Abstract:
An isolated or purified compound is provided, comprising A-GlcNAc[GlcNAc]-GalNAc-GalNAc-QuiNAc4NAc, wherein A is GlcNAc or Glc. There is further provided a vaccine based on such compound, having particular use to treat or prevent an infection caused by a  Campylobacter  organism. There is also provided an antibody or antisera against the compound, having particular use to diagnose the presence of an infection caused by a  Campylobacter  organism.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application is the U.S. National Stage filing under 35 U.S.C. §371 of International Application No. PCT/CA2011/050084, filed on Feb. 11, 2011, which claims benefit of U.S. Provisional Application No. 61/303,411, filed on Feb. 11, 2010, each of which is hereby incorporated by reference in its entirety. 
     
    
     FIELD OF THE INVENTION 
       [0002]    The invention relates to an N-linked glycan compound of Formula 1, which optionally may be fused or attached to an amino acid, peptide, protein or lipid. The invention further relates to antibodies and antisera against such compound, and the use thereof to diagnose an infection caused by a  Campylobacter  pathogen. The invention further relates to the use of the compound as a vaccine to treat or prevent infection by a  Campylobacter  pathogen. 
       BACKGROUND 
       [0003]      Campylobacter jejuni  and  Campylobacter coli  are the two most commonly isolated species of  campylobacter  that cause human infection. These organisms cause high rates of gastroenteritis worldwide, with the number of cases often exceeding that for  Salmonella, Shigella  and Enterotoxigenic  E. coli  combined (Butzler J P, Clinical Microbiology and Infection 2004). Furthermore,  C. jejuni  infection has been linked to the development of Guillain-Barré Syndrome, the most common cause of pathogen-caused paralysis since the eradication of polio (for reviews see: Kaida K, Glycobiology, 2009; Bereswill S &amp; Kist M, Current Opinion in Infectious Diseases, 2003). Other  Campylobacter  species have been recognized as emerging pathogens in human gastroenteritis ( C. upsaliensis ) were associated with inflammatory bowel disease in children and with gingivitis and periodontitis ( C. concisus ) (Zhang L S et al., Journal of Clinical Microbiology, 2009), like  C. jejuni  and  C. coli ) associated with diarrheal disease ( C. hyointestinalis ) and in causing venereal disease and infertility in livestock (especially cattle;  C. fetus venerealis ), and sheep abortions ( C. fetus fetus ) (Butzler J P, Clinical Microbiology and Infection, 2004 and references therein). 
         [0004]    Since the publication of the first  C. jejuni  genome sequence in 2000 (Parkhill J et al., Nature, 2000), several other  campylobacter  genome sequences have been reported. Unlike the majority of bacteria that have been described to date, all campylobacters contain conserved pgl genes required for N-linked protein glycosylation (Szymanski C M &amp; Wren B W, Nature Reviews Microbiology 2005; Nothaft H &amp; Szymanski C M, Nature Reviews Microbiology, 2010). 
         [0005]    In eukaryotes, glycosylated proteins are ubiquitous components of extracellular matrices and cellular surfaces. Their oligosaccharide moieties are implicated in a wide range of cell-cell and cell-matrix recognition events that are vital in biological processes ranging from immune recognition to cancer development. Glycosylation was previously considered to be restricted to eukaryotes, however through advances in analytical methods and genome sequencing, there have been increasing reports of both O-linked and N-linked protein glycosylation pathways in bacteria (Nothaft H &amp; Szymanski C M, Nature Reviews Microbiology, 2010). Since the discovery of the first general protein glycosylation pathway in bacteria (Szymanski C M et al., Molecular Microbiology 1999), the demonstration that the  C. jejuni  glycans are attached through an N-linkage en bloc (Kelly J H et al., Journal of Bacteriology 2006, Wacker M et al., Science 2002, Young N M et al., Journal of Biological Chemistry, 2002) and that the pathway not only can be functionally transferred into  Escherichia coli  (Wacker M et al., Science, 2002), but that the oligosaccharyltransferase enzyme (PglB) is capable of adding foreign sugars to protein (Feldman M et al., PNAS 2005), a surge of research activities has resulted in further characterization and exploitation of this system. 
         [0006]    The detailed structure of the unique  C. jejuni  N-linked heptasaccharide has been described (Young N M et al., Journal of Biological Chemistry, 2002). Using methods such as high resolution magic angle spinning (HR-MAS) NMR (Szymanski C M et al., Journal of Biological Chemistry, 2003), it has been shown that this heptasaccharide is conserved in structure in both  C. jejuni  and  Campylobacter coli.    
         [0007]    An intermediate in the  C. jejuni  N-linked glycosylation pathway has been described, namely a free (oligo-) heptasaccharide (fOS)—a soluble component of the  C. jejuni  periplasmic space (Liu X et al., Analytical Chemistry, 2006). This fOS has the identical structure as the N-linked oligosaccharide added onto proteins (Nothaft H et al., PNAS 2009). Under laboratory growth conditions, the ratio of fOS versus heptasaccharide N-linked to protein is approximately 9:1. The fOS in  C. jejuni  plays a role in osmoregulation similar to bacterial periplasmic glucans and this pathway can be manipulated by altering the environmental osmolyte concentration (Nothaft H et al., PNAS 2009). 
         [0008]      FIG. 1  shows N-linked protein glycosylation and free oligosaccharides in  C. jejuni . The undecaprenyl-pyrophosphate-linked heptasaccharide is assembled in the cytosol by the addition of nucleotide activated sugars (Szymanski C M et al., Journal of Biological Chemistry, 2003; Szymanski C M et al., Trends Microbiology 2003). The complete heptasaccharide is translocated across the inner membrane to the periplasm by the ABC transporter PglK (Alaimo C et al., EMBO Journal, 2006). The oligosaccharide is transferred to the amino group of asparagine in the protein consensus sequence, D/E-X1-N-X2-S/T, wherein X1, X2 can be any amino acid except proline, by PglB (Kowarik M et al., EMBO Journal 2006; Young N M et al., Journal of Biological Chemistry, 2002). In addition, large amounts of free heptasaccharide (fOS) can be found in  C. jejuni  (Liu X et al., Analytical Chemistry, 2006); the fOS to N-glycan ratio was determined to be 9:1. GlcNAc, N-acetylgalactosamine; Bacillosamine (Bac), 2,4-diacetamido-2,4,6-trideoxyglucose; GalNAc, N-acetylgalactosamine; Glc, Glucose (adapted from Szymanski C M et al., Trends Microbiology, 2003). 
       SUMMARY 
       [0009]    We have determined the N-glycan and fOS structures from a number of  Campylobacter  species, all of which possess N-linked glycans and fOS. In addition, we demonstrated that  campylobacter  N-glycans and fOS can be divided into two structural groups. The first group produces a similar structure to that published for  C. jejuni  and  C. coli  (Young N M et al., Journal of Biological Chemistry, 2002; Szymanski C M et al., Journal of Biological Chemistry, 2003). The second group produces a unique glycan structure which differs from that determined for  C. jejuni  and  C. coli  and that have never been described before.  Campylobacter  species that fall into this group include  Campylobacter fetus venerealis  (cause of venereal disease and infertility in cattle),  Campylobacter fetus fetus  (cause of sheep abortions),  Campylobacter concisus  (associated with gingivitis and periodontitis, and has been isolated from the feces of patients with gastroenteritis),  Campylobacter hyointestinalis  (like  C. jejuni  and  C. coli , is associated with diarrheal disease),  Campylobacter hyointestinalis  subspecies,  Campylobacter sputorum  and  Campylobacter sputorum  subspecies,  Campylobacter lanienae, Campylobacter ureolyticus  (an emerging enteric pathogen suggested to be involved in gastroenteritis, Bullman S et al., FEMS Immunology &amp; Medical Microbiology, 2010). 
         [0010]      Campylobacter hominis, Campylobacter gracilis, Campylobacter rectus, Campylobacter showae, Campylobacter mucosalis  and  Campylobacter curvus  are believed to be within the second group. 
         [0011]      FIG. 2  is a phylogenetic analysis of the protein sequences of the key component of this pathway, the oligosaccharyltransferase (PglB) including the genome sequenced  Campylobacter  species and other related organisms demonstrates that the Campylobacters divide into two groups. Within the  campylobacter  branch Structure 1 producing species are in the upper box, Formula 1A and Formula 1B producing strains are in the lower box (adapted from Nothaft H &amp; Szymanski C M, Nature Reviews Microbiology, 2010). 
         [0012]      FIG. 3 : illustrates N-glycan reactivity towards (A) a  C. jeuni  N-glycan-specific antiserum, (B) SBA-lectin (C) WGA-lectin reactivity and (D) mass-spectrometry-based fOS analyses showed that pgl pathway derived glycans differ among  Campylobacter  species. 
         [0013]    According to one aspect, the invention relates to a novel N-linked glycan (referred to as N-glycan) compound of Formula 1: A-GlcNAc[GlcNAc]-GalNAc-GalNAc-QuiNAc4NAc, wherein A is GlcNAc or Glc. This compound in its native form is common to several  Campylobacter  species. In its native form, the compound is soluble in the periplasm as well as attached to inner membrane and periplasmic proteins and most notably surface outer membrane proteins of many  Campylobacter  species, including pathogens. In the present invention, the compound of Formula 1 is provided in isolated and/or purified form. The compound comprises two hexasaccharides which differ from each other in a terminal sugar, which comprises either Glc or GlcNAc. The first of said compounds is: GlcNAc-GlcNAc[GlcNAc]-GalNAc-GalNAc-QuiNAc4NAc (herein Formula 1A). The second of said compounds is: Glc-GlcNAc[GlcNAc]-GalNAc-GalNAc-QuiNAc4NAc (herein Formula 1B). 
         [0014]    In the above Formula 1, QuiNAc4NAc represents an alternative signifier of the saccharide Bac, which constitutes an abbreviation of bacillosamine. 
         [0015]    In one aspect the invention relates to an isolated or purified compound comprising the compound of Formula 1 connected or linked to a single amino acid, an oligopeptide, a peptide, a protein, or a lipid. In one aspect, the oligopeptide or peptide comprises between 2 and 40 amino acids, or between 2 and 30 amino acids, or between 2 and 20 amino acids, or between 2 and 10 amino acids. 
         [0016]    The invention further relates to a method of producing an antibody or antiserum comprising the steps of providing the compound of Formula 1, inoculating an animal or humans with said compound to stimulate an immune response to said compound, withdrawing serum from said animal and optionally purifying said serum to obtain the antibody or antiserum. The resulting antibody or antiserum binds to  Campylobacter  species wherein the glycan described herein is native thereto, including  Campylobacter fetus venerealis, Campylobacter fetus fetus, Campylobacter concisus, Campylobacter hyointestinalis  and  Campylobacter hyointestinalis  subspecies,  Campylobacter sputorum  and  Campylobacter sputorum  subspecies,  Campylobacter lanienae, Campylobacter ureolyticus, Campylobacter hominis, Campylobacter gracilis, Campylobacter rectus, Campylobacter showae, Campylobacter mucosalis  and  Campylobacter curvus.    
         [0017]    The antibody or antiserum can be used for diagnostic purposes, to detect the presence of said organisms in an animal or in a human. 
         [0018]    Compounds of the present invention may be used in a vaccine formulation, with or without an adjuvant, against  Campylobacter fetus venerealis , which is a major cause of reproductive failure in cattle and for which the current vaccine is of limited use, or against other  Campylobacter  species wherein the glycan of Formula 1 is native to such organism, including the species listed above. Compounds of the present invention have possible uses in protein glycoengineering, therapeutic and diagnostic applications. The invention thus relates to a vaccine comprising the compound of Formula 1, optionally connected or linked to a single amino acid, an oligopeptide, a peptide, a protein, or a lipid. The single amino acid may comprise asparagine. 
         [0019]    The invention further relates to the use of said vaccine to treat or prevent an infection caused by a  Campylobacter  organism, wherein the compound of Formula 1 comprises a native glycan within said organism, and a method of treatment comprising said use, within a human or animal. 
         [0020]    According to another aspect, the invention relates to a method of improving the productivity and health of an animal herd by administering to said herd the vaccine as described above. 
         [0021]    The vaccines, antibodies and antisera described herein may also be used to for prevention, treatment and diagnosis in humans. 
     
    
     
       DESCRIPTION OF THE DRAWINGS 
         [0022]      FIG. 1  shows N-linked protein glycosylation and free oligosaccharides in  C. jejuni.    
           [0023]      FIG. 2  is a chart summarizing the fOS and N-glycan structures in various  Campylobacter  species. 
           [0024]      FIGS. 3A-3D  depict N-glycans and fOS analyses in select  Campylobacter  species. 
           [0025]      FIG. 4  A is the 1H NMR spectrum of purified fOS from  C. fetus fetus.    
           [0026]      FIG. 4B  overlay of 2D HSQC spectra for  C. fetus fetus  and  C. fetus venerealis    
           [0027]      FIG. 5  depicts structures of fOS and N-glycans of  C. jejuni, C. coli  and  C. upsaliensis  (structure 1) and Formula 1A and Formula 1B. 
           [0028]      FIG. 6  shows elution profiles of Formula 1A, Formula 1B, and Formula 1A and Formula 1B, under conditions described herein and the confirmation of purified Formula 1A and Formula 1B. 
           [0029]      FIG. 7  illustrates conjugation of purified Formula 1A and Formula 1B compounds to BSA. 
           [0030]      FIG. 8  illustrates immuno-blots with antiserum raised against BSA-glycoconjugates. 
           [0031]      FIGS. 9A-F  depict MS spectra of glycopeptides comprising compounds of Formula 1 as the glycan moiety. 
           [0032]      FIG. 10  depicts whole cells that were labeled with Formula 1A and Formula 1 B-specific antiserum. 
           [0033]      FIG. 11  illustrates immuno-blots with antiserum raised Formula 1A, Formula 1B and structure 1. 
       
    
    
     DETAILED DESCRIPTION 
       [0034]    The present invention relates to the glycan compound A-GlcNAc[GlcNAc]-GalNAc-GalNAc-QuiNAc4NAc, wherein A is GlcNAc or Glc. The above compound encompasses the two glycan compounds GlcNAc-GlcNAc[GlcNAc]-GalNAc-GalNAc-QuiNAc4NAc (herein Formula 1A) and Glc-GlcNAc[GlcNAc]-GalNAc-GalNAc-QuiNAc4NAc (herein Formula 1B). 
         [0035]    In the above Formulae, QuiNAc4NAc represents an alternative signifier of the saccharide Bac, which constitutes an abbreviation of bacillosamine. The compound of Formula 1 is optionally connected or linked to a single amino acid, an oligopeptide, a peptide, a protein, or a lipid. 
         [0036]    Said lipid can be isolated and purified from a bacterial, archaeal or eukaryotic source or can be chemically synthesized. Said linkage of the glycan compound to the lipid can be mediated by a phosphate, a pyrophosphate linker or by a glycosidic linkage. Examples of lipids (with various chain lengths, saturation grade and configuration) linked to N-glycans were described (Faridmoayer et al., Journal of Biological Chemistry, 2009; Chen M M et al., Biochemistry, 2007). Lipid-linked N-glycan compounds produced in the native host or in a heterologous expression system include undecaprenyl-phosphate-linked N-glycan compounds as shown for the  C. jejuni  N-glycan (Reid C W et al., Analytical Chemistry, 2008, Reid C W et al., Analytical Chemistry, 2009) and proposed for the  C. lari  N-glycan (Schwarz F et al., Glycobiology 2011)) and N-glycan-LipidA conjugates (shown for the N-glycan of  C. jejuni  (van Sorge N M et al., Cellular Microbiology, 2009)). 
         [0037]    It has been determined that the above compound is substantially conserved across multiple species of  Campylobacter.    
         [0038]      FIGS. 3A-3D  depict N-glycans and fOS in select  Campylobacter  species.  3 A) Western Blot using antiserum that recognizes the N-linked hepta-saccharide of  C. jejuni  cross-reacted with other  Campylobacter  species (open boxes) that also reacted with ( 3 B) soybean agglutinin recognizing terminal GalNAc residues, but shows little reactivity with ( 3 C) wheat-germ agglutinin (WGA) that recognizes terminal GlcNAc residues present in Formula 1A and Formula 1B. Species that did not react with the  C. jejuni -specific antiserum but reacted with WGA were highlighted.  3 D) Examples of mass spectrometry of fractions enriched for fOS of (1)  C. jejuni  (2)  C. fetus venerealis , (3)  C. concisus , (4)  C. fetus fetus , and (5)  C. hyointestinalis ; results of all species analyzed by mass spectrometry are summarized in Table 1. 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 MS-MS 2  (m/z) 
               
             
          
           
               
                 
                   Campylobacter 
                 
                 free oligosaccharide 
                   
               
               
                 species 
                 (fOS) 
                 N-linked glycan 
               
               
                   
               
               
                 
                   C. jejuni 
                 
                 GalNAc5-Glc-Bac 
                 HexNAc5-Hex-Bac- 
               
               
                   
                 (1425.0) 
                 Asn (1539.5) 
               
               
                 
                   C. coli 
                 
                 HexNAc5-Hex-Bac 
                 N/D 
               
               
                   
                 (1425.0) 
               
               
                 
                   C. upsaliensis 
                 
                 HexNAc5-Hex-Bac 
                 N/D 
               
               
                   
                 (1425.0) 
               
               
                 
                   C. fetus fetus 
                 
                 HexNAc5-Bac (1263.5) 
                 HexNAc5-Bac- 
               
               
                   
                 Formula 1A 
                 Asn (1377.5) 
               
               
                   
                 HexNAc4-Glc-Bac 
                 Formula 1A-Asn(N)-linked 
               
               
                   
                 (1222.5) 
                 On a Peptide: 
               
               
                   
                 Formula 1B 
                 HexNAc5-Bac-Asn 
               
               
                   
                   
                 Formula 1A-Asn(N)-linked 
               
               
                   
                   
                 Hex-HexNAc4-Bac-Asn 
               
               
                   
                   
                 Formula 1B-Asn(N)-linked 
               
               
                 
                   C. fetus 
                 
                 HexNAc5-Bac (1263.5) 
                 On a Peptide: 
               
               
                 
                   venerealis 
                 
                 Formula 1A 
                 HexNAc5-Bac-Asn 
               
               
                   
                 HexNAc4-Hex-Bac 
                 Formula 1A-Asn(N)-linked 
               
               
                   
                 (1222.5) 
                 Hex-HexNAc4-Bac-Asn 
               
               
                   
                 Formula 1B 
                 Formula 1B-Asn(N)-linked 
               
               
                 
                   C. concisus 
                 
                 HexNAc5-Bac (1263.5) 
                 On a Peptide: 
               
               
                   
                 Formula 1A 
                 HexNAc5-Bac-Asn 
               
               
                   
                 HexNAc4-Hex-Bac 
                 Formula 1A-Asn(N)-linked 
               
               
                   
                 (1222.0) 
                 Hex-HexNAc4-Bac-Asn 
               
               
                   
                 Formula 1B 
                 Formula 1B-Asn(N)-linked 
               
               
                 
                   C. hyointestinalis 
                 
                 HexNAc5-Bac (1263.5) 
                 N/D 
               
               
                   
                 Formula 1A 
               
               
                   
                 HexNAc4-Hex-Bac 
               
               
                   
                 (1222.0) 
               
               
                   
                 Formula 1B 
               
               
                   
               
             
          
         
       
     
         [0039]    Table 1. fOS and N-glycan structure masses determined by mass spectrometry in select  Campylobacter  strains. Numbers indicate the mass(es) of Formula 1A and Formula 1B either as free oligosaccharide (fOS) or Asn-linked. Masses were obtained in positive ion mode from whole cell lysates of the indicated strain. The structures of Formula 1A and Formula 1B were determined by NMR as shown in  FIG. 4 , Table 3 and  FIG. 5 . N/D, not determined. 
       Example 1 
     Purification of Compounds of Formulae 1A and 1B 
       [0040]      Campylobacter jejuni  11168,  C. concisus, C. hyointestinalis, C. fetus fetus  and  C. fetus venerealis  were grown under microaerophilic conditions. Whole cells obtained after centrifugation were digested with large excess of proteinase K at pH 8 (adjusted by addition of ammonia) at 37° C. for 48 hours. Products of digestion or free oligosaccharides were separated on Sephadex G-15 column (1.5×60 cm) and each fraction eluted before the salt peak was dried and analyzed by  1 H NMR. Fractions containing desired products were separated by anion exchange chromatography on a Hitrap Q column (5 mL size, Amersham) and the glycans were eluted with a linear gradient of NaCl-(0-1 M, 1 h) that resulted in the isolation of a mixture of both glycan compounds (Formula 1A and Formula 1B). Desalting was performed on Sephadex G15 prior to analysis by NMR. 
       Example 2 
     NMR Spectroscopy Analysis 
       [0041]    NMR experiments on the glycans obtained in example 1 were carried out on a Varian INOVA 500 MHz ( 1 H) spectrometer with 3 mm gradient probe at 25° C. with acetone internal reference (2.225 ppm for  1 H and 31.45 ppm for  13 C) using standard pulse sequences DQCOSY, TOCSY (mixing time 120 ms), ROESY (mixing time 500 ms), HSQC and HMBC (100 ms long range transfer delay). AQ time was kept at 0.8-1 sec for H—H correlations and 0.25 sec for HSQC, 256 increments was acquired for t1. The Results are shown in  FIG. 4 ,  FIG. 5  (NMR spectra and structures) and Table 2, corresponding chemical shifts. 
         [0042]      FIG. 4  A is the  1 H NMR spectrum of purified fOS from  C. fetus fetus .  FIG. 4B  overlay of 2D HSQC spectra for  C. fetus fetus  and  C. fetus venerealis  indicating that fOS structures from both species are identical. The NMR spectrum can also be overlaid with one obtained for  C. concisus  (not shown). The corresponding chemical shifts δ (ppm) for the purified free oligosaccharide from  C. fetus fetus  (as shown in  FIG. 4  A) are summarized in Table 2. Carbon and proton chemical shifts were referenced to an internal acetone standard (δH 2.225 ppm, δC 31.07 ppm). 
         [0043]    The  campylobacter  glycans that are either added to protein or appear in a free form (fOS) can be divided into two structural groups. The first group produces a unique glycan structure that was previously determined for  C. jejuni  and  C. coli  and herein for  C. upsaliensis. Campylobacters  which fall into the second group consist of  Campylobacter fetus venerealis  (cause of venereal disease and infertility in cattle),  Campylobacter fetus fetus  (cause of sheep abortions),  Campylobacter concisus  and  Campylobacter hyointestinalis.    
         [0044]    Structure determination by NMR using large scale purified free oligosaccharides (fOS) from  C. fetus fetus, C. fetus venerealis , and  C. concisus  demonstrated that this second group of campylobacters produced a structure different from that originally described for  C. jejuni  and  C. coli  ( FIG. 4  and  FIG. 5 ). 
         [0000]    
       
         
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                 Unit, compound 
                 Atom 
                 1 
                 2 
                 3 
                 4 
                 5 
                 6(6a) 
                 6b 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 α-QuiNAc4NAc α-A 
                   1 H 
                 5.14 
                 4.07 
                 4.00 
                 3.80 
                 3.97 
                 1.14 
                   
               
               
                   
                   13 C 
                 92.2 
                 54.3 
                 73.3 
                 58.3 
                 68.0 
                 17.6 
               
               
                 α-QuiNAc4NAc β-A 
                   1 H 
                 4.73 
                 3.78 
                 3.81 
                 3.80 
                 3.54 
                 1.17 
               
               
                   
                   13 C 
                 95.6 
                 57.2 
                 75.9 
                 58.3 
                 72.3 
                 17.6 
               
               
                 α-GalNAc B 
                   1 H 
                 5.21 
                 4.23 
                 3.83 
                 4.05 
                 3.89 
                 3.70 
                 3.75 
               
               
                   
                   13 C 
                 98.0 
                 50.7 
                 68.0 
                 77.5 
                 72.6 
                 60.9 
               
               
                 α-GalNAc C 
                   1 H 
                 5.00 
                 4.28 
                 4.14 
                 4.08 
                 4.37 
                 3.59 
                 3.64 
               
               
                   
                   
                 5.02 
               
               
                   
                   13 C 
                 99.4 
                 51.4 
                 67.7 
                 77.6 
                 72.3 
                 60.5 
               
               
                 α-GlcNAc D 
                   1 H 
                 4.90 
                 4.05 
                 3.95 
                 3.76 
                 4.33 
                 3.57 
                 4.01 
               
               
                   
                   13 C 
                 99.4 
                 54.2 
                 80.0 
                 69.3 
                 71.9 
                 65.8 
               
               
                 β-GlcNAc E 
                   1 H 
                 4.58 
                 3.70 
                 3.59 
                 3.48 
                 3.48 
                 3.77 
                 3.93 
               
               
                   
                   13 C 
                 102.5 
                 57.0 
                 74.6 
                 71.1 
                 77.0 
                 61.8 
               
               
                 α-GlcNAc F 
                   1 H 
                 4.93 
                 3.92 
                 3.78 
                 3.50 
                 3.75 
                 3.79 
                 3.85 
               
               
                   
                   13 C 
                 98.1 
                 54.9 
                 72.3 
                 71.2 
                 73.1 
                 61.7 
               
               
                 α-GalNAc C′ 
                   1 H 
                 5.00 
                 4.28 
                 4.14 
                 4.10 
                 4.37 
                 3.59 
                 3.64 
               
               
                   
                   13 C 
                 99.4 
                 51.4 
                 67.7 
                 77.7 
                 72.3 
                 60.5 
               
               
                 α-GlcNAc D′ 
                   1 H 
                 4.97 
                 4.16 
                 3.97 
                 3.75 
                 4.34 
                 3.57 
                 4.01 
               
               
                   
                   13 C 
                 99.5 
                 54.1 
                 81.7 
                 69.3 
                 71.9 
                 65.8 
               
               
                 β-Glc G 
                   1 H 
                 4.53 
                 3.29 
                 3.51 
                 3.42 
                 3.48 
                 3.74 
                 3.91 
               
               
                   
                   13 C 
                 104.3 
                 74.2 
                 76.8 
                 70.6 
                 76.8 
                 61.7 
               
               
                   
               
             
          
         
       
     
         [0045]    Table 2: Chemical shifts δ (ppm) for the purified free oligosaccharides (Formula 1A and Formula 1B) from  C. fetus fetus  (for the spectrum shown in  FIG. 4A ). Carbon and proton chemical shifts were referenced to an internal acetone standard (δH 2.225 ppm, δC 31.07 ppm). Capital letters refer to the single sugar residues as outlined in  FIGS. 4A and 5 . 
       Example 3 
     Preparation of Glycan of Formula 1 Compounds Linked to Single Amino Acid 
       [0046]    A Pronase E digest of whole cell extracts obtained after lysis of intact cells followed by mass spectrometry as described by Liu X. et al., Anal Chem, 2005 and Nothaft H. et al., Methods Mol Biol, 2010 identified the  C. jejuni  heptasaccharide (structure 1) attached to a single asparagine and Formula 1A linked to a single asparagine in  C. fetus fetus.    
       Example 4 
     Expression of Formula 1 Compounds 
       [0047]    The protein glycosylation operon encoding all the genes necessary for the production and transfer of Formula 1A and Formula 1B compounds can be cloned and expressed from an  E. coli  plasmid(s). Alternatively, the glycosyltransferases on a plasmid described by Wacker et al, Science 2002 that contains the  C. jejuni  protein glycosylation (pgl) operon can be exchanged by Formula 1A and Formula 1B producing glycosyltransferases. Expression of Formula 1A and Formula 1B compounds can be done in a heterologous system in the presence of an affinity-tagged acceptor peptide for N-linked protein glycosylation (already shown for the  C. jejuni  N-glycan and for the  C. lari  N-glycan Wacker et al., Science 2002, Schwarz et al., Glycobiology 2011). The glycan containing protein/peptide can be purified by affinity-tag purification, if necessary in combination with lectin or glycan-recognizing agent affinity chromatography to separate the glycosylated and the non-glycosylated peptides. 
       Example 5 
     Purification of Formula 1A and Formula 1B 
       [0048]    Purified Formula 1A and Formula 1B fOS were separated by high performance anion exchange chromatography with pulsed amperometric detection (HPAEC/PAD).  FIG. 6  shows the elution profile of a CarboPac® PA200 Analytical Column (3×250 mm CarboPac PA100 equipped with a Guard Column: 3×50 mm) under the following conditions: flow rate: 0.5 mL/min; eluent system, 50 mM sodium acetate in 100 mM sodium hydroxide; detection mode, pulsed amperometry, quadruple waveform, Au electrode; the ambient column temperature was set to −30° C.  6 A) Approximately 0.5 nmoles of either a mixture of Formula 1A and Formula 1B or ( 6 B) Formula 1A and ( 6 C) Formula 1B after separation using a semi preparative PA 100 column (9×250 mm) and a fraction collector (DIONEX UltiMate 3000) under the same conditions as outlined above were analyzed by HPAEC/PAD. Fractions containing either Formula 1A or Formula 1B were neutralized with equimolar amounts of 0.2 M HCl and stored at −20° C. The spectra obtained by electrospray ionization mass spectrometry (ESI-MS) of ( 6 D) purified Formula 1A and ( 6 E) Formula 1B after purification that correspond to observed masses of the mixture of Formula 1A and Formula 1B as outlined in Table 1. 
       Example 6 
     Conjugation of Formula 1A and Formula 1B to BSA 
       [0049]    Purified and neutralized Formula 1A and 1B compounds prepared in Example 5 were conjugated to BSA by reductive amination (see Gildersleeve J C., Bioconjug Chem. 2008). A mixture of Bovine serum albumin (BSA; 2 μL of a 150 mg/mL solution; fraction V), sodium borate (5.5 μL of a 400 mM solution, pH 8.5), sodium sulfate (3.7 μL of a 3 M solution, 50° C.), oligosaccharide (Formula 1A or Formula 1B) (7.0 μL of 20 mM solution for 15 eq), H2O (1.4 μL) and sodium cyanoborohydride (2.2 μL of a 3 M solution) was incubated in a 200 μL PCR tube in a PCR thermal cycler at 56° C. for 96 h with a heated lid. The reaction was diluted with H2O to a final volume of 100 μL, transferred to a 500 μl dialysis tube (MWCO 10,000) and dialyzed three times against H2O (2.51). 
         [0050]      FIG. 7  shows conjugation of Formula 1A and Formula 1B to BSA: Glyco-conjugates separated by 12.5% PAGE ( 7 A) and monitored by Western blotting using commercially available WGA-lectin conjugated with alkaline phosphatase ( 7 B). lane 1, 400 ng BSA fraction V; lane 2, 400 ng of Formula 1B coupled to BSA fraction V; lane 3, 400 ng of Formula 1B coupled to BSA fraction V. Molecular weight markers (MW in KDa) are indicated on the left. 
       Example 7 
     Rabbit Immunization with Formula 1A and Formula 1B-BSA Conjugates 
       [0051]    New Zealand White Rabbits were immunized with 2 mg of each of the glyco-conjugate compounds prepared in Example 6, using a 6 week immunization protocol (according to the animal care committee protocol No. 717). After an initial subcutaneous injection (at 3 sites, 0.5 ml were injected at each site) of 2.0 mg using Freund&#39;s complete adjuvant (in a 1:1 ratio with the antigen), a booster dose with 2.0 mg mg of each Formula 1A and Formula 1B-BSA conjugates mixed with Freund&#39;s incomplete adjuvant (in a 1:1 ratio with the antigen) was given subcutaneously (at 3 sites 0.5 ml were injected at each site) after 4 weeks. After 6 weeks serum from a 5 ml blood sample from each animal was prepared by cooling the blood sample for 60 min on ice followed by centrifugation for 20 min at 10.000×g. Individual sera were analyzed for the production of Formula 1A and Formula 1B-specific antibodies by Western Blotting with  Campylobacter  whole cell lysates ( FIG. 8 ). 
         [0052]      FIG. 8  shows an immuno-blot with antiserum that was raised against the single BSA-glycoconjugates: 120 μg of either  C. jejuni  11168 wild-type (lane 1),  C. jejuni  11168 pglB mutant strain (lane 2) or  C. fetus fetus  (lane 3) were applied to 12.5% SDS-PAGE. After transfer to a PVDF membrane the immobilized proteins were probed ( 8 A) with a 1:2000 dilution of a serum sample obtained from a rabbit that was immunized with BSA-Formula 1B compound (SZR-1) and with ( 8 B) serum of a rabbit immunized with BSA-Formula 1A compound (SZR-3). Molecular weight markers (MW in KDa) are indicated on the left. 
       Example 8 
     Formula 1A and 1B Compounds are N-Linked 
       [0053]    Cells were grown in MH broth under microaerobic conditions, harvested by centrifugation and washed twice in 50 mM Tris-HCl, pH 7.2. Pellets were freeze dried and placed in 1.5 ml Lobind tubes (Eppendorf). Pellets (10 mg) were resuspended in 1 ml ice-cold Tris-HCl (pH 7.5) in the presence of 150 units of Benozanase, vortexed to resuspend and kept on ice. After sonication (6 times 30 seconds with 1 minute on ice between) the cellular debris was removed by centrifugation at 20,000×g for 30 minutes at 4° C. The supernatant was collected in LoBind (Eppendorf) tubes and freeze dried. Sample processing, glycopeptide enrichment and mass spectrometry were applied as described (Scott N E, et al Molecular and Cellular Proteomics, 2010). Formula 1A and Formula 1B N-linked to asparagines located in polypeptides derived from proteolytic digested cell lysates were identified for  C. fetus fetus, C. fetus venerealis  and  C. concisus  (Table 3). 
         [0054]    Glycopeptides were isolated and identified from  Campylobacter fetus fetus, Campylobacter fetus venerealis , and  Campylobacter concisus  with the results shown in Table 3. The glycan portions there of all comprised the compound of Formula 1A or 1B. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Formula 1A and Formula 1B compounds containing glycopeptides 
               
             
          
           
               
                   
                   
                   
                 Area of 
                   
                 Ratio 
               
               
                   
                   
                   
                 HexNAc4- 
                 Area of 
                 (HexNAc5-Bac 
               
               
                 Accession 
                   
                   
                 Hex-Bac 
                 HexNAc5-Bac 
                 (Formula 1A)/ 
               
               
                 number 
                   
                   
                 glycoform 
                 glycoform 
                 HexNAc4-Hex-Bac 
               
               
                 (uniprot) 
                 Protein name 
                 peptide sequence 
                 (Formula 1B) 
                 (Formula 1A) 
                 (Formula 1B) 
               
               
                   
               
             
          
           
               
                 Identified glycopeptides from  Campylobacter fetus   fetus   
               
             
          
           
               
                 A0RM44_CAMFF 
                 PDZ domain 
                 109NSTEMGH 
                  9915111 
                 25419948 
                 2.563758288 
               
               
                   
                 protein 
                 IK118 
                   
                   
                   
               
               
                 A0RN17_CAMFF 
                 Putative 
                 47YAKDENVSI 
                  8804978 
                 25437438 
                 2.888983709 
               
               
                   
                 cytochrome c 
                 NVYK59 
                   
                   
                   
               
               
                   
                 family protein 
                   
                   
                   
                   
               
               
                 A0RN17_CAMFF 
                 Putative 
                 50DENVSINV 
                  1509926 
                  7656723 
                 5.070925992 
               
               
                   
                 cytochrome c 
                 YK59 
                   
                   
                   
               
               
                   
                 family protein 
                   
                   
                   
                   
               
               
                 A0RM98_CAMFF 
                 Cytochrome c 
                 374VHEYYFD 
                 19342158 
                  5383759 
                 0.278343244 
               
               
                   
                 oxidase accessory 
                 VNDTR386 
                   
                   
                   
               
               
                   
                 protein CcoG 
                   
                   
                   
                   
               
               
                 A0RP44_CAMFF 
                 Copper 
                 85SDDNETFY 
                   373909 
                  1910579 
                 5.10974328  
               
               
                   
                 homeostasis 
                 FK94 
                   
                   
                   
               
               
                   
                 protein CutF 
                   
                   
                   
                   
               
               
                 A0RRM2_CAMFF 
                 Mechano- 
                 47NASLGHDL 
                  4565871 
                 14763004 
                 3.233337955 
               
               
                   
                 sensitive ion 
                 DSLK58 
                   
                   
                   
               
               
                   
                 channel family 
                   
                   
                   
                   
               
               
                   
                 protein 
                   
                   
                   
                   
               
               
                 A0RP42_CAMFF 
                 Hydroxylamine 
                 283MSGIGDL 
                 14116580 
                 44662532 
                 3.16383515  
               
               
                   
                 oxidase 
                 NTTHNVSVR299 
                   
                   
                   
               
               
                 A0RM84_CAMFF 
                 Soluble lytic 
                 155FLNDNNIT 
                   310080 
                   638344 
                 2.058642931 
               
               
                   
                 murein 
                 SSFIPHLSSN 
                   
                   
                   
               
               
                   
                 transglycosylase 
                 WQFK177 
                   
                   
                   
               
               
                 A0RN61_CAMFF 
                 Putative 
                 68FGLGDDNN 
                  3488453 
                 20563891 
                 5.894845366 
               
               
                   
                 uncharacterized 
                 ETTK79 
                   
                   
                   
               
               
                   
                 protein 
                   
                   
                   
                   
               
               
                   
               
             
          
           
               
                 Identified glycopeptides of  Campylobacter fetus venerealis   
               
             
          
           
               
                 ACLG0100 
                 PDZ domain 
                 109NSTEMGH 
                  2200609 
                  1611018 
                 0.732078257 
               
               
                 0782.1; 
                 protein 
                 IK118 
                   
                   
                   
               
               
                 
                   C. fetus 
                 
                   
                   
                   
                   
                   
               
               
                 
                   venerealis 
                 
                   
                   
                   
                   
                   
               
               
                 Azul-94 
                   
                   
                   
                   
                   
               
               
                 Contig782 
                   
                   
                   
                   
                   
               
               
                 unknown 
                 unknown 
                 DTNQTFTK 
                  4891730 
                  7235352 
                 1.479098806 
               
               
                 unknown 
                 unknown 
                 NFHDTNK 
                  4045072 
                  2602866 
                 0.643465926 
               
               
                   
               
             
          
           
               
                 Identified glycopeptides of  Campylobacter concisus   
               
             
          
           
               
                 unknown 
                 unknown 
                 (NF)HDTNK 
                 4261136 
                 16308843 
                 3.827346276 
               
               
                   
               
             
          
         
       
     
         [0055]    List of proteins identified to be N-linked with Formula 1A and Formula 1B. The single peak areas for Formula 1A and Formula 1B were determined by multiple reaction monitoring (MRM) mass spectrometry. 
         [0056]      FIGS. 9A-F  depict MS spectra showing that both formula 1A and formula 1B compounds are N-linked (to the same peptide), as follows: 
         [0057]      9 A) MS spectrum (precursor ion scan) of tryptic digested, HILIC-LC enriched peptides; ( 9 B) Quantification of relative peak areas of the corresponding ions; ( 9 C) MS/MS of the carbohydrate portion, ( 9 D) MS/MS of the peptide portion of the m/z ion 968.44545;  9 E) MS/MS of the carbohydrate portion, and  9 F) MS/MS of the peptide portion of the m/z ion 982.12069. 
       Example 9 
     Formula 1A and 1B are Presented on the  Campylobacter  Cell Surface 
       [0058]    Cells of  C. fetus fetus, C. fetus venerealis, C. concisus, C. hyointestinalis , and  C. jejuni  were grown on MH plates for 18-24 hours under microaerophilic conditions. Cells were harvested from the plate with 2 ml MH broth, cooled on ice for 10 min, centrifuged for 5 min at 6,000×g. Cells were kept on ice for all further labeling and washing steps using pre-cooled (4° C.) solutions. Cells were washed twice with 2 ml washing buffer (50 mM potassium phosphate, 100 mM NaCl). To prevent unspecific binding cells were blocked with 1% Skim Milk in washing buffer for 30 min. Primary antibody (1:1,000 dilution in washing buffer with 0.5% Skim) was applied for 30 min. Cells were washed 3 times with 2 ml Washing buffer. Fluorescent labeled secondary antibody (anti-Rabbit-IgG-Alexa-Flour546, diluted 1:100 in washing buffer with 0.5% Skim Milk) was applied for 30 min and cells were washed 4-times in washing buffer. Cell surface labeling was monitored using a Leica DMRXA Upright Microscope equipped with an Optronics MacroFire Digital Camera (LM-MFCCD). Each picture was taken under identical software settings.  C. jejuni  that produces structure 1 served as a negative control. 
         [0059]      FIG. 10  shows fluorescent microscopy images of whole cells of  C. fetus fetus, C. fetus venerealis, C. concisus, C. hyointestinalis , and  C. jejuni  (negative control) probed with  10 A, SZR-1 (anti-Formula 1B) or  10 B, SZR-3 (anti-Formula 1A) as the primary antiserum and a fluorescent-tagged secondary antibody. 
         [0060]      FIG. 11  shows immuno-blots with antiserum that was raised either against Formula 1A or Formula 1B or with and antiserum that targets the N-glycan of  C. jejuni  (structure 1, hR6 was described by Schwarz et al., Nature Chemical Biology, 2010). 90 μg of  C. fetus fetus  (lane 1),  C. fetus venerealis  (lane 2),  C. concisus  (lane 3),  C. hyointestinalis  (lane 4) and  C. jejuni  11168 (lane 5) were applied to 12.5% SDS-PAGE. After transfer to a PVDF membrane the immobilized proteins were probed with ( 11 A) a 1:500 dilution of a serum sample obtained from a rabbit that was immunized with BSA-Formula 1B compound (SZR-1), with ( 11 B) a 1:500 dilution serum of a rabbit immunized with BSA-Formula 1A compound (SZR-3) or ( 11 C) with a 1:5,000 of an antiserum specific against the N-glycan of  C. jejuni  (hR6). Molecular weight markers (MW in KDa) are indicated on the left. 
         [0061]    The glycan compounds (Formula 1A and Formula 1B) can be attached to various glycan carriers (peptides, lipids). The resulting compounds can be used to stimulate an immune-response against the respective structure that will be protective against infection with Formula 1A and Formula 1B presenting bacterial species. 
         [0062]    Generated antisera/antibodies can be used (when i.e immobilized on a surface) as a diagnostic to detect e.g.  C. fetus venerealis  or  C. fetus fetus  in infected livestock (especially  C. fetus venerealis  cattle) or to detect human pathogenic  Campylobacter  strains (e.g  C. concisus, C. hyointestinalis, C. ureolyticus ). 
         [0063]    The compounds of the present invention can be used to immunize animals, in particular livestock, against  C. fetus venerealis, C. fetus fetus , and other  Campylobacter  species in which the glycan described herein is native to the organism. Immunization can take the form of treating or preventing disease in individual animals or on a herd-wide basis for improved productivity and health of the herd. 
         [0064]    To the extent that  Campylobacter  species in which the glycan of Formula 1 is native to the organism, the compounds described herein can be used in a similar fashion to the above for preparing vaccines to treat or prevent infection by such organisms within humans. As well, a similar diagnostic function can be obtained in humans, using the antibodies or antisera raised against such compounds. 
         [0065]    The present invention has been described by way of various embodiments thereof. It will be understood by persons skilled in the art that the invention is not limited in scope to such embodiments. Rather, the full scope of the invention encompasses and may be appreciated by reference to this patent specification in its entirety, including the claims thereof, and including modifications, variations, and alternative embodiments that would be understood to the skilled person based on said specification.