog or derivative continues to inhibit attachment of a chlamydia to a host cell, using for example, a binding assay as taught
  • a suitable oligosaccharide e.g. Man8 or Man9
  • a scaffold carrier molecule can be assembled in multivalent form by linking one or more of such molecules to a scaffold carrier molecule, thus providing a plurality of "high mannose-type” structures on a single molecule using methods known in the art.
  • Such multivalent "high mannose-type” structures are likely to have a greater effect on inhibiting binding of chlamydia organisms to host cells.
  • Carbohydrates with an initial lower inhibiting activity can find beneficial use if made multivalent, i.e. linked to a common carrier with suitable spacing.
  • the carrier can be any known inert molecule to which the carbohydrates of interest can be bound using known chemistries.
  • the carrier can be a synthetic molecule or an isolated naturally occurring molecule.
  • Such bivalent or multivalent binding sites could demonstrate an enhanced avidity for a ligand and thus inhibit binding much more efficiently. That concept is termed "monogamous ultivalency" (Klin an & Karush (1967). Immunoche istrv 4: 387-390). A monogamous multivalent structure, relative to a single site, is favored to bind to the ligand by a factor of 10 3 or 10 4
  • the instant invention therefor provides pharmaceutic compositions and methods for treating disorders normally associated with chlamydial infection, such as, optic disorders, respiratory disorders and reproductive disorders comprising:
  • a suitable oligosaccharide is one with a branched terminus comprising a plurality of mannose residues which can block the attachment of chlamydia to mammalian cells.
  • compositions and methods are applicable both for in vitro and in vivo applications.
  • the instant oligosaccharides can be included in tissue culture medium for use with fastidious or valuable cells and cultures as a means for avoiding contamination or loss.
  • Specific other uses include treatment of disorders and disease states arising from chlamydial infection.
  • the composition comprises an effective amount of an appropriate oligosaccharide and a pharmaceutically acceptable carrier, diluent or excipient.
  • the effective amount of an oligosaccharide can be determined using art-recognized methods, such as by establishing dose-response relationships in suitable animal models or in non-human primates and extrapolating to human; extrapolating from suitable in vitro data, for example, as described herein; or by determining effectiveness empirically in clinical trials.
  • Suitable doses of a composition of the instant invention depend on the particular medical application, such as the severity of the disease, the weight of the subject, age of the subject, the half-life in circulation etc. , and can be determined readily by the skilled artisan practicing known techniques. The number of doses, daily dosage and course of treatment may vary from subject to subject.
  • oligosaccharides showed inhibitory effects of infectivity at 20 ⁇ g (Table 4) , while glycopeptides of egg albumin were shown to inhibit infectivity and attachment at concentrations of 6.25 ⁇ g and 1 ⁇ g ( Figure 2A and Figure 3A) .
  • synthetic oligosaccharides may be tested for their inhibitory activities by such known methods.
  • the kinetics of achieving an appropriate and effective blood concentration depend, for example, on the route of administration, serum molecules which sequester the instant compounds, enzymes that inactivate the instant compounds and the like.
  • the phar acokinetics of the instant compounds can be determined following art-recognized methods, such as, administering radiolabelled compound to a test subject and following the time course of plasma presence, tissue distribution and the like.
  • the dose of the instant compounds administered intravenously and the number of doses are determinable by such kinetic data and generally would be adjusted to higher concentrations for an oral or topical form.
  • the oligosaccharides can be administered in a variety of ways such as orally, parenterally and topically. Suitable pharmaceutically acceptable carriers, diluents or excipients for the medicaments of the instant invention depend on the particular use of the medicament and can be determined readily by the skilled artisan. Also, the oligosaccharides can be delivered encapsulated within microspheres, such as liposomes, which can be made of, for example, phosphatidylcholine and cholesterol.
  • the medicament can take a variety of forms, such as, tablets, capsules, bulk or unit dose powders or granules; may be contained within liposomes; or may be formulated into solutions, emulsions, drops, suspensions, ointments, pastes, creams, gels, foams or jellies.
  • Parenteral dosage forms include solutions, suspensions and the like.
  • the medicament is likely to contain any of a variety of art-recognized excipients, diluents, fillers etc.
  • Such subsidiary ingredients include disintegrants, binders, lubricants, surfactants, emulsifiers, buffers, moisturizers, solubilizers and preservatives.
  • the artisan can configure the appropriate formulation comprising the oligosaccharides of interest seeking guidance from numerous authorities and references such as, Goodman & Gilman's. The Pharmaceutical Basis of Therapeutics (6th ed. , Goodman et al., eds., MacMillan Publ. Co., NY, 1980).
  • the effective doses are derived or extrapolated from in vitro studies.
  • the kinetics of achieving an appropriate and effective blood concentration depend, for example, on the route of administration, serum molecules which sequester the instant compounds, enzymes that inactivate the instant compounds and the like. But the pharmacokinetics of the instant compounds can be determined following art-recognized methods, such as, administering radiolabelled compound to a test subject and following the time course of plasma presence, tissue distribution and the like.
  • a suitable form of administration is oral, but generally higher concentrations are required as are modifications which would render the instant compounds resistant to the effects of the gastro-intestinal tract.
  • the instant compounds can be contained within microcapsules, such as liposomes, for enhanced delivery.
  • an aerator means of delivery may be preferred, although an oral or parenteral means is possible as well.
  • the instant carbohydrate may be instilled as drops or as an ointment.
  • the instant carbohydrate may be delivered by a topical means, such as, a liquid, suppository, foam or gel.
  • a topical means such as, a liquid, suppository, foam or gel.
  • Other gynecologic application means such as impregnation in a porous, inert support, such as a sponge, can be used.
  • the biologically effective amount is that amount which yields an observable beneficial change from an abnormal state.
  • the change can be curtailment or stoppage of disease progression or prophylaxis.
  • the determination of a suitable dose thus depends on the abnormal state and is obtained by an artisan practicing known methods, generally an empirical assessment built on cumulative animal and clinical studies. Determination of dose is not a critical aspect of the instant invention.
  • oligosaccharides can be administered in a suitable fashion to assure effective local concentrations.
  • oligosaccharides may be injected in a depot or adjuvant, carried in a surgically situated implant or reservoir that slowly releases a fixed amount of oligosaccharides over a period of time or may be complexed to recognition molecules with the capability of binding to the site presenting with abnormality.
  • An example of such a contemplated scenario is a recognition molecule that is an antibody.
  • Hypaque-76 was obtained from Winthrop Laboratories, Sterling Drug Inc., New York, N.Y. Structurally defined oligosaccharides were obtained from Oxford GlycoSystems, Rosedale, N.Y. N-glycanase was obtained from the Genzyme Corp., Boston, MA. Hen egg ovalbumin and pronase B were obtained from Sigma, St. Louis, MO. Concanavalin A (ConA) -Sepharose and Sephadex G-50 were from Pharmacia AB, Uppsala, Sweden. Tritiated leucine was from Du Pont NEN, Boston, MA.
  • NCS tissue solubilizer and aqueous counting scintillant were from Amersham, Arlington Heights, IL.
  • Chlamydial strains used were C. trachomatis
  • L 2 /434 BU, C. pneumoniae AR-39 and C. psittaci 6BC The organisms were grown in HeLa 229 cells and purified by Hypaque gradient centrifugation (Kuo et al. (1977) in Nongonococcal urethritis and related infections r Hobson & Holmes, ed.), pp. 328-336, American Society for Microbiology, Washington, D.C.). Two hundred fifty mg of purified organisms from five hundred 112 cm 2 culture flasks were used for preparing membrane glycoprotein.
  • the membrane glycoproteins were prepared from C . trachomatis L 2 434/BU as described previously (Swanson & Kuo (1994) Infect. Immun. 62:24-28). MOMP glycoprotein was separated from other proteins in a SDS-12.5% polyacrylamide gel. The 40 kDa band was excised, electroeluted from the gel and stored at -20°C. The material was pooled and concentrated by centrifugation at 5,000 x g at 4°C in an Ultrapure filter unit with an exclusion factor of 10,000 molecular weight (Millipore, Bedford, Mass.). The isolated glycoprotein was delipidated by methanol-chloroform fractionation according to Finne & Krusius (Methods Enzvmol.
  • the glycan was released from the glycoprotein by incubating for 48 h at 37 °C with 0.2 U of N-glycanase. Boiling for 5 min. halted the enzyme reaction. Following the addition of 3 volumes of ice-cold 95% ethanol, the mixture was centrifuged at 5,000 x g for 10 min. The supernatant was removed and saved. The pellet was washed with 75% ethanol and centrifuged again. The supernatants were combined and dried with a stream of nitrogen. The residue was used for structural analysis. Determination of N-linked oligosaccharides was performed by the two-dimensional sugar mapping technique developed by Tomiya et al. (Anal. Biochem.
  • Assays of inhibition of cell culture infectivity by glycopeptides or oligosaccharides were performed using HeLa 229 cell monolayers grown in 96-well microtiter plates (Byrne et al., supra). Serial four-fold dilutions of glycoconjugates were made. Ninety ⁇ l of each glycoconjugate dilution and 2 x 10 4 inclusion forming units/ml of organism suspensions were mixed in a microtiter well and incubated at 35°C for 30 min. Fifty ⁇ l of glycoconjugate/organism mixture were inoculated onto HeLa cell monolayers in duplicate and absorbed at 35°C for 2 h on a rocker platform. Inocula then were removed and the monolayers were washed with Hanks' balanced salt solution. Culture medium was added to the wells, the plates were sealed with parafil and incubated at 35 °C for 72 h .
  • Infectivity was assayed by counting inclusions that were stained by immunofluorescence using a fluorescein isothiocyanate-conjugated chlamydia genus-specific monoclonal antibody, such as, CF-2.
  • Positive controls included monoclonal antibodies 155-35 and RR-402 which neutralize infectivity of L j and AR-39, respectively.
  • Monoclonal antibody KK-12 which does not have neutralizing activity was used as a negative control.
  • the monoclonal antibodies have been described previously (Lucero & Kuo (1985) Infect. Immun . 50:595-597; Swanson & Kuo (1994) Infect. Immun. 62:24-28; and Puolakkainen et al. (1995) Microbiol. Immunol . 39:551-554) .
  • a reduction of more than 50% of inclusion counts is regarded as indicating positive neutralization of chlamydia (Byrne et al.
  • Chlamydial organisms were labeled metabolically by culturing with low leucine (1/10 of the normal concentration) -Eagle's minimum essential medium containing 50 ⁇ Ci of [ 3 H] -leucine per 112 cm 2 flask in the presence of 0.8 ug/ml cycloheximide (Kuo & Grayston, J.T. (1976) Infect. Immun. 13:1103-1109).
  • Tritium-labeled organisms were purified by centrifugation through a cushion of 30% Hypaque-76 and resuspended in phosphate buffered saline (PBS) . An aliquot was used for preparation of formalin-fixed organisms by addition of 0.02% formalin (final concentration) and incubation at 4°C for 72 h. Formalin was removed by centrifugation and a wash with PBS.
  • the digested tissue suspension was dissolved in 10 ml of scintillation fluid and the radioactivity counted in a scintillation counter (LS-5800 series, Liquid Scintillation System, Beckman Instrument, Inc. , Palo Alto, CA) .
  • LS-5800 series Liquid Scintillation System, Beckman Instrument, Inc. , Palo Alto, CA
  • Peaks A through F comprised about 80% of the total oligosaccharides. Peaks A through D and F were identified as "high mannose-type" by two-dimensional sugar mapping as summarized in Table 3. Peaks G and I were identified as a triantennary and biantennary oligosaccharide, respectively, that contained terminal galactose (Table 3) . Quantities of some oligosaccharides were low, such as for peaks E, H and J. The oligosaccharides were N-linked. No sialic acid was found and no O-linked oligosaccharides were detected.
  • Oligosaccharides of Man-8 (contains eight terminal mannose residues) (D1,D3) , which has a structure similar to the oligosaccharide of peak A from MOMP ( Figure 1 and Table 3) , demonstrated the strongest inhibitory effect on infectivity, followed by Man-9, then Man-6 and Man-7 in that order (Table 4) , which had structures similar to the oligosaccharides of peaks B, D and C, respectively.
  • Oligosaccharides not found in chlamydial glycan including an iso er of Man-8 and di-sialylated or galactosylated biantennary oligosaccharide also were tested. Only the "high mannose-type" oligosaccharides showed an inhibitory effect.
  • OLIGOSACCHARIDES oligomannose 9 oligomannose 8 D1,D3 oligomannose 8 (isomer) oligomannose 7 D3 oligomannose 6 asialo-, galactosylated triantennary 50, 46 asialo-, galactosylated biantennary 45, 42 d-sialylated-, galactosylated biantennary (-) 20, 34 conserved trimannosyl core (core) 0 26, 29
  • Glycopeptides from hen egg ovalbumin were separated into fractions containing "complex-type” and "high mannose-type” carbohydrate using a ConA-Sepharose column (Krusius et al. (1976) FEBS Lett. 71:117-120). Briefly, 100 g of ovalbumin was dissolved in 10 ml of 100 mM sodium bicarbonate buffer (buffer A) , passed through a ConA-Sepharose column (5 ml) and washed with 10 column volumes of buffer A.
  • Ovalbumin containing a bi-antennary structure was eluted with 15 mM ⁇ -methyl-glucoside in buffer A (fraction 1) and that containing a "high mannose-type" structure was eluted with 200 mM ⁇ -methyl-mannoside in buffer A (fraction 2) .
  • Fractions 1 and 2 were dialyzed against distilled water, concentrated to a volume of 2 ml, to which 8 mg pronase B, 10 mM CaCl 2 , 0.02% sodium azide in 0.1 M sodium borate buffer (pH 8.0) were added, and digested for 2 days. The digested material was evaporated to 1 ml and fractionated on a Sephadex G-50 column (1.5 x 50 cm). Sugar-containing fractions (monitored by phenol-sulfuric acid reaction) were collected and lyophilized.
  • oligosaccharide peaks were separated in two-dimensional sugar mapping. Three of the peaks present in fraction 2 from the ConA column were identified as "high mannose-type” (Peaks A to C in Table 5) . Oligosaccharides present in fraction 2 from the ConA column were identified as “hybrid-type” or “complex-type” (peaks D to J in Table 5) .

Abstract

La présente invention concerne des oligosaccharides ramifiés contenant du mannose qui induisent la liaison des chlamydiae aux cellules mammaliennes. On a découvert que le glycanne de type à structure riche en mannose bloque l'adhérence des chlamydiae aux cellules mammaliennes et, partant, inhibe l'infectivité. Le glycanne et ses mimétiques, y compris les dérivés multivalents, peuvent être utilisés comme agents pour traiter ou prévenir les maladies humaines dues aux chlamydiae.
PCT/US1997/013037 1996-07-25 1997-07-25 Oligosaccharides de chlamydiae WO1998004272A1 (fr)

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Cited By (1)

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US7556806B2 (en) * 2002-10-11 2009-07-07 University Of Maryland Biotechnology Institute Carbohydrate-based synthetic vaccines for HIV

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