# Patent

## Title
N-linked glycan compounds

## 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.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
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
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
Campylobacter jejuni and Campylobacter coil 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 & Kist M, Current Opinion in Infectious Diseases, 2003). Other Campylobacter species have been recognized as emerging pathogens in human gastroenteritis (C. upsaliensis, C. hyointestinalis) were associated with inflammatory bowel disease in children and with gingivitis, periodontitis, and human abortions (C. retus, C. concisus) (Zhang L S et al., Journal of Clinical Microbiology, 2009) 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).
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 & Wren B W, Nature Reviews Microbiology 2005; Nothaft H & Szymanski C M, Nature Reviews Microbiology, 2010).
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 0-linked and N-linked protein glycosylation pathways in bacteria (Nothaft H & 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.
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 C. coli. 
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).
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
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) and 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 & Medical Microbiology, 2010). Campylobacter hominis, Campylobacter gracilis, Campylobacter rectus (periodontal disease and human abortion), Campylobacter showae, Campylobacter mucosalis and Campylobacter curvus are believed to be within the second group.
FIG. 2 illustrates 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 and 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 & Szymanski C M, Nature Reviews Microbiology, 2010).
FIG. 3: illustrates N-glycan reactivity towards (A) a C. jejuni N-glycan-specific antiserum, (B) SBA-lectin (recognizing terminal GalNAc residues of structure1) (C) WGA-lectin reactivity (recognizing terminal GlcNAc residues of Formula 1A and 1B with cross-reactivity to GalNAc structures) and (D) mass-spectrometry-based fOS analyses showed that pgl pathway derived glycans differ among Campylobacter species.
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).
In the above Formula 1, QuiNAc4NAc represents an alternative signifier of the saccharide Bac, which constitutes an abbreviation of bacillosamine.
In one aspect the invention relates to an isolated or purified compound comprising the compounds 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.
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 tanienae, Campylobacter ureolyticus, Campylobacter hominis, Campylobacter gracilis, Campylobacter rectus, Campylobacter showae, Campylobacter mucosalis and Campylobacter curvus. 
The antibody or antiserum can be used for diagnostic purposes, to detect the presence of said organisms in an animal or in a human.
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 glycoprotein engineering, 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.
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.
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.
The vaccines, antibodies and antisera described herein may also be used to for prevention, treatment and diagnosis in humans.



DESCRIPTION OF THE DRAWINGS
FIG. 1 shows N-linked protein glycosylation and free oligosaccharides in C. jejuni. 
FIG. 2 is a chart summarizing the fOS and N-glycan structures in various Campylobacter species.
FIGS. 3A-3D depict N-glycans and fOS analyses in select Campylobacter species.
FIG. 4A is the 1H NMR spectrum of purified fOS from C. fetus fetus. 
FIG. 4B overlay of 2D HSQC NMR spectra for C. fetus fetus and C. fetus venerealis. 
FIGS. 5A to 5C depict structures of fOS and N-glycans of C. jejuni, C. coli and C. upsaliensis (Structure 1) and Formula 1A and Formula 1B from the other Campylobacter species described herein.
FIG. 6 shows elution profiles of Formula 1A and Formula 1B, under conditions described herein and the confirmation of purified Formula 1A and Formula 1B.
FIG. 7 illustrates conjugation of purified Formula 1A and Formula 1B compounds to BSA.
FIG. 8 illustrates immunoblots with antiserum raised against each of the BSA-glycoconjugates.
FIGS. 9A-9F depict MS spectra of glycopeptides comprising compounds of Formula 1 as the glycan moiety.
FIG. 10 depicts campylobacter cells that were labeled with Formula 1A and Formula 1B-specific antiserum.
FIG. 11 illustrates immunoblots with antiserum raised Formula 1A, Formula 1B and structure 1.



DETAILED DESCRIPTION
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).
In the above Formulae, QuiNAc4NAc represents an alternative signifier of the saccharide Bac, which constitutes an abbreviation of bacillosamine (also known as diNAcBac). The compound of Formula 1 is optionally connected or linked to a single amino acid, an oligopeptide, a peptide, a protein, or a lipid.
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)).
It has been determined that the above compound is substantially conserved across multiple species of Campylobacter. 
FIGS. 3A-3D depict N-glycans and fOS in select Campylobacter species. (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 (B) soybean agglutinin recognizing terminal GalNAc residues, but shows little reactivity with (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. (D) Examples of mass spectrometry of fractions enriched for fOS or Asn-linked 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.







TABLE 1







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.









MS-MS2 (m/z)









Campylobacter
free oligosaccharide



species
(fOS)
N-linked glycan





C. jejuni
GalNAc5-Glc-Bac
HexNAc5-Hex-Bac-Asn



(1425.0)
(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-Asn (1377.5)



Formula 1A
Formula 1A-Asn(N)-linked



HexNAc4-Glc-Bac
On a Peptide:



(1222.5)
HexNAc5-Bac-Asn



Formula 1B
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.
HexNAc5-Bac (1263.5)
N/D


hyointestinalis
Formula 1A




HexNAc4-Hex-Bac




(1222.0)




Formula 1B









Example 1
Purification of Compounds of Formulae 1A and 1B
Campylobacter jejuni 11168, C. concisus, C. hyointestinalis, C. fetus fetus and C. fetus venerealis were grown under microaerobic 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 1H 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
NMR experiments on the glycans obtained in example 1 were carried out on a Varian INOVA 500 MHz (1H) spectrometer with 3 mm gradient probe at 25° C. with acetone internal reference (2.225 ppm for 1H and 31.45 ppm for 13C) 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.
FIG. 4 A is the 1H 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).
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 of Campylobacter species 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, Campylobacter hyointestinalis, 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. 
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 FIGS. 5A-5C).















TABLE 2





Unit, compound
Atom
1
2
3
4
5
6(6a)
6b























α-QuiNAc4NAc α-A
1H
5.14
4.07
4.00
3.80
3.97
1.14




13C
92.2
54.3
73.3
58.3
68.0
17.6


α-QuiNAc4NAc β-A
1H
4.73
3.78
3.81
3.80
3.54
1.17



13C
95.6
57.2
75.9
58.3
72.3
17.6


α-GalNAc B
1H
5.21
4.23
3.83
4.05
3.89
3.70
3.75



13C
98.0
50.7
68.0
77.5
72.6
60.9


α-GalNAc C
1H
5.00
4.28
4.14
4.08
4.37
3.59
3.64




5.02



13C
99.4
51.4
67.7
77.6
72.3
60.5


α-GlcNAc D
1H
4.90
4.05
3.95
3.76
4.33
3.57
4.01



13C
99.4
54.2
80.0
69.3
71.9
65.8


β-GlcNAc E
1H
4.58
3.70
3.59
3.48
3.48
3.77
3.93



13C
102.5
57.0
74.6
71.1
77.0
61.8


α-GlcNAc F
1H
4.93
3.92
3.78
3.50
3.75
3.79
3.85



13C
98.1
54.9
72.3
71.2
73.1
61.7


α-GalNAc C′
1H
5.00
4.28
4.14
4.10
4.37
3.59
3.64



13C
99.4
51.4
67.7
77.7
72.3
60.5


α-GlcNAc D′
1H
4.97
4.16
3.97
3.75
4.34
3.57
4.01



13C
99.5
54.1
81.7
69.3
71.9
65.8


β-Glc G
1H
4.53
3.29
3.51
3.42
3.48
3.74
3.91



13C
104.3
74.2
76.8
70.6
76.8
61.7









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
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., AnalChem, 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 (Table 1).
Example 4
Expression of Formula 1 Compounds
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) or as a fusion of such a protein with a phage protein (Duerr et al., Glycobiology, 2010). The glycan containing protein/peptide/phage 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
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. (FIG. 6A). Approximately 0.5 nmoles of either a mixture of Formula 1A and Formula 1B (Fig. 6B), or Formula 1B and Formula 1A after separation (FIG. 6C) 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 purified Formula 1A (FIG. 6D) and Formula 1B (FIG. 6E) after purification that correspond to observed masses Formula 1A and Formula 1B as outlined in Table 1.
Example 6
Conjugation of Formula 1A and Formula 1B to BSA
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 15eq), 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 (2.5 L).
FIG. 7 shows conjugation of Formula 1A and Formula 1B to BSA: Glyco-conjugates separated by 12.5% PAGE (FIG. 7A) and monitored by Western blotting using commercially available WGA-lectin conjugated with alkaline phosphatase (FIG. 7B). Lane1, 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 or Formula 1B-BSA Conjugates  
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 (approved Animal Care Committee protocol No, 717). After an initial subcutaneous injection (at 3 sites, 0.5 ml was injected at each site) of 2.0 mg antigen using Freund's complete adjuvant (in a 1:1 ratio with the antigen), a booster dose with 2.0 mg of each Formula 1A-BSA and Formula 1B-BSA conjugates mixed with Freund'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).
FIG. 8 shows an immuno-blot with antiserum that was raised against the single BSA-glycoconjugates: 120 μg of whole cell lysates from 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 (FIG. 8A) 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 (FIG. 8B) 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
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).
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.







TABLE 3







Formula 1A and Formula 1B compounds containing glycopeptides















Area of
Area of






HexNAc4-
HexNAc5-






Hex-Bac
Bac
Ratio (HexNAc5-





glycoform
glycoform
Bac (Formula 1A)/




peptide
(Formula
(Formula
HexNAc4-Hex-



Protein name
sequence
1B)
1A)
Bac (Formula 1B)










Identified_glycopeptides_from_Campylobacter_fetus fetus












Accession







number







(uniprot)







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
VNDTR386






accessory







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












Accession







number







(GenBank)







ACLG01000782.1;
PDZ domain
109NSTEMGH
2200609
1611018
0.732078257


C. fetus
protein
IK118





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









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.
FIGS. 9A-F depict MS spectra showing that both Formula 1A and Formula 1B compounds are N-linked (to the same peptide), as follows:
9A) MS spectrum (precursor ion scan) of tryptic digested, HILIC-LC enriched peptides; (B) Quantification of relative peak areas of the corresponding ions; (C) MS/MS of the carbohydrate portion, (D) MS/MS of the peptide portion of the m/z ion 968.44545; 9E) MS/MS of the carbohydrate portion, and 9F) MS/MS of the peptide portion of the m/z ion 982.12069.
Example 9
Formula 1A and 16 are Presented on the Campylobacter Cell Surface
Cells of C. fetus fetus, C. fetus venerealis, C. concisus, C. hyointestinais, and C. jejuni were grown on MH plates for 18-24 hours under microaerobic 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.
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 10A, SZR-1 (anti-Formula 1B) or 106, SZR-3 (anti-Formula 1A) as the primary antiserum and a fluorescent-tagged secondary antibody.
FIG. 11 shows immunoblots with antiserum that was raised either against Formula 1A or Formula 1B or with an 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 (A) a 1:500 dilution of a serum sample obtained from a rabbit that was immunized with BSA-Formula 1B compound (SZR-1), with (B) a 1:500 dilution serum of a rabbit immunized with BSA-Formula 1A compound (SZR-3) or (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.
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.
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). Said antisera/antibodies can be used to detect compounds in any body fluid or secretion. For example, bull semen could be tested with antibodies recognizing the glycan of Formula 1 to detect Campylobacter fetus venerealis infection that may be present in the animal.
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.
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. Similarly, the compounds can be targeted by other therapeutics such as bacteriophages or their receptor binding proteins.
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.

## Claims

1. An isolated or purified compound comprising A-GlcNAc[GlcNAc]-GalNAc-GalNAc-QuiNAc4NAc, wherein A is GlcNAc or Glc and wherein said compound is free from Campylobacter sp. cell wall components.

2. An isolated or purified compound comprising A-GlcNAc[GlcNAc]-GalNAc-GalNAc-QuiNAc4NAc connected or linked to a single amino acid, an oligopeptide, a peptide, a protein, or a lipid, wherein A is GlcNAc or Glc and wherein said compound is substantially free from Campylobacter sp. cell wall components.

3. The compound of claim 2 wherein said single amino acid is asparagine.

4. An immunogenic composition comprising the compound of claim 1.

5. The immunogenic composition of claim 4, further comprising an adjuvant.

6. A method of treating or reducing an infection caused by a Campylobacter organism, comprising administering the immunogenic composition of claim 4 to an animal or human in need thereof, wherein the compound comprises a glycan that is native to said organism.

7. The method of claim 6 wherein said organism is Campylobacter fetus venerealis, Campylobacter fetus fetus, Campylobacter concisus, Campylobacter hyointestinalis, Campylobacter hyointestinalis subspecies, Campylobacter sputorum, Campylobacter sputorum subspecies, Campylobacter lanienae, Campylobacter ureolyticus, Campylobacter hominis, Campylobacter gracilis, Campylobacter rectus, Campylobacter showae, Campylobacter mucosalis, or Campylobacter curvus.