WO2012145626A1 - Oligosaccharides synthétiques destinés à un vaccin contre un staphylocoque - Google Patents

Oligosaccharides synthétiques destinés à un vaccin contre un staphylocoque Download PDF

Info

Publication number
WO2012145626A1
WO2012145626A1 PCT/US2012/034449 US2012034449W WO2012145626A1 WO 2012145626 A1 WO2012145626 A1 WO 2012145626A1 US 2012034449 W US2012034449 W US 2012034449W WO 2012145626 A1 WO2012145626 A1 WO 2012145626A1
Authority
WO
WIPO (PCT)
Prior art keywords
oligosaccharide
composition
mmol
antibody
ratio
Prior art date
Application number
PCT/US2012/034449
Other languages
English (en)
Inventor
A. Stewart Campbell
Obadiah J. Plante
Original Assignee
Ancora Pharmaceuticals Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ancora Pharmaceuticals Inc. filed Critical Ancora Pharmaceuticals Inc.
Publication of WO2012145626A1 publication Critical patent/WO2012145626A1/fr

Links

Classifications

    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/643—Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • 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 to synthetic oligo- -(1 ⁇ 6)- glucosamine structures as found in Staphylococcus and other bacterial pathogens.
  • Staphylococcus aureus capsular polysaccharides are typically comprised of long linear repeating trisaccharide units, with each repeating unit comprised of variously O-acetylated D-N- acetylmannosaminuronic acid (ManNAcA), L-N-acetyl-fucosamine (L-FucNAc) and D-Nacetyl-fucosamine (D-FucNAc) residues.
  • ManNAcA residue with a negatively charged carboxylate group at the 6-position, occurs at every third position in the sequence of the CP.
  • the specific region- and stereochemical linkages between units and the specific O-acetylation patterns dictate the particular CP type.
  • staphylococcal infections are CP Types 5 and 8, shown in Scheme 1 below (See e.g., Jean Lee, International Journal of Antimicrobial Agents 32S (2008) S71-S78).
  • dPNAG sequences including the example shown in Scheme 2, contain free amino groups that will be protonated, hence positively charged, at physiological pH. Infections involving a biofilm-producing (ica locus positive) strain of S. aureus, or vaccination with a S. aureus PNAG or dPNAG glycoconjugate, will result in the production of anti-PNAG and/or anti-dPNAG antibodies by the host. The most protective antibodies are raised against the dPNAG sequences and recognize the positively charged 2-ammonium group of the GlcNH 2 residues as a critical part of the epitope.
  • the productive epitope may contain multiple positive charges, one at each de-N-acetylated residue.
  • these anti-dPNAG antibodies are expected to have complementary functional groups in their Fv regions that are capable of recognizing, and therefore binding to, the GlcNH 3 + ammonium groups in the context of the dPNAG sequence.
  • the linear epitope may span up to two repeating units (hexasaccharide) in length.
  • PNAG molecule has been used in vaccine studies (See Pier et al., U.S. Publication 2005/01 18198). Data generated using purified PNAG- based material demonstrates the viability of this carbohydrate-based vaccine approach (Maira-Litran et al., Infect. Immun., 73:6752, 2005).
  • the described synthetic PNAGs were limited to homogenous PNAG compositions that are fully N-acetylated or fully non-N-acetylated; neither of these references, nor those cited therein, described a methodology for synthesizing homogenous mixed PNAGs having a predetermined number and arrangement of N-acetylated and N-deacetylated residues.
  • the prior art processes failed to provide oligosaccharides with spatially defined acetyl-amine positions, such as an amine every third position on the glucosamine polymer, for example.
  • the identification of a precise acetyl-amine sequence required to generate a desired immune response can neither be achieved by this method nor predicted a priori.
  • the present invention provides several benefits for vaccine development, especially against S. aureus, including specific homogenous antigen compositions with acetyl-amine sequences predicted to generate a desired immune response.
  • a vaccine against staphylococcal CP's or a vaccine against a dPNAG containing a CP-complementary sequence may result in suboptimal immunoprotection from a S. aureus infection.
  • the present invention avoids competitive binding by developing synthetic oligosaccharides (oligo-p-(1 ⁇ 6)-glucosamine structures) that will lead to antibodies that cannot bind in neutralizing head-to-head fashion.
  • the present invention is a subset of oligosaccharides have the formula 1a
  • R 1 and R 2 are each independently H or COCH 3 , where at least one at least one R 1 or R 2 in the oligosaccharide is H and at least another is COCH 3 ; n is an integer from 5-11 , X is a bond or a linker, and Y is H or a carrier; and where each occurrence of R can be the same or different.
  • the subset is defined more particularly below in the Table A below.
  • the present invention also provides compositions and methods for synthesizing the subset of oligo- -(1 ⁇ 6)-glucosamine structures.
  • the present invention further provides immunogenic and immunoprotective compositions containing the subset of synthetic oligo- ⁇ - (1 ⁇ 6)-glucosamines 1 a and antibodies derived therefrom for diagnosing, treating, and preventing infections caused by bacteria such as
  • the present invention further provides compositions consisting essentially of one of the oligosaccharides from the subset, compositions consisting essentially of two of the oligosaccharides from the subset, compositions consisting essentially of three of the oligosaccharides from the subset, etc. (including compositions consisting essentially of all or less than all of the oligosaccharides from the subset).
  • the compositions may be homogeneous and/or synthetic.
  • FIG. 1 depicts an exemplary core structure of an oligo- -(1 ⁇ 6)- glucosamine comprised of a glucosamine backbone unique to bacterial pathogens.
  • the backbone structure can be modified with N-acetyl groups with variable spacing.
  • FIG. 2A depicts naturally-derived PNAG polysaccharides and FIG. 2B depicts a synthetic PNAG oligosaccharide, including potential conjugation sites in FIGs. 2A and 2B.
  • FIG. 3 depicts a reaction scheme for synthesizing four
  • FIGs. 4A-4D depict a series of reaction schemes for assembling disaccharide building blocks from the four monosaccharide building blocks depicted in FIG. 3.
  • FIGs. 5A and 5B depict a reaction scheme for synthesizing a 6- mer thiol oligosaccharide 37.
  • FIGs. 6A and 6B depict a reaction scheme for synthesizing a 12- mer thiol oligosaccharide 40.
  • FIG. 7 depicts a reaction scheme for synthesizing an 18-mer thiol oligosaccharide 34.
  • FIGs. 8A and 8B depict a reaction scheme for conjugating the 6- mer thiol oligosaccharide 37 to BSA (A) or KLH (B) to form a 6-Mix-BSA conjugate 41 or a 6-Mix-KLH conjugate 42, respectively.
  • BSA BSA
  • KLH KLH
  • FIGs. 9A and 9B depict a reaction scheme for conjugating the 12- mer thiol oligosaccharide 40 to BSA (A) or KLH (B) to form a 12-Mix-BSA conjugate 43 or 12-Mix-KLH conjugate 44, respectively.
  • FIGs. 10A and 10B depict a reaction scheme for conjugating the 18-mer thiol oligosaccharide 34 to BSA (A) or KLH (B) to form an 8-Mix-BSA conjugate 45 or 18-Mix-KLH conjugate 46, respectively.
  • FIGs. 1 1 A-1 1 C depict IgG antibody titers as a function of antibody-antigen complex absorption (OD450) at the indicated serum dilutions obtained from 3 successive bleeds (pre-immune, 1 st bleed, and final bleed) in rabbits immunized with KLH conjugates corresponding to: (A) 6-Mix-KLH 42; (B) 12-Mix-KLH 44; and (C) 18-Mix-KLH 46.
  • the antisera were incubated on ELISA plates adsorbed with their corresponding BSA conjugate, specfically, (A) 6-Mix-BSA 41 ; (B) 12-Mix-BSA 43; and (C) 18-Mix-BSA 45.
  • FIGs. 1 1 D-1 1 F depict IgG antibody titers from antigen-specific antigen-KLH conjugate-derived antibodies recovered at three successive stages of affinity purification, including pre-affinity purification (3 rd bleed), the flow-through fraction and the antibody-enriched (purified) fraction from rabbits immunized with KLH conjugates. Results are shown as a function of antibody-antigen complex absorption (OD450) at the indicated serum dilutions obtained from the above-described antibody-enriched fractions generated against antigen-KLH conjugates corresponding to: (A) 6-Mix-KLH 42; (B) 12- Mix-KLH 44; and (C) 18-Mix-KLH 46.
  • OD450 antibody-antigen complex absorption
  • the antisera were incubated on ELISA plates adsorbed with their corresponding BSA conjugate, specifically, (A) 6-Mix-BSA 41 ; (B) 12-Mix-BSA 43; and (C) 18-Mix-BSA 45.
  • FIGs. 12A-12G depict the results of a cross-ELISA assay examining the specificity and cross-reactivity between fully non-acetylated (6-NH 2 , 12- NH 2 , I 8-NH 2 ); mixed (6-Mix, 12-Mix, 18-Mix) and fully acetylated (6-NHAc, 12-NHAc, 18-NHAc) oligosaccharides 1a and antibodies derived therefrom.
  • 6-NH 2 , 12- NH 2 , I 8-NH 2 mixed (6-Mix, 12-Mix, 18-Mix) and fully acetylated (6-NHAc, 12-NHAc, 18-NHAc) oligosaccharides 1a and antibodies derived therefrom.
  • Results are shown as a function of antibody-antigen complex absorption (OD 450 ) representing the averages from two rabbit antiseras in each case at the indicated serum dilutions, whereby total OD 450 is measured by subtracting away the background OD 450 from KLH antibodies alone.
  • OD 450 antibody-antigen complex absorption
  • FIGs. 13A-13D depict the results of a whole-cell ELISA assay examining the binding of pre-immune sera (A, C) or immune sera (B, D) generated from rabbits immunized against (left to right) KLH control, non- acetylated (12-NH 2 ); mixed (12-Mix) and non-acetylated (12-NHAc) oligo- ⁇ - (1 ⁇ 6)-glucosamines and S. epidermidis coated onto ELISA fixed with methanol (A, B) or formalin (C, D). Results are shown as a function of antibody-antigen complex absorption (OD 450 ) at the indicated serum dilutions.
  • oligosaccharide refers to a compound containing two or more monosaccharides. Oligosaccharides are considered to have a reducing end and a non-reducing end, whether or not the
  • oligosaccharides are depicted herein with the non-reducing end on the left and the reducing end on the right. All oligosaccharides described herein are described with the name or abbreviation for the non-reducing monosaccharide (e.g., Gal), preceded by the configuration of the glycosidic bond (a or ⁇ ), the ring bond, the ring position of the reducing monosaccharide involved in the bond, and then the name or abbreviation of the reducing monosaccharide (e.g., GlcNAc).
  • the linkage between two sugars may be expressed, for example, as 2,3, 2 ⁇ 3, or 2-3.
  • Each monosaccharide is a pyranose or furanose.
  • monosaccharide refers to a single sugar residue in an oligosaccharide, including derivatives therefrom.
  • an individual monomer unit is a monosaccharide which is (or can be) bound through a hydroxyl group to another monosaccharide.
  • monosaccharide unit is located three monosaccharide units from the first monosaccharide unit " refers to a substitution pattern as illustrated in the following scheme:
  • endotoxin-free refers to an oligosaccharide that does not contain endotoxins or endotoxin components normally present in isolated bacterial carbohydrate structures.
  • synthetic refers to material which is substantially or essentially free from components, such as endotoxins, glycolipids, oligosaccharides, etc., which normally accompany a compound when it is isolated.
  • synthetic compounds are at least about 90% pure, usually at least about 95%, and preferably at least about 99% pure. Purity can be indicated by a number of means well known in the art. Preferably, purity is measured by HPLC. The identity of the synthetic material can be determined by mass spectroscopy and/or NMR spectroscopy.
  • carrier refers to a protein, peptide, lipid, polymer, dendrimer, virosome, virus-like particle (VLP), or combination thereof, which is coupled to the oligosaccharide to enhance the
  • protein carrier refers to a protein, peptide or fragment thereof, which is coupled or conjugated to an oligosaccharide to enhance the immunogenicity of the resulting oligosaccharide-protein carrier conjugate to a greater degree than the oligosaccharide alone.
  • the protein carrier when used as a carrier, may serve as a T-dependent antigen which can activate and recruit T-cells and thereby augment T-cell dependent antibody production.
  • conjugated refers to a chemical linkage, either covalent or non-covalent, that proximally associates an oligosaccharide with a carrier so that the oligosaccharide conjugate has increased immunogenicity relative to an unconjugated oligosaccharide.
  • conjugate refers to an oligosaccharide chemically coupled to a carrier through a linker and/or a cross-linking agent.
  • passive immunity refers to the administration of antibodies to a subject, whereby the antibodies are produced in a different subject (including subjects of the same and different species) such that the antibodies attach to the surface of the bacteria and cause the bacteria to be phagocytosed or killed.
  • protective immunity means that a vaccine or immunization schedule that is administered to a animal induces an immune response that prevents, retards the development of, or reduces the severity of a disease that is caused by a pathogen or diminishes or altogether eliminates the symptoms of the disease.
  • Protective immunity may be predicted based on the ability of serum antibody to activate complement-mediated bactericidal activity or confer passive protection against a bacterial infection in a suitable animal challenge model.
  • immunoprotective composition refers to a composition formulated to provide protective immunity in a host.
  • Immune response indicators include but are not limited to: antibody titer or specificity, as detected by an assay such as enzyme-linked immunoassay (ELISA), bactericidal assay (e.g., to detect serum bactericidal antibodies), flow cytometry, immunoprecipitation, Ouchter-Lowry immunodiffusion; binding detection assays of, for example, spot, Western blot or antigen arrays;
  • antibody encompasses polyclonal and monoclonal antibody preparations, as well as preparations including hybrid antibodies, altered antibodies, F(ab') 2 fragments, F(ab) molecules, Fv fragments, single chain fragment variable displayed on phage (scFv), single domain antibodies, chimeric antibodies, humanized antibodies, and functional fragments thereof which exhibit immunological binding properties of the parent antibody molecule.
  • monoclonal antibody refers to an antibody composition having a homogeneous antibody population.
  • the term is not limited by the manner in which it is made.
  • the term encompasses whole immunoglobulin molecules, as well as Fab molecules, F(ab') 2 fragments, Fv fragments, single chain fragment variable displayed on phage (scFv), and other molecules that exhibit immunological binding properties of the parent monoclonal antibody molecule.
  • the specified antibody or antibodies bind(s) to a particular antigen or antigens in a sample and does not bind in a significant amount to other molecules present in the sample.
  • Specific binding to an antibody under such conditions may require an antibody or antiserum that is selected for its specificity for a particular antigen or antigens.
  • antigen refers to include any substance that may be specifically bound by an antibody molecule.
  • immunogen and “immunogenic composition” refer to an antigenic composition capable of initiating lymphocyte activation resulting in an antigen-specific immune response.
  • epitope refers to a site on an antigen to which specific B cells and/or T cells respond.
  • the term is also used interchangeably with "antigenic determinant” or "antigenic determinant site.”
  • B cell epitope sites on proteins, oligosaccharides, or other biopolymers may be composed of moieties from different parts of the macromolecule that have been brought together by folding. Epitopes of this kind are referred to as conformational or discontinuous epitopes, since the site is composed of segments the polymer that are discontinuous in the linear sequence but are continuous in the folded conformation(s). Epitopes that are composed of single segments of biopolymers or other molecules are termed continuous or linear epitopes.
  • T cell epitopes are generally restricted to linear peptides. Antibodies that recognize the same epitope can be identified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen.
  • the present invention provides a subset of oligosaccharides 1a:
  • R 1 and R 2 are each independently H or COCH 3 , where at least one at least one R or R 2 in the oligosaccharide is H and at least another is COCH 3 ; n is from 5-1 1 , X is a bond or a linker, and Y is H or a carrier; and where each occurrence of R 1 can be the same or different.
  • the subset is described below in Table A.
  • conjugates containing PNAG/dPNAG antigens as small as five or six monosaccharide units in length are immunogenic, and can be immunoprotective. While the maximum allowable size has not been established, in vivo immunoprotection using conjugates containing dPNAG antigens of 12 units in length have been shown to be protective by the present inventors (see below).
  • each monomeric unit in an oligosaccharide of n units is numbered, with position 1 being the non-reducing end.
  • position 1 being the non-reducing end.
  • the various positions in a hexasaccharide are shown below:
  • X is a bond or a linker
  • Y is H or a carrier
  • the oligosaccharides may be homogeneous and/or synthetic.
  • Suitable linkers comprise at one end a grouping able to enter into a covalent bonding with a reactive functional group of the carrier, e.g. an amino, thiol, or carboxyl group, and at the other end a grouping likewise able to enter into a covalent bonding with a hydroxyl group of an oligosaccharide according to the present invention.
  • a reactive functional group of the carrier e.g. an amino, thiol, or carboxyl group
  • a grouping likewise able to enter into a covalent bonding with a hydroxyl group of an oligosaccharide according to the present invention.
  • a biocompatible bridging molecule of suitable length e.g. substituted or unsubstituted heteroalkylene, arylalkylene, alkylene, alkenylene, or (oligo)alkylene glycol groups.
  • Linkers preferably include substituted or unsubstituted alkylene or alkenylene groups containing 1
  • Linkers able to react with thiol groups on the carrier are, for example, maleimide and carboxyl groups; preferred groupings able to react with aldehyde or carboxyl groups are, for example, amino or thiol groups.
  • Preferred covalent attachments between linkers and carriers include thioethers from reaction of a thiol with an a-halo carbonyl or a-halo nitrile, including reactions of thiols with maleimide; hydrazides from reaction of a hydrazide or hydrazine with an activated carbonyl group (e.g. activated NHS- ester or acid halide); triazoles from reaction of an azide with an alkyne (e.g.
  • amine-based conjugation chemistries could be used in principle for coupling linkers and/or spacers to the oligosaccharides described herein, these approaches would typically sacrifice uniformity inasmuch as the oligosaccharides of the present invention typically contain a plurality of amines bonded to second carbon of the respective monosaccharide units.
  • linker molecules are known to skilled workers and commercially available or can be designed as required and depending on the functional groups present and can be prepared by known methods.
  • Suitable carriers are known in the art (See e.g., Remington's Pharmaceutical Sciences (18th ed., Mack Easton, PA (1990)) and may include, for example, proteins, peptides, lipids, polymers, dendrimers, virosomes, virus-like particles (VLPs), or combinations thereof, which by themselves may not display particular antigenic properties, but can support immunogenic reaction of a host to the oligosaccharides of the present invention (antigens) displayed at the surface of the carrier(s).
  • VLPs virus-like particles
  • the carrier is a protein carrier, including but are not limited to, bacterial toxoids, toxins, exotoxins, and nontoxic derivatives thereof, such as tetanus toxoid, tetanus toxin Fragment C, diphtheria toxoid, CRM (a nontoxic diphtheria toxin mutant) such as CRM 197, cholera toxoid, Staphylococcus aureus exotoxins or toxoids, Escherichia coli heat labile enterotoxin, Pseudomonas aeruginosa exotoxin A, including recombinantly produced, genetically detoxified variants thereof; bacterial outer membrane proteins, such as Neisseria meningitidis serotype B outer membrane protein complex (OMPC), outer membrane class 3 porin (rPorB) and other porins; keyhole limpet hemocyanin (KLH), hepatitis B virus core protein,
  • OMPC Neisseria
  • albumins such as bovine serum albumin (BSA), human serum albumin (HSA), and ovalbumin; pneumococcal surface protein A (PspA), pneumococcal adhesin protein (PsaA); purified protein derivative of tuberculin (PPD); transferrin binding proteins, polyamino acids, such as
  • a carrier may display on average, for example, 1 to 500, 1 to 100, 1 to 20, or 3 to 9 oligosaccharide units on its surface.
  • Methods for attaching an oligosaccharide to a carrier are conventional, and a skilled practitioner can create conjugates in accordance with the present invention using conventional methods.
  • Guidance is also available in various disclosures, including, for example, U.S. Pat. Nos. 4,356, 170; 4,619,828; 5, 153,312; 5,422,427; and 5,445,817; and in various print and online Pierce protein cross-linking guides and catalogs (Thermo Fisher, Rockford, IL).
  • the carbohydrate antigens of the present invention are conjugated to CRM 197, a commercially available protein carrier used in a number of FDA approved vaccines.
  • CRM-conjugates have the advantage of being easier to synthesize, purify and characterize than other FDA approved carriers such as OMPC.
  • Carbohydrate antigens may be conjugated to CRM via thiol-bromoacetyl conjugation chemistry. CRM activation may be achieved by reacting the lysine side chains with the NHS ester of bromoacetic acid using standard conditions as previously described in U.S. Pat. Appl. Publ. 2007-0134762, the disclosures of which are incorporated by reference herein.
  • CRM conjugates may be purified via size exclusion
  • a minimum carbohydrate content of about 15% by weight for each conjugate may be generated.
  • a conjugate may include about 3-20 antigens per protein carrier.
  • carbohydrate antigens may be conjugated to one or more carriers suitable for development of diagnostic assays, including ELISAs and microarrays.
  • exemplary carriers for use in such assays include bovine serum albumin (BSA), keyhole limpet
  • oligosaccharide antigens may be conjugated to maleimide functionalized BSA, whereby a 20- fold molar excess of the antigen is reacted with commercially available Imject maleimide BSA (Pierce) in maleimide conjugation buffer (Pierce).
  • BSA conjugates may be purified via size exclusion
  • conjugates will contain a minimum carbohydrate content of about 10% by weight per BSA conjugate and >8 antigen copies per conjugate.
  • the invention provides a method for assembling mixed sequence oligo- -(1 ⁇ 6)-glucosamine structures 1 from four building blocks 6, 8, 14, and 47.
  • the four building blocks include donor building blocks 8 and 14, and acceptor building blocks 6 and 47 below.
  • Building blocks 6 and 8 further contain an -NPhth group for selective amine group protection of individual monosaccharide units.
  • Building blocks 14 and 47 contain a protective -NHTroc group for selective protection and subsequent acetylation of individual monosaccharide units.
  • Acceptor building blocks 6 and 47 have a linker precursor incorporated at the reducing end, which may be reacted with thioacetic acid and deblocked to form a conjugation-ready thiol for conjugation to a carrier as further described above.
  • linker precursor incorporated at the reducing end, which may be reacted with thioacetic acid and deblocked to form a conjugation-ready thiol for conjugation to a carrier as further described above.
  • the four monosaccharide building blocks 6, 8, 14, and 47 may be synthesized from a single monosaccharide 1 as shown in FIG. 3.
  • the invention provides mixed disaccharide building blocks which can be used in combination with other monosaccharide- or disaccharide building blocks to form higher-order dPNAG/PNAG structures.
  • monosaccharide donor 14 is reacted with monosaccharide acceptor 6 to form a mixed disaccharide building block 15 in which the first monosaccharide unit is protected for selective acetylation and the amine in the second monosaccharide unit is selectively protected.
  • FIG. 4A monosaccharide donor 14 is reacted with monosaccharide acceptor 6 to form a mixed disaccharide building block 15 in which the first monosaccharide unit is protected for selective acetylation and the amine in the second monosaccharide unit is selectively protected.
  • monosaccharide donor 8 is reacted with monosaccharide acceptor 47 to form a mixed disaccharide building block 49 in which the amine in the first monosaccharide unit is selectively protected and the second monosaccharide unit position is protected for selective acetylation.
  • disaccharide building block 15 can be converted into a disaccharide donor 17 or a disaccharide acceptor 48 for further couplings to other acceptors or donors, respectively.
  • disaccharide building block 49 can be converted into a disaccharide donor 51 or a disaccharide acceptor 52 for further couplings to other acceptors or donors, as well.
  • disaccharide donors 17 and 51 can be coupled with either of the monosaccharide acceptors 6 and 47 to form trisaccharides, or they can be coupled with disaccharide acceptors 48 and 52 to form
  • the present invention provides disaccharide blocks for forming consecutive acetylated residues or consecutive non- acetylated resides.
  • monosaccharide donor 14 is reacted with monosaccharide acceptor 47 to form a disaccharide building block 53 in which each of the two monosaccharide units is protected for selective acetylation.
  • monosaccharide donor 8 is reacted with monosaccharide acceptor 6 to from a disaccharide building block 18 in which each of the 2-position amines in the two monosaccharide units is protected.
  • disaccharide building block 53 can be converted into a disaccharide donor 55 or a disaccharide acceptor 56 for further couplings to other acceptors or donors, respectively.
  • disaccharide building block 18 can be converted into a disaccharide donor 20 or a disaccharide acceptor 57 for further couplings to other acceptors or donors, as well.
  • disaccharide donors 20 and 55 can be coupled with either of the monosaccharide acceptors 6 and 47 to form trisaccharides or they can be coupled with any of the above-described disaccharide acceptors 48, 52, 56, or 57 to form tetrasaccharides.
  • any of the above-described donors can be coupled to any complementary acceptor. Accordingly, by coupling the monosaccharide-, disaccharide-, or other higher order donor modules of higher length with complementary monosaccharide-, disaccharide-, or other higher order acceptor modules of higher length, any mixed sequence oligosaccharide of the present invention can be formed in which the individual acetylation positions and oligosaccharide length are engineered into a given synthesis process in a pre-determined fashion. Compositions and methods for synthesizing exemplary oligosaccharides are described in the Examples below.
  • syntheses of various oligosaccharides of the present invention proceed by a number of standard operating procedures (SOPs).
  • SOPs standard operating procedures
  • the present invention provides a number of SOPs (or reaction steps) for synthesizing dPNAG/PNAG oligosaccharides, including SOP 1 , removal of 1° TBS group(s); SOP 2, removal of allyl group(s); SOP 3, trichioroacetimidate formation; SOP 4, glycosylation using trichioroacetimidate donors; SOP 5, removal of /V-Troc group and in situ /V-acetylation; SOP 6, thiol addition to olefin; and SOP 7, removal of O-acetate, /V-phthaloyl and S- acetate groups.
  • SOPs 1 -7 are further detailed in the Examples below.
  • the above-described protecting groups may be substituted with other protecting groups customarily considered in
  • the present invention provides compositions containing dPNAG/PNAG oligosaccharides 1a and a pharmaceutically acceptable vehicle.
  • the compositions are preferably immunogenic and immunoprotective.
  • compositions may be homogeneous and/or synthetic, and may contain one or more of the oligosaccharides of formula 1 b/Table A described above (e.g., a homogeneous composition consisting essentially of one of the oligosaccharides of formula 1 b/Table A; a homogeneous composition consisting essentially of two of the oligosaccharides of formula 1 b/Table A; etc.).
  • the present invention contemplates the use of single- and multivalent vaccines comprising any of the synthetic oligosaccharides described herein.
  • compositions may contain a single oligosaccharide 1a.
  • the present invention further contemplates multi-antigen vaccine candidates and vaccines thereof.
  • the invention provides a composition containing two, three, four or more different oligosaccharides 1a.
  • Suitable vehicles and their formulation, inclusive of other human proteins, e.g., human serum albumin, are described, for example, in
  • Pharmaceutically acceptable vehicles may include any vehicle that does not itself induce the production of antibodies harmful to the individual receiving the composition.
  • Suitable vehicles may include large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers; inactive virus particles, insoluble aluminum compounds, calcium phosphate, liposomes, virosomes, ISCOMS, microparticles, emulsions, and VLPs.
  • compositions of the present invention may further include one or more adjuvants.
  • An oligosaccharide-protein conjugate composition may further include one or more immunogenic adjuvant(s).
  • An immunogenic adjuvant is a compound that, when combined with an antigen, increases the immune response to the antigen as compared to the response induced by the antigen alone so that less antigen can be used to achieve a similar response.
  • an adjuvant may augment humoral immune responses, cell- mediated immune responses, or both.
  • adjuvant can overlap to a significant extent.
  • a substance which acts as an "adjuvant” may also be a “carrier,” and certain other substances normally thought of as “carriers,” for example, may also function as an “adjuvant.”
  • a substance which may increase the immunogenicity of the synthetic oligosaccharide or carrier associated therewith is a potential adjuvant.
  • a carrier is generally used in the context of a more directed site-specific conjugation to an oligosaccharide of the present invention, whereby an adjuvant is generally used in a less specific or more generalized structural association therewith.
  • Exemplary adjuvants and/or adjuvant combinations may be selected from the group consisting of mineral salts, including aluminum salts, such as aluminum phosphate and aluminum hydroxide (alum) (e.g.,
  • TLR toll-like receptor
  • ssRNA single-stranded RNA genomes of such viruses as influenza, measles, and mumps; and small synthetic guanosine-base antiviral molecules like loxoribine and ssRNA and their analogs
  • agonists of TLR-8 e.g. binds ssRNA
  • agonists of TLR-9 e.g. unmethylated CpG of the DNA of the pathogen and their analogs
  • agonists of TLR-10 function not defined
  • TLR-1 1 - e.g. binds proteins expressed by several infectious protozoans (Apicomplexa), specific toll-like receptor agonists include monophosphoryl lipid A (MPL®), 3 De-O-acylated
  • MPL monophosphoryl lipid A
  • OM-174 E. coli lipid A derivative
  • OM triacyl lipid A derivative and other MPL- or lipid A-based formulations and combinations thereof, including MPL®-SE, RC-529 (Dynavax Technologies), AS01 (liposomes+MPL+QS21 ), AS02 (oil-in-water PL + QS-21 ), and AS04 (Alum + MPL)(GlaxoSmith Kline, Pa.), CpG-oligodeoxynucleotides (ODNs) containing immunostimulatory CpG motifs, double-stranded RNA,
  • ODNs CpG-oligodeoxynucleotides
  • polyinosinic:polycytidylic acid poly l:C
  • other oligonucleotides or polynucleotides optionally encapsulated in liposomes
  • oil-in-water emulsions including AS03 (GlaxoSmith Kline, Pa.), MF-59 (microfluidized detergent stabilized squalene oil-in-water emulsion; Novartis), and Montanide ISA-51 VG (stabilized water-in-oil emulsion) and Montanide ISA-720 (stabilized water/squalene; Seppic Pharmaceuticals, Fairfield, NJ); cholera toxin B subunit; saponins, such as Quil A or QS21 , an HPLC purified non-toxic fraction derived from the bark of Quillaja Saponaria Molina (STIMULONTM (Antigenics, Inc., Lexington, Mass.) and saponin-based adjuvants, including immunostimulating complexes (ISCOM
  • coli heat-labile enterotoxin LT
  • immune-adjuvants including cytokines, such as IL-2, IL-12, GM-CSF, Flt3, accessory molecules, such as B7.1
  • mast cell (MC) activators such as mast cell activator compound 48/80 (C48/80); water-insoluble inorganic salts; liposomes, including those made from DNPC/Chol and DC Choi; micelles; squalene; squalane; muramyl dipeptides, such as N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP) as found in U.S. Pat. No.
  • adjuvant potency may be enhanced by combining multiple adjuvants as described above, including combining various delivery systems with immunopotentiating substances to form multi- component adjuvants with the potential to act synergistically to enhance antigen-specific immune responses in vivo.
  • immunopotentiating substances include the above-described adjuvants, including, for example, MPL and synthetic derivatives, MDP and derivatives, oligonucleotides (CpG etc), double-stranded RNAs (ds RNAs), alternative pathogen-associated molecular patterns (PAMPs)(E.
  • coli heat labile enterotoxin coli heat labile enterotoxin
  • flagellin saponins (QS-21 etc)
  • small molecule immune potentiators SMIPs, e.g., resiquimod [R848]
  • cytokines e.g., IL-12, IL-12, and chemokines.
  • the present invention provides
  • an immunogenic or immunoprotective composition will include a "sufficient amount” or “an immunologically effective amount” of a dPNAG/PNAG -protein conjugate according to the present invention, as well as any of the above mentioned components, for purposes of generating an immune response or providing protective immunity, as further defined herein.
  • the compositions may be homogeneous and the oligosaccharides may be synthetic.
  • Administration of the oligosaccharide- or oligosaccharide conjugate compositions or antibodies, as described herein may be carried out by any suitable means, including by parenteral administration (e.g., intravenously, subcutaneously, intradermal ⁇ , or intramuscularly); by topical administration, of for example, antibodies to an airway surface; by oral administration; by in ovo injection in birds, for example, and the like.
  • parenteral administration e.g., intravenously, subcutaneously, intradermal ⁇ , or intramuscularly
  • topical administration of for example, antibodies to an airway surface
  • oral administration by in ovo injection in birds, for example, and the like.
  • they are administered intramuscularly.
  • compositions may be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection.
  • An aqueous composition for parenteral administration may include a solution of the immunogenic component(s) dissolved or suspended in a pharmaceutically acceptable vehicle or diluent, preferably a primarily aqueous vehicle.
  • An aqueous composition may be formulated as a sterile, pyrogen-free buffered saline or phosphate-containing solution, which may include a preservative or may be preservative free.
  • Suitable preservatives include benzyl alcohol, parabens, thimerosal, chlorobutanol, and benzalkonium chloride, for example.
  • Aqueous solutions are preferably approximately isotonic, and its tonicity may be adjusted with agents such as sodium tartrate, sodium chloride, propylene glycol, and sodium phosphate.
  • auxiliary substances required to approximate physiological conditions including pH adjusting and buffering agents, tonicity adjusting agents, wetting or emulsifying agents, pH buffering substances, and the like, including sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc. may be included with the vehicles described herein.
  • compositions may be formulated in a solid or liquid form for oral delivery.
  • nontoxic and/or pharmaceutically acceptable solid vehicles may include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
  • a pharmaceutically acceptable nontoxic composition may be formed by incorporating any of the normally employed excipients, including those vehicles previously listed, and a unit dosage of an active ingredient, that is, one or more compounds of the invention, whether conjugated to a carrier or not.
  • Topical application of antibodies to an airway surface can be carried out by intranasal administration (e.g., by use of dropper, swab, or inhaler which deposits a pharmaceutical formulation intranasally).
  • Topical application of the antibodies to an airway surface can also be carried out by inhalation administration, such as by creating respirable particles of a pharmaceutical formulation (including both solid particles and liquid particles) containing the antibodies as an aerosol suspension, and then causing the subject to inhale the respirable particles.
  • Methods and apparatuses for administering respirable particles of pharmaceutical formulations are well known, and any conventional technique can be employed.
  • Oral administration may be in the form of an ingestable liquid or solid formulation.
  • the concentration of the oligosaccharides of the invention in the pharmaceutical formulations can vary widely, i.e., from less than about 0.1 %, usually at or at least about 0.1 % to as much as 20% to 50% or more by weight, and may be selected on the basis of fluid volumes, viscosities, stability, etc. , and/or in accordance with the particular mode of administration selected.
  • a human unit dose form of the compounds and composition is typically included in a pharmaceutical composition that comprises a human unit dose of an acceptable vehicle, preferably an aqueous vehicle, and is administered in a volume of fluid that is known by those of skill in the art to be used for administration of such compositions to humans, and is adjusted according to commonly understood principles for a particular subject to be treated.
  • the invention provides a unit dosage of the vaccine components of the invention in a suitable amount of an aqueous solution, such as 0.1 -3 ml, preferably 0.2-2 ml_.
  • compositions of the present invention may be administered to any animal species at risk for developing an infection by a microbial species expressing a PNAG and/or PNAG antigen.
  • the present invention can also be used to treat or prevent other bacteria infections where the bacterium is known or suspected to express PNAG or dPNAG.
  • Suitable bacteria that can be treated with the present invention include Staphylococcus species, such as S. aureus and S.
  • dPNAG/PNAG oligosaccharides may be modified, depending on the specific compositional makeup, including acetylation profiles of these antigens in their respective bacterial species.
  • the treatment may be given in a single dose schedule, or preferably a multiple dose schedule in which a primary course of treatment may be with 1 -10 separate doses, followed by other doses given at subsequent time intervals required to maintain and or reinforce the response, for example, at 1-4 months for a second dose, and if needed, a subsequent dose(s) after several months.
  • suitable treatment schedules include: (i) 0, 1 month and 6 months, (ii) 0, 7 days and 1 month, (iii) 0 and 1 month, (iv) 0 and 6 months, or other schedules sufficient to elicit the desired responses expected to reduce disease symptoms, or reduce severity of disease.
  • the amounts effective for inducing an immune response or providing protective immunity will depend on a variety of factors, including the oligosaccharide composition, conjugation to a carrier, inclusion and nature of adjuvant(s), the manner of administration, the weight and general state of health of the patient, and the judgment of the prescribing physician.
  • the amounts may generally range for the initial immunization (that is for a prophylactic administration) from about 1.0 pg to about 5,000 pg of oligosaccharide for a 70 kg patient, (e.g., 1.0 pg, 2.0 pg, 2.5 pg, 3.0 pg, 3.5 pg, 4.0 pg, 4.5 pg, 5.0 pg, 7.5 pg, 10 pg, 12.5 pg, 15 pg, 17.5 pg, 20 pg, 25 pg, 30 pg, 35 pg, 40 pg, 45 pg, 50 pg, 75 pg, 100 pg, 250 pg, 500 pg, 750 pg, 1 ,000 pg, 1 ,500 pg, 2,000 pg, 2,500 pg, 3,000 pg, 3,500 pg, 4,000 pg, 4,500 pg or 5,000 pg
  • a primary dose may optionally be followed by boosting dosages of from about 1.0 to about 1 ,000 of peptide (e.g. , 1 .0 pg, 2.0 pg, 2.5 pg, 3.0 pg, 3.5 pg, 4.0 pg, 4.5 pg, 5.0 pg, 7.5 pg, 10 pg, 12.5 pg, 15 pg, 17.5 pg, 20 pg, 25 pg, 30 pg, 35 pg, 40 pg, 45 pg, 50 pg, 75 pg, 100 pg, 250 pg, 500 pg, 750 pg, 1 ,000 pg, 1 ,500 pg, 2,000 pg, 2,500 pg, 3,000 pg, 3,500 pg, 4,000 pg, 4,500 pg or 5,000 pg) pursuant to a boosting regimen over weeks to months depending upon the patient's response and condition
  • the immunogenic compositions comprising a compound of the invention may be suitable for use in adult humans or in children, including young children or others at risk for contracting an infection caused by a dPNAG/PNAG-expressing bacterial species.
  • a composition may be administered in combination with other pharmaceutically active substances, and frequently it will be administered in combination with other vaccines as part of a childhood vaccination program.
  • the invention provides an antibody preparation against one or more oligo-p-(1— >6)-glucosamine 1a in accordance with the present invention.
  • the antibody preparation may include any member from the group consisting of polyclonal antibody, monoclonal antibody, mouse monoclonal IgG antibody, humanized antibody, chimeric antibody, fragment thereof, or combination thereof.
  • compositions may be used in a method for providing passive immunity against a bacterial target species of interest, including S. aureus and other dPNAG/PNAG-expressing bacteria.
  • a pharmaceutical antibody composition may be administered to an animal subject, preferably a human, in an amount sufficient to prevent or attenuate the severity, extent of duration of the infection by the bacterial target species of interest.
  • the administration of the antibody may be either prophylactic (prior to anticipated exposure to a bacterial infection) or therapeutic (after the initiation of the infection, at or shortly after the onset of the symptoms).
  • the dosage of the antibodies will vary depending upon factors as the subject's age, weight and species. In general, the dosage of the antibody may be in a range from about 1 -10 mg/kg body weight.
  • the antibody is a humanized antibody of the IgG or the IgA class.
  • the route of administration of the antibody may be oral or systemic, for example, subcutaneous, intramuscular or intravenous.
  • the present invention provides compositions and methods for inducing production of antibodies for diagnosing, treating, and/or preventing one or more infections caused by dPNAG/PNAG expressing bacteria.
  • Antisera to dPNAG/PNAG conjugates may be generated in New Zealand white rabbits by 3-4 subcutaneous injections over 13 weeks.
  • a pre- immune bleed may generate about 5 mL of baseline serum from each rabbit.
  • a prime injection (10 ⁇ g antigen equivalent) may be administered as an emulsion in complete Freund's adjuvant (CFA).
  • CFA complete Freund's adjuvant
  • Subsequent injections (5 ⁇ g antigen equivalent) may be given at three week intervals in incomplete Freund's adjuvant (IFA).
  • Rabbits may be bled every two weeks commencing one week after the third immunization.
  • Approximately 25 - 30 mL of serum per rabbit may be generated from each bleeding event and frozen at -80°C.
  • Serum may be analyzed by ELISA against the corresponding dPNAG/PNAG conjugate as described below.
  • antisera from later bleeds may be affinity purified as further described below.
  • the oligosaccharides and antibodies generated therefrom can be used as diagnostic reagents for detecting dPNAG-PNAG structures or antibodies thereagainst, which are present in biological samples.
  • the detection reagents may be used in a variety of immunodiagnostic techniques, known to those of skill in the art, including ELISA- and microarray-related technologies.
  • these reagents may be used to evaluate antibody responses, including serum antibody levels, to immunogenic oligosaccharide conjugates.
  • the assay methodologies of the invention typically involve the use of labels such as fluorescent, chemiluminescent, radioactive, enzymatic labels or dye molecules, and/or secondary immunologic reagents for direct or indirect detection of a complex between an antigen or antibody in a biological sample and a corresponding antibody or antigen bound to a solid support.
  • labels such as fluorescent, chemiluminescent, radioactive, enzymatic labels or dye molecules
  • secondary immunologic reagents for direct or indirect detection of a complex between an antigen or antibody in a biological sample and a corresponding antibody or antigen bound to a solid support.
  • Such assays typically involve separation of unbound antibody in a liquid phase from a solid phase support to which antibody-antigen complexes are bound.
  • Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e.g., in membrane or microtiter well form); polyvinylchloride (e.g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like.
  • a solid support is first reacted with a first binding component (e.g., an anti- dPNAG-PNAG antibody or dPNAG-PNAG oligosaccharide) under suitable binding conditions such that the first binding component is sufficiently immobilized to the support.
  • a first binding component e.g., an anti- dPNAG-PNAG antibody or dPNAG-PNAG oligosaccharide
  • mobilization to the support can be enhanced by first coupling the antibody or oligosaccharide to a protein with better binding properties, or that provides for immobilization of the antibody or antigen on the support without significant loss of antibody binding activity or specificity.
  • Suitable coupling proteins include, but are not limited to, macromolecules such as serum albumins including bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), immunoglobulin molecules, thyroglobulin, ovalbumin, and other proteins well known to those skilled in the art.
  • BSA bovine serum albumin
  • KLH keyhole limpet hemocyanin
  • Other molecules that can be used to bind antibodies the support include polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and the like.
  • Such molecules and methods of coupling these molecules are well known to those of ordinary skill in the art and are described in, for example, U.S. Pat. No. 7,595,307, U.S. Pat. Appl. No. US 2009/0155299, the disclosures and cited references therein of which are incorporated by reference herein.
  • Molecules generated with the first analog core in the initial PNAG library represented by 58, 59, 60 can serve as a control group and to mimic functionally the major secreted component of staphylococcal biofilm.
  • the second analog core containing a partially acetylated PNAG molecule exemplified by oligosaccharides 37, 40, 34, and conjugates thereof represents one possible product of chemical or ica B-mediated deacetylation (16.7% /V-acetyl), which was shown by the semi-synthetically prepared version to confer opsonic and immunoprotective capability.
  • the synthetic antigens herein are structurally defined molecules wherein the N- acetyl groups are regularly spaced every 6 glucosamine units. A significant portion of the multiple free amine groups in these antigens are likely to be protonated at physiologic pH and will have a major impact on the physical properties, such as tertiary structure and solubility.
  • the third analog core exemplifying a fully deacetylated PNAG molecule as represented by 61 , 62, 63 in FIG. 2B.
  • the third analog core presents a three-dimensional structure or tertiary structure distinguished from naturally derived deacetylated PNG structures and is similar to synthetic PNAGs recently described (Gening et al., Infect. Immun., 78:764, 2010, epub 1 1/30/2009).
  • Each protected antigen was reacted with thioacetic acid to install a thioacetate at the reducing end as a conjugation site (See Scheme 2 of Buskas et al., J. Org. Chem., 65:958, 2000). Removal of the protecting groups provided two sets of compounds, the 100% poly-NH 2 sequences (by 61 , 62, 63) and the 6.7% /V-acetyl substituted structures (thiol
  • oligosaccharides 37, 40, 34 Reaction of a portion of the 100% poly-NH 2 sequences with acetic anhydride under aqueous conditions provided the 100% poly-/V-acetyl sequences ( 58, 59, 60). All synthetic antigens were purified via size exclusion chromatography (BioGel P2 or P4) and fully characterized by 1 H-NMR, 13 C-NMR and mass spectroscopy.
  • the PNAG-based library set contains 9 structures comprised of molecules varying by the 3 core unit analogs used and by 3 different molecule lengths (FIG. 2B). Complete analytical characterization (NMR, MS, HPLC, elemental analysis) demonstrated that each molecule was over 98% pure compound. [00126]
  • SOP Standard Operating Procedure
  • Example 5 - SOP 4 Glycoslyation using trichloroacetimidate donors
  • Zinc activation Zinc (50g, powdered) was washed with 200 mL each: 2M HCI(aqueous), H 2 0, EtOH and THF. The solids were dried in vacuo overnight to a constant weight.
  • Example 8 - SOP7 Removal of O-acetate, N-phthaloyl, and S- acetate groups
  • FIG. 3 outlines the reaction scheme for synthesizing the monosaccharide building blocks, which proceeds according to the following steps.
  • Glucosamine 1 (90 g, 0.23 mol), (prepared as described in Tetrahedron 1997, 53, 12, 4159) was dissolved in pyridine (405 mL) and triethylamine (36.5 mL). The solution was stirred for 15 minutes followed by the addition of phthalic anhydride (22 g, 0.15 mmol). The reaction mixture was maintained at room temperature in a water bath for 30 minutes.
  • Triethylamine (40.5 mL) and phthalic anhydride (22 g, 0.15 mmol) were added and stirred at room temperature for an additional 45 minutes.
  • the reaction mixture was heated to 90°C and acetic anhydride (121.5 mL) was added.
  • the reaction mixture was maintained at 90-95°C for 10 minutes followed by concentration in vacuo to a thick, yellow syrup.
  • the crude product was dissolved in CH 2 CI 2 (1.0 L) and washed with H 2 0 (3 x 500 mL). The organic solution was dried over Na 2 S0 4 , filtered and concentrated to a syrup.
  • the product was recovered via recrystallization from ethanol (300 mL, 190 proof, 0.1 % MeOH).
  • TBSCI tert-butyldimethylsilyl chloride
  • Glycosyl trichloroacetimidate 14 was formed as described in SOP 3 using 13 (21 g, 38 mmol), trichloroacetonitrile (30 ml_) and K 2 CO 3 (20 g). Product 14 was formed in 97% yield (25.5 g).
  • FIG. 4A-4D outline reaction schemes for synthesizing various disaccharide building blocks, including those depicted in FIG. 4A which proceeds according to the following steps.
  • coupling was performed as described in SOP 4 using trichloroacetimidate 14 (13.2g, 19.1 mmol), acceptor 6 (6.34 g, 14.7 mmol) and TMSOTf (2.94 mL, 0.5 M in CH 2 CI 2 , 1.47 mmol).
  • Product 15 was formed in 80% yield (1 1.04 g).
  • reaction schemes depicted in FIGs. 4B-4D employ monosaccharide building block starting materials (as shown), but essentially the same steps and SOPs (-1 , -2, -3, and -4) as described above.
  • SOPs monosaccharide building block starting materials
  • FIG. 4C coupling was performed as described in SOP 4 using: trichloroacetimidate 14 (12 g, 17.2 mmol), acceptor 47 (6.86 g, 14.3 mmol) and TMSOTf (1.43 mL, 1.0 M in CH 2 CI 2 , 1.43 mmol).
  • Product 48 was formed in 98% yield (14.2 g, 14 mmol).
  • FIG. 5A-5B depict a reaction scheme for synthesizing a mixed-/V- acetyl oligo- -(1 ⁇ 6)-glucosamine 6-mer (37 in FIG. 2B), which proceeds according to the following steps.
  • Utilization of monosaccharide and disaccharide building blocks described in FIGs. 3 and 4A-4D in conjunction with the indicated SOPs additionally described in Examples 2-8 exemplify the methodologies and reagents for synthesizing mixed-/V-acetyl oligo- -(1 ⁇ 6)- glucosamine structures, including oligosaccharides 37, 40, and 34 in FIG. 2B, the syntheses of which are outlined in FIGs. 5A, 5B (6-mer, 37), FIGs. 6A, 6B (12-mer, 40), and FIGs. 7A, 7B (18-mer, 34) and further described below.
  • Glycosyl trichloroacetimidate 26 was formed from 25 as described in SOP 3b using 25 (7.0 g, 2.8 mmol), trichloroacetonitrile (10 mL) and K 2 C0 3 (7 g). Product 26 was formed i n 86% yield (6.3 g, 2.4 mmol). TBS removal of was performed as described in SOP 1 using 24 (4.55 g, 1.8 mmol) and Sc(OTf) 3 (60mg, 0.12 mmol). Product 27 was formed in 83% yield (3.6 g, 1.5 mmol).
  • TBS removal in 30 was performed as described in SOP 1 using 30 (2 g, 0.29 mmol) and Sc(OTf) 3 (40mg, 0.08 mmol).
  • Product 31 was formed in 90% yield (0.18 g, 0.26 mmol).
  • Exchange of N-Troc for N-Acetate groups in 31 was performed as described in SOP 5 using 31 ( 0.73g, 0.10 mmol) and Zn (1 g) in THF:Ac 2 0:AcOH (20 ml_).
  • Product 32 was formed in 92% yield (0.6 g, 0.092 mmol). Thiol addition to 32 was performed as described in SOP 6 using 32 (0.6 g, 0.092 mmol), HSAc (0.5 mL) and AIBN (50 mg). Product 33 was formed in 83% yield (0.5 g, 0.076 mmol). Deprotection of 33 and formation of 34 was performed as described in SOP 7 using 33 (0.5 g, 0.076 mmol) and hydrazine (3 mL). Product 34 was formed in 79% yield (0.19 g, 0.06 mmol).
  • Table 1 provides supporting characterization data for selected antigens and intermediates described in Example 1 1.
  • FIGs. 8A and 8B depict reaction schemes for conjugating a mixed-/V-acetyl oligo-p-(1 ⁇ 6)-glucosamine 6-mer thiol 37 to BSA and KLH as follows.
  • a conjugation stock solution of hexamer thiol 37 was prepared by dissolving the hexamer thiol 37 (4.2 mg, 3.81 ⁇ ) in water (0.300 mL).
  • a solution of tris(2-carboxyethyl)phosphine (TCEP) in water (40 ⁇ , 0.05 M, 1 .95 ⁇ ) was added and stirred for 1 hour.
  • Imject® Conjugation Buffer (Pierce, 300 ⁇ ) was added to provide a stock solution for conjugation to BSA (FIG. 8A) and KLH (FIG. 8B).
  • the conjugation stock solution of hexamer thiol 37 (500 ⁇ , 3.0 ⁇ ) was added to a solution of maleimide- activated bovine serum albumin (Imject® BSA, Pierce, Rockford, IL) (5 mg, ⁇ 1.5 ⁇ maleimide) in Imject® Conjugation Buffer (Pierce, 250 ⁇ diluted with 250 ⁇ water) and the resulting solution stirred for 18 hours at room temperature.
  • the reaction mixture was purified by de-salting on D-Salt P- 6000 10 mL column (Pierce, Rockford, IL). The column was pre-equilibrated with 30 mL of purification buffer (Pierce, Prod. No.
  • the conjugation stock solution of hexamer thiol 37 (140 ⁇ _, 0.86 ⁇ ) was added to a solution of maleimide-activated keyhole limpet hemocyanin (Imject® KLH, Pierce, Rockford, IL) (5 mg, ⁇ 0.43 ⁇ maleimide) in water (0.5 mL) was added and the resulting solution stirred overnight at room temperature.
  • the reaction mixture was purified by de-salting on D-Salt P-6000 10 mL column (Pierce, Rockford, IL). The column was pre-equilibrated with 30 mL of purification buffer (Pierce, Prod. No.
  • FIGs. 9A and 9B depict reaction schemes for conjugating mixed- /V-acetyl oligo ⁇ -(1 ⁇ 6)-glucosamine 6-mers (Ag 5 in FIG. 2B) to BSA and
  • a conjugation stock solution of 12-mer thiol 40 was prepared by dissolving the 12-mer 40 (8.1 mg, 3.84 ⁇ ) in water (300 ⁇ ).
  • the conjugation stock solution of 12- mer thiol 40 (500 ⁇ , 3.0 ⁇ ) was added to a solution of maleimide- activated bovine serum albumin (Imject® BSA, Pierce, Rockford, IL) (5 mg, ⁇ 1.5 ⁇ maleimide) in Imject® Conjugation Buffer (Pierce, 250 ⁇ diluted with water, 250 ⁇ ) and the resulting solution stirred for 18 hours at room temperature.
  • the reaction mixture was purified by de-salting on D-Salt P- 6000 10 mL column (Pierce, Rockford, IL). The column was pre-equilibrated with 30 mL of purification buffer (Pierce, Prod. No.
  • FIGs. 10A and 10B depict reaction schemes for conjugating mixed-/V-acetyl oligo- -(1 ⁇ 6)-glucosamine 18-mers (Ag 6 in FIG. 2B) to BSA and KLH as follows.
  • a conjugation stock solution of 18-mer thiol 34 was prepared by dissolving the 18-mer 34 (12 mg, 3.8 ⁇ ) in water (0.3 mL).
  • a suspension of tris(2-carboxyethyl)phosphine (TCEP)-bound agarose resin in water (200 ⁇ , ⁇ 1 ⁇ ) was added and stirred for 1 hour.
  • TCEP tris(2-carboxyethyl)phosphine
  • the TCEP-resin was filtered and to the filtrate was added Imject® Conjugation Buffer (Pierce, 300 ⁇ ) to provide a stock solution for conjugation to KLH (FIG. 10A) and BSA (FIG. 10B).
  • Imject® Conjugation Buffer Pieris, 300 ⁇
  • the conjugation stock solution of 18- mer thiol 34 (0.5 mL, 3.0 ⁇ ) was added to a solution of maleimide- activated bovine serum albumin (Imject® BSA, Pierce, Rockford, IL) (5 mg, ⁇ 1.5 ⁇ maleimide) in Imject® Conjugation Buffer (Pierce, 250 ⁇ _ diluted with water, 2500 ⁇ _) and the resulting solution stirred for 18 hours at room temperature.
  • the reaction mixture was purified by de-salting on D-Salt P- 6000 10 mL column (Pierce, Rockford, IL). The column was pre-equilibrated with 30 mL of purification buffer (Pierce, Prod.
  • Table 2 provides supporting characterization data for the antigen conjugates described in Example 12.
  • Antisera to antigen-KLH conjugates were raised in New Zealand white rabbits by four subcutaneous injections of antigen-KLH conjugate over 13 weeks. A pre-immune bleed generated 5 mL of baseline serum from each rabbit. The prime injection (10 ⁇ g antigen equivalent) was given as an emulsion in complete Freund's adjuvant (CFA). Subsequent injections (5 ⁇ g antigen equivalent) were given at three week intervals in incomplete Freund's adjuvant (I FA). Rabbits were bled every two weeks commencing one week after the third immunization. Approximately 25 - 30 mL of serum per rabbit was generated for each bleeding event, and was aliquoted into 1 -mL aliquots and frozen at -80°C. Serum was analyzed by ELISA against the
  • Affinity purification of antisera was conducted with serum from the third bleed from each rabbit. Affinity purification was carried out by coupling of antigen-BSA conjugates to CNBr-activated Sepharose 4B. Briefly, CNBr- activated Sepharose 4B (0.8 g, 2.5ml of final gel volume) was washed and re- swelled on a sintered glass filter with 1 mM HCI, then coupling buffer (0.1 M NaHC0 3 , 0.25M NaCI, pH 8.5). Antigen-BSA conjugate (1 mg) was dissolved in coupling buffer, mixed with the gel suspension and incubated overnight at 40°C.
  • An oligosaccharide-BSA conjugate solution was prepared by dissolving the conjugate in carbonate buffer (1.59 g Na 2 C0 3 , 2.93 g NaHC0 3 , 0.20 g NaN 3 , dissolved and diluted to 1 L in H 2 0, final pH 9.5) at a
  • Serum samples were prepared by 1 :5 serial dilutions starting from a 1 : 1 ,000 dilution of serum in 0.1 % BSA in PBS with 0.02% thimerosal. Diluted serum (100 ⁇ .) was added to each well and incubated for 2 hours at room temperature in a humidity chamber. The serum solution was removed, the wells were rinsed twice with water and dried on a paper towel. Goat anti- rabbit-HRP conjugate solution (100 was added and incubated for 2 hours at room temperature in a humidity chamber.
  • the HRP-conjugate solution was removed, and wells were washed three times with PBS/0.02% thimerosal/0.05% tween-20, twice with water, and dried on a paper towel.
  • TMB solution 100 ⁇ /weW
  • the reaction was stopped by the addition of 1 N HCI (100 ⁇ - ⁇ / ⁇ ) and the wells were read immediately at A450 (absorbance at 450 nm).
  • the titer of the test serum was designated as the dilution which gave an optical density (OD 450 ) reading of 0.1 above background.
  • the antisera were incubated on ELISA plates adsorbed with their corresponding BSA conjugate, specfically, (A) 6-Mix-BSA 41 ; (B) 12-Mix-BSA 43; and (C) 18-Mix-BSA 45 as described the ELISA protocol above (Example 14).
  • FIGs. 1 1 D-1 1 F depict antigen-specific IgG antibody titers from antigen-KLH conjugate-derived antibodies recovered at three successive stages of purification, including the pre-affinity purification fraction (3 rd bleed), the flow-through fraction, and the antibody-enriched (purified) fraction.
  • Results are shown as a function of antibody-antigen complex absorption (OD450) at the indicated serum dilutions obtained from the above-described antibody-enriched fractions generated against antigen-KLH conjugates corresponding to (A) 6-Mix-KLH 42; (B) 12-Mix-KLH 44; and (C) 18-Mix-KLH 46.
  • the antisera were incubated on ELISA plates adsorbed with their corresponding BSA conjugate, specifically, (A) 6-Mix-BSA 41 ; (B) 12- Mix-BSA 43; and (C) 18-Mix-BSA 45.
  • Affinity purification of 10 mL of 3 rd bleed sera yielded: 5.5 mL of a purified 6-Mix Ab solution at 2.3 mg/mL (12.7 mg purified Ab total); 5.4 mL of a purified 2-Mix Ab solution at 5.5 mg/mL (29.7 mg purified Ab total); and 5.4 mL of purified 8-Mix Ab solution at 1.7 mg/mL (9.2 mg purified Ab total).
  • FIGs. 12A-12G depict the results of a cross-ELISA assay examining the specificity and cross-reactivity between fully non-acetylated (6- NH 2 61 , 12-NH 2 62, I 8-NH2 63;); mixed (6-Mix 37, 12-Mix 40, 18-Mix 34) and fully acetylated (6-NHAc 58, 12-NHAc 59, 18-NHAc 60;) oligo- -(1 ->6)- glucosamines and antibodies derived therefrom.
  • 6- NH 2 61 , 12-NH 2 62, I 8-NH2 63; mixed (6-Mix 37, 12-Mix 40, 18-Mix 34) and fully acetylated (6-NHAc 58, 12-NHAc 59, 18-NHAc 60;) oligo- -(1 ->6)- glucosamines and antibodies derived therefrom.
  • Results are shown as a function of antibody-antigen complex absorption (OD 450 ) representing the averages from of antisera from two rabbits in each case at the indicated serum dilutions, whereby total OD450 is measured by subtracting away the background OD 450 from KLH antibodies alone.
  • OD 450 antibody-antigen complex absorption
  • FIGs. 13A- 3D depict the results of a whole-cell ELISA assay examining the binding of pre-immune sera (A, C) or immune sera (B, D) generated from rabbits immunized against (left to right) KLH control, fully non- acetylated antigen (12-NH 2 ) 62; mixed antigen (12-Mix) 44 and fully acetylated antigen (12-NHAc) 59 and Staphylococcus epidermidis coated onto ELISA fixed with methanol (A, B) or formalin (C, D). Results are shown as a function of antibody-antigen complex absorption (OD 450 ) at the indicated serum dilutions.
  • Example 18 - Opsonophagocytic Assay The opsonophagocytic (bacterial killing) activity of serum samples will be determined in an assay using S. aureus ATCC strain 25904 in the presence of phagocytic cells and complement.
  • HL-60 cells human promyelocytic cells; ATCC Cat #CCL240
  • the cells will be in differentiation medium for 5-7 days prior to use (RPMI 1640 with 15% heat-inactivated fetal bovine serum and 1.25% dimethylsulfoxide).
  • RPMI 1640 heat-inactivated fetal bovine serum and 1.25% dimethylsulfoxide
  • Approximately 50 ul of a stock solution of target bacteria will be grown on tryptic soy agar plates with 5% sheep red blood cells (blood agar plates) and incubated overnight at 36-37°C.
  • the bacterial lawn will be transferred to a sterile 50 ml conical containing 30 mis of tryptic soy broth with 1 % (w/v) glucose.
  • the bacteria will be grown in a shaking water batch set for 80 strokes per minute at 36-37°C.
  • the bacterial suspension will be adjusted to a %T of 72-75% (1 cm light path) and 2.7 - 3.0 ul of this suspension was mixed with 1.4 mis of TSB for a final concentration of approximately 5-6 X 104 cfu/ml.
  • Ten ul of the bacterial suspension will be mixed with 40 ul of heat- inactivated serum samples or reference antibody in a 96-well, round-bottom assay plate and incubated at 36-37°C in a shaking incubator at ⁇ 100 rpm for 30-40 minutes.
  • Antibody dilutions, if required, will be made in DMEM/F12 medium buffered with 10 mM HEPES to maintain a pH of 7.2-7.6.
  • Differentiated HL-60 cells will be pelleted by centrifugation at ⁇ 1000 X g at room temperature for 10 minutes and the supernatant removed.
  • the cells will be resuspended in DMEM/F12 medium buffered with 10 mM HEPES to maintain a pH of 7.2 - 7.6 and pelleted twice more to remove residual DMSO. After the final centrifugation the supernatant will be removed to near dryness and the cells suspended to a final concentration of 5 X 107viable cells per ml.
  • the complement will be derived from human serum treated with protein A and protein L to extract inherent antibodies reactive with the target bacteria.
  • the reagents will be mixed by rapid pipetting up and down 20-25 times using a multichannel pipettor set at 10 ul. After mixing a sample will be removed from each well, diluted 20-fold in water containing 0.1 % BSA and 0.01 % Tween20. These samples will be designated the T 0 samples and 100 ul of each T 0 sample will be transferred to a blood agar plate, allowed to dry, inverted and incubated overnight at 36-37°C.
  • the assay plate After transfer of the T 0 samples the assay plate will be incubated at 36-37°C in an orbital shaking incubator at 250-300 rpm for an additional 90 minutes. At the end of this incubation period samples will be taken from each well, diluted and plated as described above (T 90 samples).
  • Assay controls included HL-60's alone, HL-60's with complement and reference antibody. The percentage of bacterial killing will be calculated using the formula: (Number of colonies Tn - Number of colonies Tgn) * 100
  • SC subcutaneous
  • each mouse will be challenged via intravenous tail injection (IV) route with Staphylococcus aureus Newman strain at a concentration of approximately 4 x 109 CFU/mL in a dose volume of 0.2 ml_.
  • mice On Days -3, 10, 21 and 28 mice will be bled via retro-orbital sinus (approx. 0.2mL) into serum separator tubes for processing of sera (stored frozen at -20°C). On Days 30, 31 and 32, mice will be bled in the submandibular region (approx. 0.1 mL) onto solid media for bacteremia analysis. All surviving animals will be euthanized via C0 2 asphyxiation on Day 36. The percentage of survival and mortality for each group will be determined and microbiological analyses of blood were expressed as ⁇ bacteremia.
  • Example 20 Each of the synthetic, mixed-N-acetyl oligo- ⁇ - (1 ⁇ 6)-glucosamine hexamers and decamers listed in Tables 3 and 4 below were synthesized from building blocks 8, 14, 6 and 47 using methods exemplified in Example 1 1 as described below. Characterization data (NMR and mass spec) for each individual compound synthesized are listed below in Tables 3 and 4.
  • the decamer set was assembled via a 4 + 4 + 2 approach from the reducing end.
  • the appropriately designed tetramer acceptors were assembled using the strategy outlined above for the hexamer set.
  • a set of tetramers (assembled as above) was treated according to SOP 2 to remove each allyl group.
  • SOP 3 formation with K2C03
  • SOP 4 formation with K2C03

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Polymers & Plastics (AREA)
  • Biochemistry (AREA)
  • Materials Engineering (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

La présente invention concerne des structures synthétiques d'oligo-β-(1,6)-glucosamine et un procédé qui permet essentiellement la synthèse de n'importe quel type d'oligo-β-(1,6)-glucosamine présentant un nombre défini d'unités monosaccharide, y compris une configuration définie de résidus acétylés et non acétylés. L'invention concerne en outre des anticorps dirigés contre ces oligo-β-(1,6)-glucosamines synthétiques ainsi que des compositions associées et des procédés de traitement et de prévention d'infections provoquées par les bactéries exprimant des poly-β-(1,6)-glucosamines, telles que Staphylococcus aureus.
PCT/US2012/034449 2011-04-22 2012-04-20 Oligosaccharides synthétiques destinés à un vaccin contre un staphylocoque WO2012145626A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201161478238P 2011-04-22 2011-04-22
US61/478,238 2011-04-22

Publications (1)

Publication Number Publication Date
WO2012145626A1 true WO2012145626A1 (fr) 2012-10-26

Family

ID=47041937

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2012/034449 WO2012145626A1 (fr) 2011-04-22 2012-04-20 Oligosaccharides synthétiques destinés à un vaccin contre un staphylocoque

Country Status (1)

Country Link
WO (1) WO2012145626A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8852885B2 (en) 2011-02-02 2014-10-07 Solazyme, Inc. Production of hydroxylated fatty acids in Prototheca moriformis
WO2014186358A2 (fr) 2013-05-14 2014-11-20 Antonio Digiandomenico Sous-unités oligosaccharidiques de synthèse de l'exopolysaccharide psl de pseudomonas aeruginosa et applications associées
CN110511257A (zh) * 2019-09-23 2019-11-29 济南山目生物医药科技有限公司 一种四-O-乙酰基-2-邻苯二甲酰亚氨基-beta-葡萄糖的制备方法
US11203633B2 (en) 2011-11-07 2021-12-21 Medimmune Limited Polynucleotides encoding antibodies or antigen binding fragments thereof that bind pseudomonas perv
EP4061411A4 (fr) * 2019-11-22 2023-11-29 Alopexx, Inc. Procédés pour fournir une thérapie continue contre des microbes contenant pnag

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040191834A1 (en) * 1999-10-28 2004-09-30 Laferriere Craig Antony Joseph Novel method
US20090281058A1 (en) * 2005-06-14 2009-11-12 Genis Ehf Compositions of partially deacetylated chitin derivatives
WO2010011284A2 (fr) * 2008-07-21 2010-01-28 The Brigham And Women's Hospital, Inc. Procédés et compositions se rapportant à des bêta-1,6-glucosamine oligosaccharides synthétiques

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040191834A1 (en) * 1999-10-28 2004-09-30 Laferriere Craig Antony Joseph Novel method
US20090281058A1 (en) * 2005-06-14 2009-11-12 Genis Ehf Compositions of partially deacetylated chitin derivatives
WO2010011284A2 (fr) * 2008-07-21 2010-01-28 The Brigham And Women's Hospital, Inc. Procédés et compositions se rapportant à des bêta-1,6-glucosamine oligosaccharides synthétiques

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8852885B2 (en) 2011-02-02 2014-10-07 Solazyme, Inc. Production of hydroxylated fatty acids in Prototheca moriformis
US11203633B2 (en) 2011-11-07 2021-12-21 Medimmune Limited Polynucleotides encoding antibodies or antigen binding fragments thereof that bind pseudomonas perv
WO2014186358A2 (fr) 2013-05-14 2014-11-20 Antonio Digiandomenico Sous-unités oligosaccharidiques de synthèse de l'exopolysaccharide psl de pseudomonas aeruginosa et applications associées
CN105829342A (zh) * 2013-05-14 2016-08-03 米迪缪尼有限公司 铜绿假单胞菌的psl胞外多糖的合成寡糖亚基及其用途
EP3001838A4 (fr) * 2013-05-14 2017-02-08 Medlmmune, LLC Sous-unités oligosaccharidiques de synthèse de l'exopolysaccharide psl de pseudomonas aeruginosa et applications associées
US10870710B2 (en) 2013-05-14 2020-12-22 Medimmune Limited Synthetic oligosaccharide subunits of the Psl exopolysaccharide of pseudomonas aeruginosa and uses thereof
CN110511257A (zh) * 2019-09-23 2019-11-29 济南山目生物医药科技有限公司 一种四-O-乙酰基-2-邻苯二甲酰亚氨基-beta-葡萄糖的制备方法
CN110511257B (zh) * 2019-09-23 2023-08-04 济南山目生物医药科技有限公司 一种四-O-乙酰基-2-邻苯二甲酰亚氨基-beta-葡萄糖的制备方法
EP4061411A4 (fr) * 2019-11-22 2023-11-29 Alopexx, Inc. Procédés pour fournir une thérapie continue contre des microbes contenant pnag

Similar Documents

Publication Publication Date Title
US20220175904A1 (en) Methods and compositions relating to synthetic beta-1,6 glucosamine oligosaccharides
US20140170151A1 (en) Synthetic Oligosaccharides for Staphylococcus Vaccine
WO2012082635A1 (fr) Streptocoque du groupe a à base d'oligosaccharides synthétiques
WO2012145626A1 (fr) Oligosaccharides synthétiques destinés à un vaccin contre un staphylocoque
WO2011149778A1 (fr) Oligosaccharides synthétiques pour un vaccin contre neisseria meningitidis
EP3016685B1 (fr) Oligosaccharides de synthèse pour vaccin contre p. aeruginosa
US20160375120A1 (en) Synthetic oligosaccharides for moraxella vaccine
WO2012138698A1 (fr) Synthèse d'oligosaccharides d'acide bêta-mannuronique
EP2884842A1 (fr) Oligosaccharides synthétiques pour vaccin anti-p. aeruginosa
WO2012103058A1 (fr) Procédé pour synthétiser des glucanes avec des liaisons bêta-1,3 et bêta-1,6

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 12774601

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 12774601

Country of ref document: EP

Kind code of ref document: A1