Anthrax carbohydrates, synthesis and uses thereof

#Bacillus anthracis is a gram-positive, spore-forming bacterium that causes anthrax in humans and other mammals. Because of the high resilience of B. anthracis spores to extremes of their environment they can persist for many years until encountering a signal to germinate. When spores of B. anthracis are inhaled or ingested they may germinate and establish populations of vegetative cells which release anthrax toxins often resulting in the death of the host. The relative ease by which B. anthracis may be weaponized and the difficulty in early recognition of inhalation anthrax due to the non-specific nature of its symptoms were demonstrated by the death of four people who inhaled spores from contaminated mail (Jernigan et al., 2002, Emerg Infect Dis; 8:1019-1028; Jernigan et al., 2001, Emerg Infect Dis; 7:933-944; Webb, 2003, Proc Natl Acad Sci; 100:4355-4356). Consequently, considerable efforts are being directed towards the development of early disease diagnostics and improved anthrax vaccines.

#B. anthracis belongs to the Bacillus cereus group which includes the closely related B. cereus, B. anthracis, and B. thuringiensis species. Bacillus cereus strains can be potent food-borne pathogens, while B. thuringiensis are insect pathogens, and B. anthracis is the causative organism of anthrax. Although differentiation amongst B. cereus, B. thuringiensis and B. anthracis in practice is not difficult, the speed and specificity of confirmatory identification of virulent B. anthracis are of great importance in the context of bioterrorism preparedness and emergency response. This need is all the more pressing as the existence of non-pathogenic B. anthracis strains is well established and recent studies have shown the potential for B. cereus strains to harbor functional B. anthracis virulence genes (Hoffmaster et al., 2004, Proc Natl Acad Sci 101:8449-8454).

#Thus, there is a need for improved diagnostic assays that can reliably identify anthrax in its early stages and quickly distinguish it from other flu-like or febrile illnesses. And, in view of the potential use of B. anthracis as a weapon in a bioterrorism attack, there is a need for improved vaccines.

#FIGS. 1A-1B show gas chromatographic-mass spectrometric (GC-MS) sugar profiles obtained from B. anthracis Sterne 34F2 vegetative cell walls after hydrolysis of the total cell wall preparations and derivatisation into trimethylsilyl (TMS) methylglycosides. FIG. 1A is a GC-MS sugar profile obtained from a B. anthracis Sterne cell wall sample. FIG. 1B is a GC-MS sugar profile obtained from a B. anthracis Sterne purified sample released from cell walls through HF treatment and purified on BioGel P2 columns. “1” identifies Gal; “2” identifies Glc; “3” identifies ManNAc; “4” identifies GlcNAc; “6” identifies Inositol (internal standard); and “7” identifies N-acetylmuramic acid.

#FIGS. 2A-2B show gas chromatographic-mass spectrometric (GC-MS) sugar profiles obtained from B. cereus ATCC 10987 vegetative cell walls after hydrolysis of the total cell wall preparations and derivatisation into trimethylsilyl (TMS) methylglycosides. FIG. 2A is a GC-MS sugar profile obtained from a B. cereus ATCC 10987 cell wall sample. FIG. 2B is a GC-MS sugar profile obtained from a B. cereus ATCC 10987 purified sample released from cell walls through HF treatment and purified on BioGel P2 columns. “1” identifies Gal; “2” identifies Glc; “3” identifies ManNAc; “4” identifies GlcNAc; “5” identifies GalNAc; “6” identifies Inositol (internal standard); and “7” identifies N-acetylmuramic acid.

#FIG. 3 is a matrix-assisted laser desorption ionization-time of flight mass spectrum (MALDI-TOF MS) (positive mode) of the HF-PS from B. anthracis Ames. The spectra of the HF-PSs from B. anthracis Sterne, UT60, and Pasteur were all identical to this spectrum.

#Anthrax is a disease of caused by the spore-forming bacterium Bacillus anthracis. The present invention includes the isolation, characterization, and synthesis of B. anthracis carbohydrates and various diagnostic and therapeutic applications.

#The present invention includes methods of identifying B. anthracis by determining the glycosyl composition of a cell wall carbohydrate preparation. For example, the present invention includes methods of identifying B. anthracis by determining the glycosyl composition of a cell wall carbohydrate preparation, wherein a cell wall carbohydrate preparation from B. anthracis includes glucose (Glc), galactose (Gal), N-acetyl mannose (ManNAc), N-acetyl glucosamine (GlcNAc) and does not includes N-acetylgalactosamine (GalNAc). The present invention include methods of identifying B. anthracis by determining the glycosyl composition of a phosphate bound cell wall polysaccharide preparation, wherein a phosphate bound cell wall polysaccharide preparation from B. anthracis includes galactose (Gal), N-acetyl mannose (ManNAc) and N-acetyl glucosamine (GlcNAc) in a ratio of about 3:1:2. In some aspects, the phosphate bound cell wall polysaccharide preparation is released from the cell wall by treatment with aqueous hydrogen fluoride (HF). Determinations of the glycosyl composition of a cell wall carbohydrate preparation and/or a phosphate bound cell wall polysaccharide preparation, and ratios thereof, can be used in methods of determining the clade and/or lineage of a member the B. cereus group of species and in methods of identifying pathogenic members of the Bacillus cereus group.

#The present invention describes the isolation, characterization, and synthesis of novel oligosaccharide structures that are present in vegetative cells of B. anthracis. These structures are specific to B. anthracis and differ from that of other closely related Bacillus species. These structures can be conjugated to protein carriers or chemically synthesized and conjugated to protein carriers, to be used as a vaccine antigen for the prevention of anthrax, as a moiety for the distinction of B. anthracis from other bacteria, and as a diagnostic tool to detect B. anthracis infections. This novel oligosaccharide is a hydrogen fluoride released polysaccharide (also referred to herein as “HF-PS”) released from the vegetative cell wall of B. anthracis and has an amino sugar backbone of →6)-α-GlcNAc-(1→4)-β-ManNAc-(1→4)-β-GlcNAc-(1→. In some embodiments, the α-GlcNAc residue is substituted with α-Gal at O3, the α-GlcNAc residue is substituted with β-Gal at O4, and/or the β-GlcNAc is substituted with α-Gal at O3. The present invention includes an isolated oligosaccharide having the amino sugar backbone of →6)-α-GlcNAc-(1→4)-β-ManNAc-(1→4)-β-GlcNAc-(1→, wherein the α-GlcNAc residue is substituted with α-Gal at O3, the α-GlcNAc residue is substituted with β-Gal at O4, and/or the β-GlcNAc is substituted with α-Gal at O3. The present invention includes, but is no limited to, isolated monosaccharides, disaccharides, trisaccharides, and tetrasaccharides of this oligosaccharide, and synthetic analogs thereof. The present invention also includes polysaccharides that include repeating units such oligosaccharides, monosaccharides, disaccharides, trisaccharides, tetrasaccharides, or synthetic analogs.