CA2957430C - Immunogenic composition against campylobacter jejuni - Google Patents

Abstract

The inventive subject matter relates to an immunogenic composition against Campylobacter jejuni comprising isolated capsule polysaccharide from selected pathogenic Campylobacter jejuni strains. The inventive subject matter also relates to methods of using the polysaccharide compositions in inducing and anti-C. jejuni immune response.

Description

IMMUNOGENIC COMPOSITION AGAINST CAMPYLOBACTER JEJUNI
BACKGROUND OF INVENTION
Field of the Invention [0001] The inventive subject matter relates to an immunogenic composition capable of conferring protection against diarrhea caused by Campylobacter jejuni and a method of inducing an immune response against C. jejuni using the immunogenic composition.
Description of Related Art

[0002] Campylobacter jejuni is estimated to cause 2.5 million cases annually in the United States and >400 million cases worldwide. In developing countries C.
jejuni is, like ETEC, primarily a pediatric disease. The symptoms of campylobacter enteritis include diarrhea, abdominal pain, fever and sometimes vomiting. Stools usually contain mucus, fecal leukocytes and blood, although watery diarrhea is also observed.
The disease is zeonetic, and wild and domesticated birds represent a major reservoir. C.
jejuni is a major foodborrie infection, most alien being associated with contaminated poultry, but major outbreaks have been associated with water or raw milk contamination (44). C. jejuni is also associated with Reiter's syndrome and inflammatory bowel syndrome, but the major complication of C. jejuni enteritis is Guillain-Banre Syndrome (GBS), a post-infectious polyneuropathy that can result in paralysis (Allos, B.M., J.
Infect, Dis 176 (Stipp' 2):5125-128 (1997)). The association is due to molecular mimicry between the sialic acid containing-outer core of the iipooligosaccharide (LOS) and human gangliosides (Moran, et al., J. Endotox. Res. 3: 521 (1996)). Thus, antibodies generated against LOS cores result in an autoimrnune response to human neural tissue.

[0003] C. jejuni capsular moieties are important in serodetermination.
However, despite over 47 Penner serotypes of C. jejuni having been identified, most Campylobacter diarrhea' disease is caused by C jejuni expressing only a limited number of serotypes.
Therefore, only selected strains of C. jejuni, predicated on epidemiological studies, provides suitable candidate strains for development of vaccine compositions.
However, despite the importance of this organism to human disease, there are no licensed vaccines against C. jejuni.
[0002] LOS synthesis in Campylobacter is controlled by a number of genes, including genes encoding enzymes involved in biosynthesis of sialic acid for incorporation into LOS. Thus, C jejuni is one of a limited number of bacteria that can endogenously synthesize sialic acid, a 9 carbon sugar that is found in many mammalian cells. This is consistent with the observed molecular mimicry of LOS and human gangliosides important in GBS (Aspinall, et al., Eur. J. Biochem,, 213: 1029 (1993);
Aspinali, et al., Infect. Immun. 62: 2122-2125 (1994); .Aspin.all, et al., Biochem., 33: 241 (1994);
Salloway et al.; Infect. Irninun., 64: 2945 (1996)).
[0003] An interesting recent revelation regarding the Campylobacter genome sequence was the presence of a complete set of capsule transport genes similar to those seen in type 11/1.11 capsule loci in the Enterobactericeae (Parkhill et al., Nature, 403:
665 (2000);
Karlyshev et al., Mol. Mierobiol., 35: 529 (2000)). Subsequent genetic studies in which site.-specific mutations were made in several capsule transport genes indicated that the capsule was the serodeterminant of the Penner serotyping scheme ((arlysliev et al., Mol.
I'vficrobiol., 35: 529 (2000)). The Penner scheme (or HS for heat stable) is one of two major serotyping schemes of campylobacters and was originally thought to he based on lipopolysaccharide 0 side chains (Moran and Penner, J. App!. Microbia, 86: 361 (1999)). Currently it is believed that the structures previously described as 0 side chains are, in fact, capsules.
SUMMARY OF THE INVENTION

[0004] The inventive composition relates to an immunogenic composition comprising polysaccharide antigens comprising isolated capsule polysaccharides from a Campyiobacterjejuni strain, linked to form polysaccharide polymers. The polysaccharides are isolated from lipooligosaccharide structures and other structures associated with Guillain Barre Syndrome or autoimmune disorders. The embodied composition comprises one or more polysaccharide antigens each comprising isolated 3.

polysaccharides from the C. jejuni strains selected from the group consisting of HS1, HS1/HS44, HS44, HS2, HS3, HS4, HS5, HS13, HS4/13/64, and HS50.

[0005] Another embodiment is a method of inducing an immune response by administering an immunogenic composition comprising one or more polysaccharide antigens with each antigen comprising an isolated polysaccharides or polysaccharide polymer derived from a C. jejuni strain where the C. jejuni strains are selected from the group consisting of: HS1, HS1/HS44, HS44, HS, HS3, HS4, HS5, HS13, HS4/13/64, and HS50. The composition is devoid of lipooligosaccharide structures and other structures associated with Guillain Barre Syndrome or other autoimmune disorders.

[0006] Another embodiment is a method of immunizing against C. jejuni strains HS4, HS13, 11S4/13/64 and 11S50 by administering one or more antigens, wherein each antigen comprises an isolated polysaccharide or polysaccharide polymers derived from a C. jejuni strain selected from the group consisting of HS4, HS13, and HS50.

[0007] Another embodiment is a method of immunizing against C. jejuni strains HS I, HS1/H544, HS44 by adminisntering one or more antigens, wherein each antigen comprises an isolated polysaccharide or polysaccharide polymer derived from a C.
jejuni strain selected from the group consisting of C. jejuni strains HS4, HS13, HS4/13/64.

[0007a] Accordingly, in one aspect of the present invention there is provided an immunogenic composition against Campylobacter jejuni comprising one or more polysaccharide antigens and an adjuvant, wherein each of said polysaccharide antigens comprises an isolated Campylobacter jejuni capsule polysaccharide from a Campylobacter jejuni strain linked to form a repeating polysaccharide polymer comprising 2 or more of said capsule polysaccharides, wherein said Campylobacter jejuni strain is selected from the group consisting of: HS4, HS5, HS4/13/64, and HS50, wherein said immunogenic composition does not contain Campylobacter jejuni lipooligosaccharide structures associated with Guillain Barre Syndrome, wherein said capsule polysaccharide or said polysaccharide polymer is conjugated to a protein carrier, wherein the structure of HS4 is ¨43)-L-13-D-ido-Hep-(1-4)-13-D-GleNAc-(1¨>, with non-stoichiometric MeOPN at C-4 of LD-ido-Hep, wherein the structure of HS4/13/64 is [¨>3)-6d-13-D-ido-Hep-(1¨>4)43-D-G1eNAc-(1¨dn, with non-stoichiometric MeOPN at C-2 and/or C-7 of 6d-ido-IIep, and wherein the number of repeats of the capsule polysaccharide "n" is 1 to 100.
[0007b] According to another aspect of the present invention there is provided a use of the immunogenic composition described herein for inducing an immune response in a mammal against a Campylobacter jejuni strain selected from the group consisting of H54, HS5, HS4/13/64, and HS50, wherein the use comprises the immunogenic composition described herein at a dose range of 0.1 tg to 10 mg per dose and a boosting immunogenic composition comprising the immunogenic composition described herein at a dose range of 0.1 jig to 10 mg per dose.
4a [0007c] According to yet another aspect of the present invention there is provided a use of the immunogenic composition described herein for inducing an anti-Campylobacter jejuni immune response in a mammal against a Campylobacter jejuni strain selected from the group consisting of HS4, HS4/13/64, HS5, and HS50, wherein the use comprises 3 doses of the immunogenic composition described herein at a dose range of 0.1 p,g to 10 mg per dose.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. Alignment of variable CPS loci from C. jejuni HS1 and HS44 Penner type strains. Genes are as indicated in the figure and include: methyl phosphoramidate (MeOPN) biosynthesis and transferase; CPS transport and assembly; putative methyl 4b transferase; Heptoseldeoxyheptose biosynthesis; putative glycosyl transferase;
sugar biosynthesis; arid hypothetical.
FIG. 2. Structure of HSI teichoic acid-like capsule.
FIG. 3. 21) sH-3P HMBC NMR spectrum of C. jejuni HS:1/44 teichoic acid CPS.
This NI'vIR spectrum shows the connections between the ivleOPN moieties and positions 3 of the Fru units, and between the diester-phosphate and position 4 of Gal and position I of Gro.
FIG. 4. GC-MS and NMR of C. jefuni HS44 CPS material. (A) GC-MS profile of the alditol acetate derivatives from the two CPSs of C. jejuni ILS44, showing (i) the backbone units of the teichoic acid CPS, glycerol (Gro) and galactose (Gal), and (ii) those emanating from the second heptose-rich CPS, 6-deoxy-3-0-Methyl-a/tro-heptose (6d-3-O-Me-altro-H ep), 6-deoxy-a/tro-heptose (6d-a/tro-Hep) and 6-deoxy-ga/acto-lieptose (6d-gal-Hep). (B) 1H Ni'vIR spectrum of HS44 CPS material showing the 0.-anoincric resonances emanating from 6d-altro-Hepf, 6-deoxy-galacto-Hepf and 6d-3-0-Me-a/fro-Heplof the heptose-rich CPS and from Gal of the teichoic acid CPS.
FIG, 5. Characterization of mutants in the HSI CPS locus. A. Alcian blue stained 12.5% SDS PAGE of crude CPS preparations. Lane I, Precision Plus protein standards;
lane 2, HSI wildtype; lane 3, HSI. 1.08 mutant; lane 4, HSI 1.08 mutant complemented;
lane 5, HSI 1,09 mutant; lane 6, HSI 1.09 mutant complemented, lane 7, HSI
wildtypc.
B. 31P NMR of CPS from HSI .08 complement; C. 31P NMR of CPS from HS1.09 complement; D. 31P .NIVIR of CPS from IHS I wildtype.
FIG. 6. The GC-MS profile of the alditol acetate derivatives of C jejuni CG2995 CPS.
FIG. 7. 'The 1H NMR spectrum of C. jejuni CG2995 CPS.

FIG. 8. (A) The 2D 1-11-13C HSQC NMR spectrum of C. jejuni CO2995 CPS; (B) The ID selective LH NOEs of the C. jejuni CO 2995 CPS, Irradiated peaks are denoted with an "*". Mixing time of 0.250 tis was used.
FIG. 9. The 31P NMR spectrum of C. jejuni CG2995 CPS.
FIG. 10. The 21) IFI-31P HMBC NMR spectrum of C. jejuni CG2995 CPS.
FIG. 11. Structure, of HS5 CPS showing four variations: it) The main PS
structure of C.
jejuni CG2995, ii) variation 1, iii) variation 2, and iv) variation 3.
FIG. 12. The GC-MS profile (top) of the alditol acetate derivatives of C.
jejuni CG2995 CPS, following TEMPO oxidation that shows a reduction in abundance of the 3,6-dideoxy-ribo-heptose.
FIG. 13. TEMPO oxidation that shows a reduction in abundance of the 3,6-dideoxy-ribo-heptose, indicating that its C-7 primary hydroxyl (free of MeOPN) is the site of preferred oxidation in this CPS, and that which will be mostly involved in the conjugation of C.
jejuni CG2995 CPS to carrier protein CRM197.
FIG. 14. Characterization of the HSI conjugate vaccine. A. 3 P NMR of HS1 cPS:

CR.M 197 conjugate vaccine showing the presence of MeOPN in the conjugate CPS.
B.
Gel code blue stained 12% SDS-PAGE gel. Lane 1, CRM197; lane 2, HSI-CRM197 conjugate. The mass of protein standards are shown on the left.
FIG, 15. NMR of HS:13 CPS. (A) ID LH NMR; and (B) ID 3IP NMR spectra of C.
jejuni 3019 CPS (serotype HS:13).
FIG, 16. Linkage determination of MeOPN group by NMR. 21) 11-1-31P HMBC NMR
spectrum of C jejuni BH-01-0142 CPS (A': 1,2,3-linked 6d-ido-flep/LD-ido-flep with C
residue; C: MeOPN).

FIG. 17. NMR analysis showing that non-sugar moiety was 3-hydroxypropanoyl.
(A) 2D iH-13C FIMBC NMR spectrum of C. jejuni BH-01-0142 CPS (B': 1,3,4-linked Gal with residue D, (B) D: 3-hy-droxypropanoyl group.
FIG. 18. Immunogenieity of IS1-CRM 9 7 conjugate in mice. A. ELIS A titers to BSA two weeks after three doses. B. Dot blot of C jejuni cells immunodetected with mouse sera at a final dilution of 1:1000.
FIG. 19, Immune response to HS5-C,RMI97conjugate. Mice were immunized with three (3) doses (10 p,g and 50 &g by weight of conjugate) given at 4 week intervals.
The mice were bled two weeks after the last doses.
FIG, 20, .13110142 (HS3) conjugate vaccine is immunogenic in mice. Data represent the mean ( SEM) reciprocal IgG endpoint titer per treatment group.
FIG, 21. Dot blot demonstrating inummogenicity of an HS1-CRM197 vaccine.
Purified capsules (1 mg/m1) were dot blotted in triplicate (2 ul each) to nitrocellulose and immunodetected with rabbit polyelonal antiserum to an HS1-CRM197 vaccine. HS
1, wildtype 1181 capsule; MS1.08, capsule from a fructose transferase mutant of HS/ that lacks the fructose branch and the MeOPN; HS23/36, capsule from 81-176 which expresses a heterologous capsule (I1S23/36).
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0008] The term "polysaccharide antigen" as used herein refers to a capsule polysaccharide derived from Campylobacterjejuni (C. jejuni or Campylobacter jejuni) capsule. As used, herein, each polysaccharide antigen comprises a polysaccharide or polysaccharide polymer derived from one C'. jejuni strain. The inventive composition can he comprised of multiple polysaccharide antigens. As used herein, "polysaccharide"
refers to two or more monosacchatide units composing a carbohydrate polymer molecule.
A "polysaccharide polymer" refers to two or more polysaccharide molecules connected together, As used herein, "n" in the polysaccharide structure refers to the number of polysaccharide repeats in the polymer and is 1 or more and can be up to 100.

[0009] An embodiment of the current invention comprises polysaccharide antigens comprising a polysaccharide or polysaccharide polymer derived from the capsule of a C.
jejuni strain. The strains from which the capsule polysaccharides are isolated are selected from the group consisting of HSI, HS44, HS2, HS3, HS4, HS5, HS13, I-IS4/13/64, and HS50. A capsule polysaccharide polymer comprises 1 to 100 copies of a polysaccharide structure, derived from an individual C. jejuni strain, connected together to form a polysaccharide polymer. The inventive immunogenic composition one or more polysaccharide antigens with each polysaccharide antigen comprising an isolated C
Muni polysaccharide structure or polysaccharide polymer from a C jejuni strain. The polysaccharides are isolated or purified away from lipooligosaccharide, or other structures associated with GBS or other autoimmune disorders.
[00101 A large number of the C. jejuni strains are identified. An embodiment of the current invention includes only capsule polysaccharides derived from C. jejuni strains, which have been shown to result in disease in humans.

Example 1: HSI/11544 and 11,544 polysaccharide structure [0011] Vaccine strategies against C jejuni have been largely limited due to the molecular mimicry between lipooligosaccharide (LOS ) cores of many strains of C. jejuni and human gangliosides (Moran, et al., J. Endotox. Res., 3: 521 (1996). This mimicry is thought to be a major factor in the strong association of C. jejuni infection with Guillain Bane Syndrome (GBS), a post-infectious polyneuropathy (Allos, J. Infect. Dis,, 176(Suppl.): S125-128 (1997)), Thus, antibodies generated against LOS cores result in an autoimmune response to human neural tissue. It has been estimated that as many as 1/3000 cases of earnpylobaeter enteritis results in GBS. Therefore, the possibility of developing GBS could be associated with any whole cell vaccine against C.
jejuni that includes ganglioside mimicry.
[0012] Recent development of a molecular CPS typing system re-enforced the strong correlation between CPS and Penner types (Poly, et al., I. Clin. Microbial.
49: 1750 (2011)), Both Penner serotyping and molecular CPS typing have revealed the predominance of a handful of CPS types worldwide. Among CPS types, the HS 1 complex is one of the most common, accounting for 8.2% of C jejuni induced diarrhea worldwide ((Poly, et al., J. Clin. Microbiol. 49: 1750 (2011); Pike, et al., plOs One 8:
e67375 (2013)). This complex is composed of HSI and HS44 types, and strains can serotype as HS1, H544 or HSI/44. So far, only the CPS structure of the HS1 type strain has been described, which is composed of repeating units composed of 4-substituted a-D-galactose (Gal) and glycerol (Gm) linked by phosphate (P) in a teichoic acid-like structure [-4)-a-D-Ga1p-(1-2)-Gro-(1-P-1õ (Aspinall, et al, 'Eur. Biochem.
216: 880 (1993)). The HS I CPS backbone may be decorated by fi-D-fructofuranoses (Fm) branches, at C-2 and C-3 of the Gal unit, which in turn may be decorated at C-3 with Me0PN (Fig. 1; (McNally, et al., FEES J. 272: 4407 (2005)). Both the fructofuranose branches and MeOPN are found in non-stoichiometric amounts, presumably due to phase variation at homopolymeric tracts of bases in the genes encoding their respective transferases (McNally, et al., FEES J. 272: 4407 (2005)). The ¨15 kb HSI CPS
locus encoding eleven genes for the syrithesis of this polysaccharide (BX545859) is the smallest CPS locus identified to date in C jejuni (Karlyshev, et al., Appl.
Eriviron.
Microbiol. 71: 4004 (2005))(Fig. I).
[0013] The HS I type strain used was MSC57360 and the HS44 strain (ATCC 43463) was obtained from the American Type Culture Collection (ATC,C)(Manassas, VA).
C
jejune strain CG98-U-77 was isolated from a diarrhea case from Thailand and was obtained from the Armed Forces Research Institute of Medical Sciences (AFRIMS). C.
jejuni strains were routinely cultured at 37 C under microaerobic conditions (5% 02, 10%
C07, and 85% N2) on Mueller Hinton (MH) agar plates, supplemented with the appropriate antibiotic, if required. E. coil strains were grown in LB.media supplemented with the appropriate antibiotics.
[0014] C. jejuni genomic DNA was extracted from 16 hour cultures. Sequencing of the CPS loci was performed as previously described (Karlyshev, et al., Mol.
Microbiol. 55:
90 (2005); Poly, et al. J. Clin. IVIicrobiol. 49: 1750 (2011); , Karlyshev, et al., Gene 522:
37 (2013)).
[0015] The CPS was extracted from cells by hot water¨phenol extraction for 2 hours at 70 C. The aqueous layer was dialyzed (1000 Da) against water followed by ultracentrifugation to separate the CPS from the LOS. The supernatant material containing the CPS was subjected to size-exclusion chromatography (Sephadex G50) for further purification to yield the intact CPSs.
[0016] Determination of monosaccharide composition was performed using a procedure amenable to the alditol acetate method (Chen, et al., Carbohydr. Res. 343:
1034 (2008)) with the alditol acetates being analyzed in a ThenrnoFinnigan POLARISTm-Q
(Thermo Fisher Scientific, Inc, Waltham, MA) gas chromatograph/mass spectrometer (GC/MS) using a DB-17 capillary column. The sugar linkage types were characterized by characterization of the permethylated alditol acetates by GC/MS as previously described (Chen, et al., Carbohydr. Res. 343: 1034 (2008)). The NMR experiments were performed on a Bruker 400 MHz spectrometer (Bruker Corporation, Billeria, MA) equipped with a :Broker cryo platform at 295 K with deuterated trimethylsily1 propanoic acid and orthophosphoric acid as external standards.
[0017] The variable region containing the genes for synthesis of the polysaccharide are located between the conserved genes encoding the ABC transporter involved in capsule synthesis and assembly (FIG. 1), which also shows the variable region of the HSI CPS
locus (McNally, et al., FEBS 1, 272: 4407 (2005)). The DNA sequence of the capsule locus of the H844 type stain contained homologs of 10 of the 11 genes found in HS1, missing only HS1,08, a gene of unknown function (FIG. 1). The gene content of capsule biosynthesis locus is summarized in Table 1. All shared hornologs were >96%
identical, except for the putative MeOPN transferase (H544.07) which showed only 47%
identity to that of HS]
11.

Table 1 Size Locus Identity with (amino Tag Putative function Relationship HS1b acid) HS44.01 MeOPN biosynthesis HS1.01 164/170(96%) 170 HS44.02 MeOPN biosynthesis 1-131.02 252/253(99%) 253 1-1344.03 MeOPN biosynthesis HS1.03 197/200 (98%) 200 H344.04 MeOPN biosynthesis HS 1.04 775/779 (99%) 779 HS44.05 Methyl transferase HS1.05 (100%) 253 HS44.06 Methyl transferase HS1.06 255/257(99%) 257 H344.07 MeOPN transferase H31.07 308/642(47%) 609 sugar-phosphate HS44.08 nucleotidyltransferase 224 sedoheptulose 7-phosphate HS44.09 isomerase 201 D-glycero-D-manno-heptose 7-H344.10 phosphate kinase 360 GDP-mannose 4.6-HS44.11 dehydratase 343 HS44.12 GDP-fucose synthetasE. (fcl) 381 HS44.13 GDP-fucose synthetase (fcl) 385 H344.14 C/1429 like 310 Nucleotide-sugar HS44.15 epimeraseidehydratase 181 Nucleotidyl-sugar pyranose 1-1S44.16 mutase 416 H344.17 Heptosyl transferase 1202 CDP glycerol 1067/1095 HS44.18 giycerophosphotransferase HS1.09 (97%) 1100 H844.19 Unknown 1-131,10 390/396(98%) 397 Glycerol-3-phosphate HS44.20 cytidylyltransferase HS1.11 128/129(99%) 129 'Function attributed based on Blastp performed on non-redundant protein sequences database.
bNumbers in parenthesis are the percentage of identity between the FIS1 and H344 proteins.
[0018] The HS44 locus included an insertion of 10 additional genes between HS1.07 and HS1.09 encompassing 9,258 bp (Table 1, FIG. 1). These include 4 genes encoding 12.

enzymes predicted to be involved in deoxyheptose biosynthesis (HS44.08 to HS44.11) and three genes (HS44.12, HS44.13 and HS44.15) encoding proteins that are homologous to epimerase red uctases that have been recently demonstrated to be involved in 6-deoxy-cdtro-heptose biosynthesis. The CPS locus of H844 also includes a gene (HS44.14) similar to 01429c coding for a protein of unknown function in NCTC 11168 (HS2), a nucleotidyl-sugar pyranose mutase (HS44.16) and a putative heptosyltansferase (1-1S44.17, Table 1 and FIG. 1). In contrast, the DNA sequence of the variable CPS locus of a clinical isolate that typed as HS1/44 was identical with that of the type strain of HS I.
The minimum protein homology predicted from the 11 genes in these two capsule loci was >99%.
[0019] Fine structural analysis revealed that the polysaccharide structure of HS1144 is similar to that of the previously described teichoic acid capsule polysaccharide (CPS) of HS1 strain (Aspinall. McDonald et al. 1993, McNally, Jarrell et al. 2005): -44)-[MeOPN-43)-13-D-Fru-(1--A-ct-Gal-(1-2)-Gro-(1¨P-4 (FIG. 2).
[0020] FIG. 3 shows the pb.osphorous-proton connections detected in HS1/44 CPS
that emanate from the linkages of the teiehoie-acid diester-phosphate Op 0.5 and 1.5) to position 1 of Gm and position 4 of Gal, and from the attachment of the MeOPN
(op 14.3) to position 3 (OH 4.83) of Fru residues. The H-4 resonance of the 4-linked Gal carrying the Fru branches appeared at 0 4.68, whereas that of the defructosylated 4-linked Gal resonated at 5 4.49 (FIG. 3). A similar pattern was observed for the H-1 resonances of Gro. Simultaneous analysis of the HSI type strain and HSI/44 CPSs, suggested that the HSI/44 CPS contained a lower degree of fmetosylation, as judged by the lower intensities of the 23,4-trisubstituted Gal linkage (GC-MS) and MeOPN resonance (31P
NMR).
[0021] Analysis of HS44 CPS material identified two distinct polysaccharide capsule structures, One CPS was analogous to the aforementioned teichoic-acid CPS of HSI and HS1144 (FIG. 2), but in which no MeOPN-containing Fru branches were observed.
The second CPS was rich in heptoses, being composed of repeating blocks of 6-deoxy-galacto-heptose (6d-gal-Hep), 6-deoxy-a/tro-heptose (6d-a/tro-Hep) and, in lesser amounts, 6-deoxy-3-0-methyl-a/tro-beptose (6d-a/tra-3-0-Me-Hept). The heptose configurations were characterized by comparison with well defined synthetic standards by GC. The linkage-type analysis (GC-MS) (FIG. 4A) revealed that the deoxy-heptoses were present in part as terminal and 2-substituted units in the furanose form.
[00221 Accompanying MAR studies (FIG. 4B) confirmed the presence of deoxy-heptoses (6 H 1.5 ¨ 2,0) and revealed that these units were present in the a anomeric configuration p 5.15 ¨ 5.42), A new MeOPN moiety Op 14,0), different from that expressed by and FIS1144 was associated with HS44 CPS material. This is consistent with the divergence of the putative MeOPN transferase observed in this strain [0023] The product of the HS1.08 gene encodes a predicted protein of 849 amino acids, annotated as a putative sugar transferase (Karlysheev, et al., Mol. Microbiol.
55: 90 (2005)). Because the HS44 teichoic acid-like CPS lacked the non-stoichiomettie fructose branch and the HS1.08 gene was missing from the capsule locus, we hypothesized that HS1.08 encoded a fructose transferase, A mutant in this gene expressed a lower MW
capsule as on an Alcian blue stained gel and the MW was restored to that of wild type in the complement as shown by gel; NMR analysis also confirmed complementation, but the lower intensity of the MeOPN resonance in the 31P NMR (FIG. 5B) suggested that complementation in this ease was partial. Thus, 1-IS1.08 appears to encode a transferase that can transfer Fru to Gal.
[0024] Gene HSI .09 was annotated as a putative CDP glycerol transferase (Karlyshev et al., 2005). Mutation of this gene in I-IS I resulted in the loss of CPS as detected by Alcian blue staining of an SDS-PAGE gel (Fig. 5A). Gel analysis of the complement of the mutant showed a faint CPS band (Fig. 5A), but restoration of CPS expression was confirmed by the 31F NMR spectrum which indicated the presence of MeOPN (Fig, 5B).
[0025] In one embodiment an immunogenic composition, useful for inclusion in a vaccine composition against HSI , HS 1/H S44 and H544 C jejuni strains, comprises polysaccharide antigen, comprising the structure:
[¨>4)-a-D-Galp-(1---42)-Gro-0 "A 2 I' Fri.V:(34¨[MeOPN], or a polymer comprising a repeating of the polysaccharide structure, wherein "n" is 1 to 100. The polysaccharide structure of H544 comprises the above structure without "[MeOPN]---43)-Fruf " unit connected at the 2 or 3 position of Gal. Therefore, in another embodiment, an immunogenic composition would comprise a polysaccharide antigen with a repeating polysaccharide structure, derived from 11544 that comprises the structure:
wherein "n" is 1 to 100.

C'ampylobacterjejuni strain PG 3588 (HS:1):
[0026] Upon treatment of Campylobacterjejuni strain PG 3588 (HS:1) capsule polysaccharide (CPS) with mild acetic acid (5%), the fructose (Frill) side branches and.
their accompanying MeOPN units were removed. The 1H NMR. of the defruetesylated CPS showed the anomeric.: resonance at 8 5,21that corresponds to the a-D-Gal residue (without the Fnif substitutions). H5 5 4.18 was assigned from the H6 6 3.75 proton resonance, Gm resonances were found to be at H111'6 4.05/4.12, H2 8 3.98, and H3/3' 3.78/3.82.
[0027] All carbon resonances of Campylobacter jejuni strain PG 3588 (HS:1) capsule polysaccharide were assigned using a 2D H-13C .FISQC are summarized in Table 2. A
2D t.H-3P 1-1MBC (Figure 4) showed a strong cross peak at OH 4.54/ 41,14), and (6u 4,05, 4,11/ Op1.14) which confirmed the presence of the phosphodiester and its attachment to the Gro and to the C4 of Gal through a phosphodiester. Another resonance was detected in the 2D 111-31P IIMBC at 5 14.04 for a MeOPN moiety, and it showed a cross peak at 8H 3.75/8r 14.04, identifying the attachement of MeOPN at the C-6 of Gat.
Table 2: 1H, 13C chemical shifts of Cjejuni CPS PG 3588 Sugar H 1 ' H2 I H3/3' H4 1. H5 H616' residue Cl C2 C3 C4 1 C5 C6 a-D-Gal 5.21 3.88 3.90 4.54 4.18 3.75 100,84 71.05 71.10 77.31 73,45 63.42 Gm o 4.05/4%12 3.98 3.78/3.82 67.23 79.81 __ 63.95 trift,no a-Gal Ho OR
P _______________________________________________ 0 p-Frqf OH
OH OH
HO.

OR
OH
R: MeOPN, or H OH p-Fruf Example 2: LISS derived polysaccharide structure [0028] One embodiment is an immunogenic. composition against C jejuni that contains an isolated capsule polysaccharide structure or polymers of the structure derived from HS5. The polysaccharide structure comprises four variants, with the structures as follows:

[MeOPN] [MeOPNr-pc 7 K
a-Dideoxy-Hep a-Dideoxy-Hp 7)a-DD-Hep(I 3)Glucitol(6-4 P ---,7)a-DD-Hep(1--4-2)G1ucito1(6 a-Dideoxy-Hep ex-Dideoxy-Hep et-Dideoxy-Hep fMeOPNr. [MeOPNr- [MeOPN)-"
7)a-DD-Hep(I 2)G1ucitol(6---* P 7)a-DD-Hep(I 3)Ghwitol(6--t=P

a-Dideoxy-Hp a-Dideoxy-flep a-Dideoxy-Hep I N 7 s 7 L
iMeOPNr- [MeOPN]--- [Me0Fr7)---[0029] Results from monosaccharide composition analysis revealed that the capsule polysaccharide (CPS) of strain CG2995 (FIS:5) contained 3,6-dideoxy-ribo-heptose, glucitol, and D-glycero-D-manno-heptose (FIG. 6). Multiple linkages of each residue were observed; terminal 3,6-dideoxy-ribo-heptose, 2,6-disubstituted Glucitol, 2,3,6-trisubstituted Giucitol, 2-monosubstituted n-glycero-D-manno-heptose, 2,6-disubstituted o-glycero-o-manno-heptose, 2,7-disubstituted D-glycero-D-manno-heptose, and 2,6,7-trisubstituted D-glycero-D-mann o-heptose.
[0030] The ID 1H NMR of the CPS revealed six anomeric peaks, three of which are associated with D-glycero-D-manno-heptose residues at 5.20 ppm, 5.18 ppm, and 5,16 ppm (A,B,C respectively), and 3 of which are associated with 3,6-dideoxy-ribo-heptose residues at 5.21 ppm, 4.96 ppm, and 4.87 ppm (K,L,N respectively) (FIG. 7).
Linkages and ring resonances were then confirmed via 2D 1H-1H COSY, TOCSY, and NOESY

experiments. Linkages found through i\TMR experiments coincided with the linkages assigned by GC-MS.
[0031] With the aid of 2D 111-13C FISQC and HMBC the Glucitol residues (X,Y,Z) could are assigned, along with the ring region resonances from the six heptose residues. As expected carbons involved in the glycosidic linkages, C2 (6 78.1) of the D-glycero-D-manno-heptose A, B and C, C6 (3 76.8) of D-glyeero-D-rnanno-heptose A, C2 (6 81.6) of Glucitol. Y and Z, C2 (6 32.5) of Ghicitol X, and C3 (6 78.8) of Glucitol Y
and Z, were found to be down-field resonances (FIG. 8 (A)). The deoxy resonances associated with the 3,6-dideoxy-ribo-heptose were easily observed at 8 37.1 (C3) and 6 36.1 (C6).
Selective ID nOe experiments (FIG. 8(8)) also showed the presence of the linkages aforementioned.
[0032] The ID 31P and 2D 1H-31P HMBC 'NMR revealed resonances at 0.96 and 1.30 ppm, indicating that the capsular polysaccharide repeats were linked with a phosphate bridge (FIG. 9). This bridge links through the 6-position of the Glucitol and the 7-position of the D-glyeero-D-manno-heptose (Fig. 9). The 1D 31P spectra also gave rise to a peak. 6 14.5 indicating MeOPN, and through. the 2D III-31P HMBC the I'vleOPN
could be linked to being a 7-subsituted 3,6-dideoxy-riboaheptose (FIG. 10).
[0033] One main capsular polysaccharide was observed with a backbone of [-7)-a-D-glycero-D-rnanno-heptose-(1-3)-Glucitol-(6-)-P-1 with 2,6-disubstitution of the D-glycero-D-manno-heptose, and 2-monosubstitution of the Glucitol with a-3,6-dideoxy-ribo-heptose (FIG. Ili). Three other variations of the capsular polysaccharide repeat were also noted; variation I with 2-monosubstitiated D-glycero-D-rnanno-b.eptose and Glucitol linked through the 2-position instead of 3 to D-glycero-D-manno-heptose (FIG.
I iii), variation 2 with 2,6-disubstitution of the D-glycero-o-manno-heptose and Glucitol linked through the 2-position instead of 3 to n-glycero-o-rnanno-heptose (FIG. and variation 3 with 2-monosubstituted n-glycero-n-manno-heptose and 2-monosubstituted Glucitol (FIG. Hill).
Example 3: conjugation of CPS polysaccharide to protein carrier [0034] One or more polysaccharides or polysaccharide polymers can he conjugated to a carrier molecule to improve itranunity. The carrier, in one embodiment, is a protein carrier molecule. As an example protein carrier, CRM197 can be conjugated to the polysaccharide or polysaccharide polymer. The GC-MS profile of the alditol acetate derivatives of C. jejuni CO2995 CPS, following TEMPO oxidation is shown in FIG. 12.
Conjugation is illustrated in FIG. 13.
Conjugation of HS5 polysaccharide [0035] Isolated C. jejuni HS5 polysaccacharide was conjugated to a protein structure and is described here as an illustration of conjugation of the polysaccharide or polysaccharide polymers. The overall scheme for conjugation is illustrated in FIG, 13. Any protein carrier is envisioned to be conjugated. Furthermore, conjugation to a protein carrier can be by any number of means.
[0036] As an illustrative example, in FIG. 13 the polysaccharide was conjugated to CR1197 by TEMPO-mediated oxidation. In this method, as shown in FIG. 13, the first step is oxidation of approximately 10% of the plrimary hydroxyls of the intat CPs to carboxylic acids via TEMPO-mediated oxidation. The scheme in FIG. 13 illustrates conjugation using the primary hydroxyl of the DD-Hep as one of the sites of oxidation.
Non-stoichiometric oxidation may also occur at C-6 of Glc and at the CH2-0H of the side-chain substituent. Following activation of the CPS, conjugation to the carrier protein (e.g., CRM197) is accomplished, in the TEMPO-mediated method shown in FIG. 13, through carbodiimide coupling. Visualization of conjuation is by any means, such as gel electrophoresis.
Conjugation of 'HSI polysaccharide [0037] A giycoconjugate composed of HSI teichoic acid CPS and the protein carrier CRM m was created through a conjugation scheme, similar to that used for HS5, based on stoichiometric oxidation of 10% of the available primary hydroxyls in the CPS. After oxidation of primary hydroxyls, the activated HS1 CPS was then conjugated to CRM.197 by carbodiirnide-type coupling of the newly created carboxylic acid functionalities in the CPS and exposed CRM197 lysine units. Importantly, analysis of the HS1 CPS-conjugate vaccine by NMR confirmed that the MeOPN and phosphate moieties remained intact during the conjugation manipulations. These results are shown in FIG.
14. A
comparison of the intensities of the anomeric resonances in the partially oxidized HSI
CPS indicated that half of the backbone Gal residues were branched by the Fru-cntaining MeOPN units, Example 4: Polysaccharides in IIS complexes [0038] Polysaccharide structures were identified in C. jejuni Penner serotype complexes. For example, anti-H.S4 serum results in cross-reaction to other strains strains of the HS4 complex, including HSU, HS4/13164 and HS50 capsules. Isolation and analysis of the polysaccharides from these strains resulted in identification of dissacharides containing a common ido-heptose unit. The strains and isolated polysaccharide derived from the strains are listed in Table 3.
Table 3: HS4 complex capsule polysaccharide structures Serotype Structure /Strain IIS4 type --)3)-L-p-D-ido-Hep-(1¨.4)+D-GIcNAc-(1--4 strain (non-stoichiometric MeOPN at C-4 of LD-ido-Hep) HS13 (50%) (non-stoichiometric MeOPN at C-2 and/or C-7 of 6d-ido-Hep); and/or ______________________________________________________ (50%
HS4,13,64 -43)-6d-15-0-ido-Hep-(1-44)-p-n-GIcNAc-(1--k (80%) (e.g., strain (non-stoichiometric MeOPN at C-2 and/or C-7 of 6d-ido-Hep);
and/or C68486) -43)-L-p-nsido-Rep-(1--.4)-p-D-GIcNAc-(1--* (20%) ........ (non-stoichiometric MeOPN at C-2 of LD-ido-Hep) HS50 (85%) (non-stoichiometric MeOPN at C-4 of LD-ido-Hep); and/or -43)-64-0-0-ido-Hep-(1--+4)-11-0-Gic-(1-+ (15%) (non-stoichiometric MeOPN at C-7 of 6d-ido-Hsa) 1.00391 Illustrated in Table 3, the common, surprising feature of these isolated capsule polysaccharides is the ido-heptose unit. As such, an embodiment is an immunogenic composition comprising one or more polysaccharide antigens, each comprising polysaccharide structures derived from these strains of C. jejuni.
[0040] The previously described CPS structure of C. fejuni strain CG8486 (HS:
4:13:64) consisted mainly of a disaccharide repeating unit [-0)-6d-i3-D-ido-Flep-(l¨*4)-GIcNAc-(1-4], with non-stoichiometric 0-methyl phosphoramidate substituent attached to 0-2 and C-7 positions of ido-heptose. A minor component of L-glycero-D-ido-heptose (LD-ido-Hep) was detected by GL0-MS, using alditol acetate derivatives for compositional analysis and permethylated alditol acetate derivates for lingag,c analysis of, and was newly found in this strain. The sugar ring configuration of 6-deoxy-heptose and L-g/ycero-D-heptose were assigned as idose. The traces of 1,7-anhydro-L-glycero-D-ido-heptese (1,7-anhydro-LD-ido-l-lep) and 1,6-anhydro-L-g/ycero-D-ido-heptose (1,6-anhydro-w-ido-Hep) originated from LD-ido-Hep during acid hydrolysis.
[0041] In addition to previously reported linkage types in C. jejuni 0G8486 CPS (3-substistuted 6d-ido-heptose [--->3)-6d-ido-I-lep-(1-4] and 2,3-di-substistuted 6d-ido-heptose 3,7-di-substistuted 6d-ido-heptose [--)6,7)-6d-ido-Hep-(1-4], 4-substituted N-acetyl-glucosamine [-44)-GleNAc-(1--->]), the GLC
profile of GLC-MS of pennethylated alditol acetate derivatives of C jejuni HS:4:13:64 CPS

showed two additional linkage types from LD-ido-Hep which were not detected in previously reported structure, including 3-substistuted L-glycero-D-ido-heptose [-43)-LD-ido-Hep-(1--->] and 2,3-di-substistuted L-giycero-D-ido-heptose H2,3)-LD-ido-Hcp-(1-->].
[0042] The ID NMR of C. jejuni CG8486 CPS showed two resonances of two 0-[0043] glycosides at 8 4,94 and 8 4.66 which were 6d-ido-Hcp/LD-ido-Hep and GicNAc, respectively. The presence of two anomeric proton resonances for three rnonosaccharide residues (64-icio-Fiep, LD-ido-Hep, and GicNAc) suggested that both 6d-Wo-liep and LD-ido-Hep may contain the same chemical shifts through the sugar ring system except the 1-1-6 position since the only difference between them was at the C-6 position with or without a hydroxyl group. The 'H NMR spectrum also revealed one methyl singlet at 5 2.07 which was characteristic of the N-acetyl moiety from GicNAe and methylene signals at 5 1.77 and 6 2,03 which were 6-deoxy-moiety from 6d-ido-Hep. In addition, NMR detected a characteristic MeOPN signal at Op 14.7. It was determined that the CPS of C. jejuni serotype HS4:13:64 (see Table 3) contained both 6-d-ido-Hep and LD-ido-Hep within its CPS:
[-->3)-6d-13-ido-I-Iep-(1---).4)-13-GicNAe-(1--->] (with non-stoichiometric MeOPN at C-2 and/or C-7 of 6d-ido-Hep) as a major repeat; and [-43)-LD-13-ido-Hep-(1---->4)-P-G1c.NAe-( I -41 (with non-stoichiometric MeOPN
at C-2 of 1.3)-ido-Hep) as a minor repeat.
CPS determination of C. jejuni HS:4 Type Strain (Strain MK7) [00441 The CPS isolated from C jejuni strain MK7 (HS4) was composed of L-glycero-D-ido-heptose (LD-ido-Hep) and N-acetyl-glucosamine (Glt-,NAc) by GC-MS profile determination of alditol acetate derivatives. The above CPS composition of C.
jejuni HS:4 type strain was similar to previously reported CPS of serotype HS:4 complex (HS:4,1.3,64; strain CG8486), which contains mostly 6-deoxy-ido-heptose (6d-ido-Hep) instead of LD-ido-Hep. GC-MS of pennethylated alditol acetate derivatives showed the following linkage types of each monosaceharide: 3-substituted L-glycero-D-ido-heptose [-43)-LD-ido-Hep-(1-4] and 4-substituted N-acetyl gincosamine [0045] The H NMR spectrum of the C. jejuni strain MK7 (type strain HS:4) CPS

showed two P-anorneric proton resonances at 8 4.70 and 6 4,94 for GleNAc and LD-ido-Hep, respectively, The 111 NMR spectrum also revealed one methyl singlet at 6 2.07 which was characteristic of the N-acetyl moiety from GIcNAc and a broad range of overlapping sugar ring proton resonances between 63.50 and 5 4.55. In addition, 11) 3 NMR detected a weak trace of MeOPN signals at 6p 14.3. The substituted sites of MeOPN could not be detected due to the small amount of MeOPN substitution in this HS:4 type strain.
CPS determination of Cejejuni C. jejuni Serotyrie HS:13 (Strain MK16) [0046] C. jejuni 1-1S4 type-strain (MK7) contains a CPS composed of the following disaccharide repeat: [--->3)-L-P-D-ido-Hep-(1-44)-13-GICNAc-(1--->]. The monosaccharide composition analysis, using GC-MS of alditol acetate derivatives of C. jejuni strain MK16 (serotype HS:13) revealed the presence of glucose (Gle), 6-deoxy-ido-heptose (6d-ido4Hep), and L-glyeero-D-ido-heptose (LD-ido-Hep) by GS-MS detenninationof alditol acetate derivative profiles. Linkage analysis of profiles of permethylated alditol acetate derivatives showed that these units were present as 4-substituted glucose [-44)-G1c-(1¨*], 3-substituted 6-deoxy-ido-heptose [¨)3)-6d-ido-Hep-(1--->], 2,3-di-substituted 6-deoxy-ido-heptose [-42,3)-6d-ido-Hep-(1--)1, substituted L-glycero-D-ido-heptose +3)-i,D-ido-Hep-(1-4], and 3,7-di-substituted 6-deoxy-ido-heptose [--3,7)-6d-ido-Hep-(1-eq In addition, a small amount of terminal glucose [Glc-(1--->] was detected as the non-reducing end of the CPS. C.
jejuni serotype HS:13 contains 4-substituted Gic as backbone instead of 4-substituted GIcNAc (seen in serotypes HS: 4:13:64 and HS:4), [0047] The 'H NMR spectrum of C jejuni serotype HS:13 CPS showed two p-anomeric proton resonances at 8 4.63 and 5 4.92 which assigned as Glc and 6d-ido-Hepl LD-ido-Hep, respectively (Fig. 15A). The 'H NMR spectrum also revealed the methylene sigials (multiplet) at 5 1.86 and 6 2,00 which were characteristic of the 6-deoxy moiety from 6d-ido-Hep and a broad range of overlapping sugar ring proton resonances between 6 3.30 and 6 4.55. ID 31P NMR detected two resonances at 6p 14.1 and 5p 14.4 which were typical of MeOPN signals (Fig. 15B), [0048] It was determined that C jejuni strain MK16 (serotype HS:13) CPS
consists of the following disaccharide repeats in quasi equal concentrations (with MeOPN
non-stoichionietrically attached to C-2 and C-7 of 6d-P-ido-Hcp):
[-- 3)-6d-13-D-ido-Hep-(1--.1.4)-13-G1c-(1--ild; and C_tiefuni F,erot:Te HS3/13/50 [0049] HS: 3:13:50 complex has been identified predicated on a quantitatively low level immune-crossrcactivity. C. jejuni strain BH-01-0142 (serotype HS: 3:13:50) was composed of galactose (Gal), 6-deoxy-ido-beptose (6d-ido-Hep), and L-glycero-D-ido-heptose (LD-ido-Hep) using GS-MS using alditol acetate derivative profile determination for compositional analysis of C jejuni BH-01-0142 CPS (serotype HS:3:13:50)).
[0050] The sugar linkage types: 4-substituted galactose [-44)-Gal-(1-4], 3-substituted 6-deoxy-heptose [--->3)-6d-Hep-(1--->j and 3-substituted L-glycero-D-ido-hcptose ido-Hep-(1--->] were found to make up the CPS of serotype HS:3:13:50, using GC-MS

profile analysis of pemiethylated alditol acetate derivatives of C jejuni BH-(serotype (HS: 3:13:50)), In addition, minor components, 3,4-di-substituted galactose [-43,4)-Gal-(1-4], 2,3-di-substituted 6-deoxy-heptose [-*2,3)-6d-Hep-(1--->], and 2,3-di-substituted L-g/yeero-D-ido-heptose [--->2,3)4D-ido-Hep-(1-->] were also characterized.
The above results suggested that the backbone units of C. jejuni serotype HS:
3,13,50 CPS were [--44)-Gal-(1-4], [-,)3)-6d-Hep-(1-+], and [-+3)-LD-ido-Hep-(1--->], with three other non-sugar components were non-stoichionietrically attached to the C-3 of Gal, and C-2 of 6d-ido-Hep and LD-ido-Hep. Also, a terminal Gal [Gal-(1-->] was also determined and was suggested as a non-reducing end.
[0051] The 1H NMR spectrum of the C. jejuni serotype HS:3:13:50 CPS showed broad overlapping peaks between 8 5,00 ppm and 5 530 ppm representing the anomeric proton signals. These overlapping peaks suggested the presence of a-anonieric sugars. In addition, the 1H NMR spectrum also revealed a methylene signal at 6 1.80 and 8 2.02 which are characteristic of 6-deoxy moiety from 6d-ido-Hep. Another proton resonance at 6 2.72 was later confirmed as a methylene signal which also revealed in the spectrum.
[0052] In order to obtain the information for the non-sugar component, 31P NMR
of the C.jejuni BH-01-0142 CPS was performed to determine any phosphorus substituents. The phosphorus resonances at 6p 15.3 revealed the presence of an 0-methyl phosphoramidate groups (MeOPN) or CI-130P(0)NH2(OR), which was involved in the structural moiety in the serotype HS:3,13,50 of C. jejuni CPS, The appearance of one MeOPN signal suggested this unique component was partially attached to one of the monosaccharide residues in the CPS of C. jejuni strain BH-01-0142, since the results of sugar linkage type analysis revealed the presence of minor component of 1,3,4-linked Gal, 1,2,3-linked 6d-filo-Hen and 1,2,3-linked LD-ido-Hep.
[0053] 21)111-31P ITIMBC NMR of C.jejuni BH-01-0142 CPS was carried out to elucidate the linkage site of the MeOPN group (FIG, 16). The cross-peak at 8p 15.3/51.1 3.78 represented the correlation between the phosphorus and the methyl group of the MeOPN. A strong proton-phosphorus correlation between op 15.3 and 0e 4,56 suggested the linkage site of the MeOPN group, with also a weak proton-phosphorus correlation between Op 15.3 and the anomeric proton at 011 5.10, Thus, the combination of the results from monosaccharide linkage type analysis and 21) 1H-3 IP HMIBC NMR showed that the 0-methyl-phosphorainidate group (residue C) was attached to the C-2 position of 6d-ido-liep and 1.,D-ido-Hep (residue As).
[0054] A 2D I H-13C HMBC NMR experiment (FIG. 17) showed that a second non-sugar moiety was that of 3-hydroxypropanoyl. The cross-peaks at SH 3.89/5c 173.0 and 8112.72/0c 173.0 showed three-bond and two-bond connectivities of the carbonyl ester C-I with H-3 and H-2 of 3-hydroxypropanoyl group (residue D), respectively. The hydroxypropanoy1 group was observed to be connected to the C-3 of Gal, by interpreting the cross-peak at OH 5.20/8c 173.0, and also by taking into account the results from linkage type analysis that showed a minor peak of 1,3,4-linked Gal.
[0055] We determined that C jejuni serotype HS:3 has a CPS with the disaccharide repeat (with non-stoichiometric substitutions of 0-methyl phosphoramidate at C-2 of 6d-a-ido-Heplt,-a-D-ido-Hep and 3-hydroxypropanoyl ester at C-3 of a-Gal):
[--->3)L-a-D-ido-Hep-(I-*4)-a-Gal-(1-41; and [---->3)- 6d-a-ido-flep-(1 Example 5: Immunogenic composition [0056] An immunogenic composition against C jejuni is can comprise one or more isolated C. jejuni polysaccharides or polysaccharide polymers. The composition contains the polysaccharides or polysaccharide polymers free of LOS, which is associated with Guillain-BarTe Syndrome. An embodiment is a composition comprising one or more isolated C. jejuni derived polysaccharides or polysaccharide polymers, with the polysaccharide polymer comprising 1 to 100 polysaccharides linked together (i.e., "n"
greater than or equal to 1). The structures of the isolated C jejuni polysaccharide are derived from one or more of the strains HS5, HS1,1152, HS.3, HS4, HS4/13/64, and HS13.
[0057] In one embodiment, the composition comprises one or more polysaccharide structures selected from the group consisting of:
NeOPNI--a-Didioxy-Hep 7)a-DD-Hep(1 --+ 3)Glucito1(6 a.Dideoxy.-Hep tx-laideoxy-Hep [MeOPNr. [114e0PN]-leOPN]'-ct-Didry-ficp 7)a-DD-Hep(1 ¨02)Giucito1(6 P

a-Did.toxy-Piep [MeOPNr 7)a-DD41ep(1 ¨0 2)Gtucitol(6 -4P

a-Dideoxy-liep [MeOPN)--7)a-DD-Hep( I 3)Glucito1(6 ¨+P

a-Dideoxyglep a-Dideoxy-tirp [MeOPN)-- [MeOPNr-derived from C. jejuni strain FIS44; and T

[MeOPM---1.3)-FrIal Fruf:(3--[MeOPN], derived from C. jejuni strain HS1 and/or HS 1!44;
[--,3)-L-3-D-ido-Hep-( I->4)43-D-CilcNAc-(I-Hr,, derived from Cjejuni strain HS4/HS I 3SHS64, with non-stoichiornetric substitution of 0-methyl-phosphorarnidate at position 2 of L-D-ido-heptose;
[--43)-6d-O-D-ido-.Hep-(1.--44)-P-D-GleNAc-(1--4]0, derived from C. jejuni strain HS4113164, with non-stoichiometric MeOPN at C-2 and/or C-7 of ód-icio-Hep;
[--43)-L43-D-ido-Hep-(1.---44)43-D-GleN.Ao-0.---4],, derived from C. jejuni strain HS4, with non-stoichiometrie MeOPN at C-4 of LD-ido-Hep;
[-.43-6d-3-D-ido-Hep-(1---44)-P-D-Gle-(1---11,, derived from C. jejuni strain HS13, without MeOPN or with non-stoichiornetric MeOPN at C-2 and/or C-7 of 6d-ido-Hep;
[MeOPN]

(3,6,-O-Me)-D-glyeero-a-L-gle-Eiepp )-1.1-D-GaljNAc-(1.--4-a-D-GlepA6-0 --5]õ

MeOPN] [NGro/Etn], whererin NGro =
aminoglyeeroi; Etn = ethanolamine, derived from HS2;
[-03)-L-a1pha-D-ido-Hep-(1->4)-alplia-Gal-(1. -Hi, derived from C. jejuni strain HS3, with non-stoichiomoetric substitution 0-methy1-phosphorarnidate at position 2 of 6-deoxy-alpha-D4do-heptose with or without a 3-hdroxyproparioyi ester at C-3 of a-Gat;

[-3.3)-L-11-D-ido-Hep-(1---04)*D-Gle-(1--->b, derived from HS50, with non-stoichiometric Me01'N at C-4 of LD-ido-Hep;
and derived from C. jejuni strain HS50, with non-stoichiometric MeOPN at C-7 of 6d-ido-Hep, wherein the same polysaccharide is linked to form a polysaccharide polymer comprising 1 to 100 polysaccharides linked together (i.e., "n" greater than or equal to I), [00581 The polysaccharides or polysaccharide polymers of the decomposition can be linked to a carrier, wherein said carrier can be a protein. In one embodiment, the protein carrier is CIRM197.
Example 6: Induction of immune response by CPS conjugates.
Induction of immune response against US I. HS1/1-IS44 and IIS44 [0059] In one embodiment an immunogenic composition, useful for inclusion in a vaccinc composition against HSI, HS ItHS44 and BS411. C. jejuni strains, comprises a polysaccharide, comprising the structure:
[--44.)-a-D-Galp-(1--42)-Gro-(1.--413-41õ

I I
I I
[MeOPN]---1.3)-Fruf Fruf-(34¨[MeOPN1, or a polymer comprising a repeating of the polysaccharide structure, where "n". In an alternative embodiment, the immunogenic composition can comprise the HS44 composition, as in Example 1, which does not contain the "[MeOPM--->3)-Fruf "
unit.
[0060] Surprisingly, the above structure found in HS1 and HS1/I1544 strains induces an immune response against I-1844 strains. In the study, mice were immunized with escalating amounts of vaccine administered with Alhydrogel (Clifton, NJ). Two weeks following the final immunization, all immunized animals exhibited significant levels of serum IgG antibodies specific against HS1 CPS (P<0.05) compared to pre-immune sera.
Furthermore, this effect was dose dependent as mice immunized with 50 pig of vaccine (by weight) per dose had a significantly higher endpoint titer (P<0.05) than mice receiving 10 f.tg per dose. These results illustrate that HS I is capable of generating high levels of anti-CPS antibodies in mice. The results of these studies is illustrated in HG.
18. Also, shown in FIG. 19, a dot blot demonstrating immunogenicity of an HSI-GRIM] 97 vaccine. Purified capsules (I mg/m1) were dot blotted in triplicate (2 ul each) to nitrocellulose and inununodetected with rabbit polyelonal antiserum to an HS1-vaccine. HS1, wildtype HSI capsule; HS1.08, capsule from a fructose transferase mutant of HSI that lacks the fructose branch and the MeOPN; HS23/36, capsule from 81-which expresses a heterologous capsule (H823/36).
Induction of an immune response using an HS-5 polysaccharide composition [0061] The ability of isolated HS5 polysaeccharide to induce an immune response was evaluated. It is contemplated that isolated HS5 polysaccharide could be used conjugated to any of a number of protein carriers. However, as an illustration, CRMI97-conjugated HS5 polysaccharide was evaluated.
[0062] In this study, HS5 was conjugated to CRN1197 predicated on the method in Example 3, BALB/c mice were given three doses each of 10 itg or 50 ug of HS5 polysaccharide-conjugate at 4 week intervals, with 200 pg of ALHYD.ROGEL
(Brenntag AG, Germany). The mice received a total of throe injections. Two weeks after the last dose, the mice were bled and the sera evaluated by ELISA. The results of this study are shown in FIG. 19 showing CPS-specific IgG responses.
[0063] The immune response of HS3 conjugated to CRM197 was also examined.
Female BALBIc mice were immunized via subcutaneous injection with conjugate vaccine (HS3 from BH0142 conjugated to CRM 97) in aluminum hydroxide 3 times at 4-week intervals.
Vaccine was given by weight. A dose of 5 ug corresponded to approximately 0.5 jig of conjugated polysaccharide and a dose of 25 ug corresponded to approximately 2.5 pg of conjugated polysaccharide. Serum was collected 2-weeks following each immunization.
Capsule-specific IgG responses were determined by ELISA. The results are shown in FIG. 20, [0064] Additionally, the immune cross-reactions among members of the HS4 complex were evaluated. in these studies, whole cell proteinase K digested samples of various members of the HS4 complex were electrophoresed on 12.5% SDS-PAGE gels and irnmunoblotted with rabbit polyclonal antisera made against fomialin killed whole cells of members of the HS4 complex. 1-1S4 anti-serum was found to cross react to H513 and.
HS4. Anti-HS4113/64 serum was found to crossreact with HS64 and HS4 and to a small extent to 1-1550.

[0065] In similar studies, rabbit anti-HS13 serum was found to cross react with HS4 and HS13 and anti-H564 serum was found to cross react with 1-1S4, 1-IS13, HS4113/64 and HS50. Similarly, rabbit polyclonal antiserum made to conjugate vaccine composed of the capsule of HS4/13/64 strain conjugated to CR.M197 was used in an immunoblot to determine the cross reactivity of the vaccine to proteinase K digested whole cell preparations of other members of the HS4 complex. Antibodies to the vaccine cross-reacted to HS4 and 1-1S64, but not to HS13 or HS50.
Example 7: Method for inducing an anti-C. jejuni immune response in mammals [0066] An embodiment of the invention is the induction of an immune response against capsule polysaccharide. The embodied method comprising administering an immunogenic composition comprising one or more polysaccharide antigens, wherein each polysaccharide antigen comprises a C. jejuni capsule polysaccharide polymer. The Campylobacter jejuni capsule polysaccharide polymers comprise of C. jejuni strains, as in Examples 1-4. As such, a capsule polysaccharide polymer comprises I to 100 copies of a polysaccharide structure, derived from an individual C. jejuni strain, connected together to form a polysaccharide polymer. Induction of immunity can be against one or more strains of C jejuni.
[0067] The capsule polysaccharide are derived from one or more C. jejuni strains selected from the group consisting of 1-IS1 and HS1 complex (HSI, HS1/1-1S44 or HS44), I-1S2, HS3, 11S4, HS5, I-1513, HS4/13/64, and HS50, The inventive immunogenic compositions would comprise isolated C jejuni polysaccharide structures or polysaccharide polymers of the structures, without lipooligosaccharide, or other structures associated with GBS. The polysaccharide polymers can be conjugated or unconjugated to a carrier molecule and the composition administered at a dose range of 0.1 ng to 10 mg per dose with or without an adjuvant.
[0068] Another embodiment is a method of to induce an immune response against C
jejuni by administering isolated C'. jejuni capsule polysaccharide derived from HSI, HS1/HS44 or HS44, In the inventive method, the composition is used to induce an immune response against HSI, HS1/HS44 or HS44. As an example, a composition comprising isolated C jejuni capsule polysaccharide, isolated away from or purified from LOS components and other components that can Cause autoirnmune responses such as Guillain-Barre syndrome, such as derived from HSI, are used to induce an immune response against HSI, HSI /1-I544 and HS44 C jejuni strains, [0069] In another embodiment, a composition comprising one or more of the polysaccharide comprising one or more of polysaccharides derived from IIS4, HS13, HS411S13/HS64 or HS50 can be used in a method to induce immunity against any of the C jejuni strains of the HS4 complex, comprising HS4, HS13, 1-IS4/11S13/H64 or HS50.
[0070] In the above described compositions, the polysaccharides or polysaccharide polymers can be linked to a carrier, wherein said carrier can be a protein..
In one embodiment, the protein carrier is CRIV1197.
[0071] As an example, the embodiment method, comprises the steps:
a. administering an immunogenic composition comprising one or more C. jejuni isolated capsule polysaccharide polymers derived from capsules of C jejuni strains selected from the group consisting of: HSI and HSI complex (HSI, HSI/HS44 or HS44), 1-IS2, HS3, 'HS4, HSS, HS13, HS4/13/64, and HS50, wherein capsule polysaccharides of a strain can be linked to form a polysaccharide polymer comprises 1 to 100 copies of a polysaccharide structure, derived from an individual C.
jejuni strain, connected together to form a polymer and wherein said composition would comprise isolated C. jejuni polysaccharide structures or polymers of the structures, without lipooligosaccharide, or other structures associated with GBS and wherein the polysaccharide or polysaccharide polymers can be conjugated or unconjugated to a carrier molecule and the composition administered at a dose range of 0.1 lig to 10 mg per dose with or without an adjuvant, and wherein the polysaccharide structures include one or more of the following structures selected from the group consisting of:
IMeOPNr=

eaktioxy-llep ¨67)roDD-Hep(1 3)Glueitol(6 ¨*P

oDideoxy-flep a-Dideoxy-Rep [MeOPNr (MeOPNI¨

(MeOPNr=

a-Didrucy-lkg 7)a-DD41ep(1 2)GlucitoK6 a-Dideoly-Hep [NleOPNr-s=-== 7)a-DD-Rep(1 2)Glucitoi(6 P

a-Dicleoxy-Plep riVfeOPN)-----07)a-Dts-Hep(1 ¨03)61116.010 P

deenty-Hp a-Dideory-Elep rIVIeOPNr [MeOPNr= =
derived from the derived from the C.
jejuni strain HS44;

T

[MeOPN1--43)-Fruf Frt.11-(3,--{MeOPN1, derived from the C. jejuni strain HSI
and/or HS1/44;
[----3)-L43-D-ido-Hep-(1->4)-P-D-G1eNAe-(1H0, derived from HS4/HS13/1-1S64, with non-stoichiometric substitution of 0-methyl-phosphoramidate at position 2 of L-D-ido-heptose;
[---3)-6d-13-D-ido-Hep-(1---04)-fi-D-G1eNAc-(1-41õ, derived from HS4/13/64, with non-stoichiometric MeOPN at C-2 and/or C-7 of 6d-ido-Hep;

[---+3)-L4i-D-iclo-Ilep-(1---4)-p-D-GleNAe-(1-H,, derived from HS4, with non-stoichiornetric MeOPN at C-4 of ID-ido-Hep;
derived from HS13, without MeOPN
or with non-stoichiornetric MeOPN- at C-2 and/or C-7 of 6d-ido-flep;
[MeOPN]

(3,6,-O-Me)-D-glycero-a-L-gic4Hepp [MeOPN] [NGroNEtn], derived from HS2, whererin NGro = aminoglyeerol; Em = etha_nolarnine;
[--->3)-L-a1pha-D-ido-Hep-(1->4)-alpha-Ga1-OHõ; derived from HS3, with non-stoiehiornoetrie substitution O-methyl-phosphoramidate at position 2 of 6-deoxy-alpha-D-ido-heptose with or without a 3-hdroxypropanoyl ester at C-3 of ot-Gal;
[--6)-L-p-D-ido-Hep-(I--+4)-p-D-Gic-(I-H,, derived from HS50, with non-stoiehiometrie MeOPN at C-4 of LD-ido-Hep;
and derived from HS50, \kith non-stoichiometrie MeOPN at C-7 of od-ido-Hep, wherein the same polysaccharide is linked to form a polysaccharide polymer comprising 1 to 100 polysaccharides linked together (i.e., "n" greater than or equal to 1);
b. administering a boosting dose of the composition as described in step (a), with or without adjuvant at a dose range of 01 ug to 10 mg per dose.
[0072] Another embodiment comprises a method of immunizing against Campylobacter ,jejuni strains 1-151; US 1/1-1544 and/or 14S44 by the administration of a composition comprising one or more isolated C jejuni capsule polysaccharides. The method comprises the steps:
a. administering an immunogenic composition comprising one or more C. jejuni capsule polysaccharide polymers. The C. jejuni capsule polysaccharide polymers comprise polysaccharide structures derived from capsules of C'. jejuni strains selected from the group consisting of HS1, HS 1/H544, .HS44, wherein a capsule polysaccharide polymer comprises 1 to 100 copies of a polysaccharide structure, derived from an individual C. jejuni strain, connected together to form a polymer, without lipooligosaccharide, or other structures associated with GBS administered at a dose range of 0.1 lig to 10 mg per dose with or without an adjuvant. The polysaccharide structures include one or more of the following structures selected from the structures:

2)-Gro-(1----->P---H0, derived from the derived from the C.
jejuni strain HS44; or [---4)-o.-D-Cialp-(1--,2)43ro-(1--->PH

ii [MeOPM-e.3)-Fruf Fruf-(34----[MeOPN1, derived from the Cl jejuni strain HS 1 and/or HS1/44;
wherein the same polysaccharide is linked to folio a .polysaccharide polymer comprising 1 to 100 polysaccharides linked together (i.e., "n" Dealer than or equal to 1);
b. administering a boosting dose of the composition as described in step (a), with or without adjuvant at a dose range of 0.11.ig to 10 mg per dose.
[0073] Another embodiment comprises a method of immunizing against Campylobacter jejuni strains 11S4, HS13, HS4/HS13/H64 or HS50 by the administration of a composition comprising one or more isolated C. jejuni capsule polysaccharides derived from HS4, HS13, HS4/HS13/H64 or HS50. The method comprises the steps:
a. administering an immunogenic composition comprising one or more C. jejuni capsule polysaccharides derived from HS4, HS13, HS4THS13/1-164 or HS50, wherein a capsule polysaccharide polymer compriseing 1 to 100 copies of a polysaccharide structure, connected together to form a polymer, without lipooligosaccharide, or other structures associated with GBS, administered at a dose range of 0.1 to 10 mg per dose 41.

with or without an adjuvant and wherein the polysaccharide structures include one or more of the following structures selected from the structures:
[¨+3)-L-P-D-ido-fiep-(1>4)-fi-D-Gicl\TAc-(1----*in, derived from HS4IHS13/1-1564, with non-stoichiometric substitution of 0-methyl-phosphoramidate at position 2 of L-D-ido-heptose;
[----3)-6d-P-1)-ido-liep-(1---3-1)13-D-GicNAc-(1-4],, derived from HS4,113164, with non-stoichiometrie MeOPN at C-2 and/or C-7 of 6d-ido-Hep;
[¨.3)-L-P-D-ido-Hep-(1--.4)-3-D-G1eNAc-(1--01, derived from HS4, with non-stoichiometrie MeOPN at C-4 of LD-ido-Hep;
[¨+3-6d-p-D-ido-Hep-(1---44)-p-D-Cilc-(11,õ derived from HS13, without MeOPN
or with non-stoiehiometric MeOPN at C-2 and/or C-7 of 6d4do-Hep;
)1,, derived from HS50, with non-stoichiometric MeOPN at C-4 of LD-ido-Ilep; and [---03-6d-13-D-ido-Hep-(1--4.4)-P-D-Cilc-(1-4],õ derived from HS50, with non-stoichiometrie MeOPN at C-7 of 6d-ido-Hep, wherein the same polysaccharide is linked to form a polysaccharide polymer comprising 1 to 100 polysaccharides linked together (i.e., ''n" greater than or equal to I);
b. administering a boosting dose of the composition as described in step (a), with or without adjuvant at a dose range of 0.1 tg to 10 mg per dose.
[0074] The polysaccharide polymers can be conjugated or =conjugated to a carrier molecule and the composition. In the above method, immunogenic composition can be administered orally, nasally, subcutaneously, intradermally, tra.nsdermally, transcutaneously, intramuscularly or rectally. Also, the carrier molecule can be a protein, for example CRM1,7, or a non-protein molecule. Adjuvants can be any of a number of adjuvants. Examples of adjuvants include: LTR 192G, Aluminum hydroxide, RC529E, QS21, E294, olgodeoxyriticleotides (ODN), CpG-containing oligodeoxynucleotides, and aluminum phosphage.
[0075] Obviously, many modifications and variations of the present invention are possible, in light of the above teachings. It is therefore to be understood that, within the scope of the appended claims, the invention may be practiced otherwise than as specifically described.

Claims (15)

What is claimed is:

1. An immunogenic composition against Campylobacter jejuni comprising one or more polysaccharide antigens and an adjuvant, wherein each of said polysaccharide antigens comprises an isolated Campylobacter jejuni capsule polysaccharide from a Campylobacter jejuni strain linked to form a repeating polysaccharide polymer comprising 2 or more of said capsule polysaccharides, wherein said Campylobacter jejuni strain is selected from the group consisting of: HS4, HS5, HS4/13/64, and HS50, wherein said immunogenic composition does not contain Campylobacter jejuni lipooligosaccharide structures associated with Guillain Barré Syndrome, wherein said capsule polysaccharide or said polysaccharide polymer is conjugated to a protein carrier, wherein the structure of HS4 is .fwdarw.3)-L-.beta.-D-ido-Hep-(1.fwdarw.4)-.beta.-D-GlcNAc-(1.fwdarw., with non-stoichiometric MeOPN at C-4 of LD-ido-Hep, wherein the structure of HS4/13/64 is [.fwdarw.3)-6d-.beta.-D-ido-Hep-(1.fwdarw.4)-(3-D-GlcNAc-(1.fwdarw.]n, with non-stoichiometric MeOPN at C-2 and/or C-7 of 6d-ido-Hep, and wherein the number of repeats of the capsule polysaccharide "n" is 1 to 100.

2. The immunogenic composition of claim 1, wherein said isolated Campylobacter jejuni capsule polysaccharide comprises a polysaccharide structure selected from the group consisting of:
[.fwdarw.3)-L-.beta.-D-ido-Hep-(1.fwdarw.4)-.beta.-D-Glc-(1.fwdarw.]n, from HS50, with non-stoichiometric MeOPN at C-4 of LD-ido-Hep; and [.fwdarw.3-6d-.beta.-D-ido-Hep-(1.fwdarw.4)-.beta.-D-Glc-(1.fwdarw.]n, from HS50, with non-stoichiometric MeOPN at C-7 of 6d-ido-Hep, and wherein the number of repeats of the capsule polysaccharide "n" is 1 to 100.

3. The immunogenic composition of claim 1 or claim 2, wherein said protein carrier is CRM197.

4. The immunogenic composition of claim 1, wherein said adjuvant is selected from the group consisting of LTR192G, aluminum hydroxide, RC529, QS21, oligodeoxynucleotides (ODN), and aluminum phosphate.

5. The immunogenic composition of claim 4, wherein said oligodeoxynucleotide (ODN) is a CpG-containing oligodeoxynucleotide.

6. Use of the immunogenic composition of claim 1 or claim 2 for inducing an immune response in a mammal against a Campylobacter jejuni strain selected from the group consisting of HS4, HS5, HS4/13/64, and HS50, wherein the use comprises the immunogenic composition of claim 1 or claim 2 at a dose range of 0.1 µg to 10 mg per dose and a boosting immunogenic composition comprising the immunogenic composition of claim 1 or claim 2 at a dose range of 0.1 µg to 10 mg per dose.

7. The use of claim 6, wherein said protein carrier is CRM197.

8. The use of claim 6, wherein said adjuvant is selected from the group consisting of LTR192G, aluminum hydroxide, RC529, QS21, oligodeoxynucleotides (ODN), and aluminum phosphate.

9. The use of claim 6, wherein said immunogenic composition is for administration by a route selected from the group consisting of orally, nasally, subcutaneously, intradermally, transdermally, transcutaneously, intramuscularly and rectally.

10. The use of claim 8, wherein said oligodeoxynucleotide (ODN) is a CpG-containing oligodeoxynucleotide.

11. Use of the immunogenic composition of claim 1 or claim 2 for inducing an anti-Campylobacter jejuni immune response in a mammal against a Campylobacter jejuni strain selected from the group consisting of HS4, H54/13/64, H55, and HS50, wherein the use comprises 3 doses of the immunogenic composition of claim 1 or claim 2 at a dose range of 0.1 µg to 10 mg per dose.

12. The use of claim 11, wherein said adjuvant is selected from the group consisting of LTR192G, aluminum hydroxide, RC529, QS21, oligodeoxynucleotides (ODN), and aluminum phosphate.

13. The use of claim 12, wherein said oligodeoxynucleotide (ODN) is a CpG-containing oligodeoxynucleotide.

14. The use of claim 11, wherein said immunogenic composition is for administration by a route selected from the group consisting of orally, nasally, subcutaneously, intradermally, transdermally, transcutaneously, intramuscularly and rectally.

15. The use of claim 11, wherein said protein carrier is CRM197.