AU2018204255A1

AU2018204255A1 - Immunogenic composition against Campylobacter jejuni

Info

Publication number: AU2018204255A1
Application number: AU2018204255A
Authority: AU
Inventors: Patricia Guerry; Mario Artur Monteiro
Original assignee: US Department of Navy
Current assignee: US Department of Navy
Priority date: 2014-08-07
Filing date: 2018-06-14
Publication date: 2018-07-05
Anticipated expiration: 2035-07-31
Also published as: AU2015301368A1; EP3188748A4; WO2016022412A8; CA2957430A1; CA2957430C; WO2016022412A1; AU2018204255B2; JP2017524710A; JP6530051B2; WO2016022412A9; AU2015301368B2; EP3188748A1

Abstract

IMMUNOGENIC COMPOSITION AGAINST CAMPYLOBACTER The inventive subject matter relates to methods of inducing an immune response in a mammal against a Campylobacterjejuni strain comprising administering to such a mammal an immunogenic composition against Campylobacterjejuni comprising isolated capsule polysaccharide from selected pathogenic Campylobacterjejuni strains. The inventive subject matter also relates to uses of such polysaccharide compositions for the manufacture of medicaments for inducing an anti-C. jejuni immune response.

Description

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

2018204255 14 Jun 2018 mucus, fecal leukocytes and blood, although watery diarrhea is also observed. The disease is zoonotic, and wild and domesticated birds represent a major reservoir. C. jejuni is a major foodborne infection, most often 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-Barre Syndrome (GBS), a post-infectious polyneuropathy that can result in paralysis (Alios, B.M., J.

Infect. Dis 176 (Suppl 2):S 125-128 (1997)). The association is due to molecular mimicry between the sialic acid containing-outer core of the lipooligosaccharide (LOS) and human gangliosides (Moran, et al., J. Endotox. Res. 3: 521 (1996)). Thus, antibodies generated against LOS cores result in an autoimmune 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 diarrheal 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

2018204255 14 Jun 2018 consistent with the observed molecular mimicry of LOS and human gangliosides important in GBS (Aspinall, et al., Eur. J. Biochem., 213: 1029 (1993); Aspinall, et al., Infect. Immun. 62: 2122-2125 (1994); Aspinall, et al., Biochem., 33: 241 (1994); Salloway et al., Infect. Immun., 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 II/III capsule loci in the Enterobactericeae (Parkhill et al., Nature, 403: 665 (2000); Karlyshev et al., Mol. Microbiol., 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 (Karlyshev et al., Mol.

Microbiol., 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 be based on lipopolysaccharide O side chains (Moran and Penner, J. Appl. Microbiol., 86: 361 (1999)). Currently it is believed that the structures previously described as O 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

Campylobacter jejuni 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

2018204255 14 Jun 2018 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, HS4/13/64 and HS50 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, HS4/13/64 and HS50. [0007] Another embodiment is a method of immunizing against C. jejuni strains HS1, HS1/HS44, 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.

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

2018204255 14 Jun 2018 transferase; Heptose/deoxyheptose biosynthesis; putative glycosyl transferase; sugar biosynthesis; and hypothetical.

FIG. 2. Structure of HS1 teichoic acid-like capsule.

FIG. 3. 2D *H-³¹P HMBC NMR spectrum of C. jejuni HS:l/44 teichoic acid CPS. This NMR spectrum shows the connections between the MeOPN moieties and positions 3 of the Fru units, and between the diester-phosphate and position 4 of Gal and position 1 of

Gro.

FIG. 4. GC-MS and NMR of C. jejuni HS44 CPS material. (A) GC-MS profile of the alditol acetate derivatives from the two CPSs of C. jejuni HS44, 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-O-Methyl-a//ro-heptose (6d-3-O-Me-a//ro-Hep), 6-deoxy-a/PO-heptose (6d-a//ro-Hep) and 6-deoxy-ga/actoheptose (6d-ga/-Hep). (B) *H NMR spectrum of HS44 CPS material showing the aanomeric resonances emanating from 6d-a//ro-Hep/j 6-deoxy-ga/acto-Hep/and 6d-3-OMe-a/PO-Hep/of the heptose-rich CPS and from Gal of the teichoic acid CPS.

FIG. 5. Characterization of mutants in the HS1 CPS locus. A. Alcian blue stained

12.5% SDS PAGE of crude CPS preparations. Fane 1, Precision Plus protein standards; lane 2, HS1 wildtype; lane 3, HS1 1.08 mutant; lane 4, HS1 1.08 mutant complemented; lane 5, HS1 1.09 mutant; lane 6, HS1 1.09 mutant complemented; lane 7, HS1 wildtype. B. ³¹P NMR of CPS from HS1.08 complement; C. ³¹P NMR of CPS from HS1.09 complement; D. ³¹P NMR of CPS from HS1 wildtype.

FIG. 6. The GC-MS profile of the alditol acetate derivatives of C. jejuni CG2995 CPS. FIG. 7. The *H NMR spectrum of C. jejuni CG2995 CPS.

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FIG. 8. (A) The 2D ‘H-¹³C HSQC NMR spectrum of C. jejuni CG2995 CPS; (B) The ID selective *H NOEs of the C. jejuni CG 2995 CPS. Irradiated peaks are denoted with an Mixing time of 0.250 ps was used.

FIG. 9. The ³¹P NMR spectrum of C. jejuni CG2995 CPS.

FIG. 10. The 2D *Η-³¹Ρ HMBC NMR spectrum of C. jejuni CG2995 CPS.

FIG. 11. Structure of HS5 CPS showing four variations: i) 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 3,6dideoxy-rz'ho-heptose.

FIG. 13. TEMPO oxidation that shows a reduction in abundance of the 3,6-dideoxyrzho-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 CRM 197.

FIG. 14. Characterization of the HS1 conjugate vaccine. A. ³¹P NMR of HSIcpsCRM 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 *H NMR; and (B) ID ³¹P NMR spectra of C. jejuni 3019 CPS (serotype HS:13).

FIG. 16. Linkage determination of MeOPN group by NMR. 2D *Η-³¹Ρ HMBC NMR spectrum of C. jejuni BH-01-0142 CPS (A: 1,2,3-linked 6d-z'i/o-Hep/LD-z'i/o-Hep with C residue; C: MeOPN).

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FIG. 17. NMR analysis showing that non-sugar moiety was 3-hydroxypropanoyl. (A)

2D Ή-¹³Ε HMBC NMR spectrum ofC. jejuni BH-01-0142 CPS (B': 1,3,4-linked Gal with residue D; (B) D: 3-hydroxypropanoyl group.

FIG. 18. Immunogenicity of HSI-CRM197 conjugate in mice. A. ELIS A titers to HS 1 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-CRMi97 conjugate. Mice were immunized with three (3) doses (10 pg and 50 pg by weight of conjugate) given at 4 week intervals. The mice were bled two weeks after the last doses.

FIG. 20. BH0142 (HS3) conjugate vaccine is immunogenic in mice. Data represent the mean (±SEM) reciprocal IgG endpoint titer per treatment group.

FIG. 21. Dot blot demonstrating immunogenicity of an HS1-CRM197 vaccine. Purified capsules (1 mg/ml) were dot blotted in triplicate (2 ul each) to nitrocellulose and immunodetected with rabbit polyclonal antiserum to an HS 1 -CRM 197 vaccine. HS 1, wildtype HS1 capsule; HS1.08, capsule from a fructose transferase mutant of HS1 that lacks the fructose branch and the MeOPN; HS23/36, capsule from 81-176 which expresses a heterologous capsule (HS23/36).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS [0008] The term “polysaccharide antigen” as used herein refers to a capsule polysaccharide derived from Campylobacter jejuni (C. jejuni or Campylobacter jejuni) capsule. As used, herein, each polysaccharide antigen comprises a polysaccharide or

2018204255 14 Jun 2018 polysaccharide polymer derived from one C. jejuni strain. The inventive composition can be comprised of multiple polysaccharide antigens. As used herein, “polysaccharide” refers to two or more monosaccharide 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 HS1, HS1/HS44, HS44, HS2, HS3, HS4, HS5, HS13,

HS4/13/64, 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. jejuni 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.

[0010] 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.

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Example 1: HS1/HS44 andHS44 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

Barre Syndrome (GBS), a post-infectious polyneuropathy (Alios, 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 Campylobacter 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., J. Clin. Microbiol. 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 HS1 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 HS1 and HS44 types, and strains can serotype as HS1, HS44 or HS1/44. 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-Dgalactose (Gal) and glycerol (Gro) linked by phosphate (P) in a teichoic acid-like structure [-4)-a-D-Gal/?-(l-2)-Gro-(l-P-]_n (Aspinall, et al., Eur. J. Biochem. 216: 880 (1993)). The HS1 CPS backbone may be decorated by β-D-fructofuranoses (Fru) ίο

2018204255 14 Jun 2018 branches, at C-2 and C-3 of the Gal unit, which in turn may be decorated at C-3 with MeOPN (Fig. 1; (McNally, et al., FEBS 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., FEBS J. 272: 4407 (2005)). The ~15 kb HS1 CPS locus encoding eleven genes for the synthesis of this polysaccharide (BX545859) is the smallest CPS locus identified to date in C. jejuni (Karlyshev, et al., Appl. Environ.

Microbiol. 71: 4004 (2005))(Fig. 1).

[0013] The HS1 type strain used was MSC57360 and the HS44 strain (ATCC 43463) was obtained from the American Type Culture Collection (ATCC)(Manassas, VA). C. jejuni 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% O2, 10% CO2, and 85% N2) on Mueller Hinton (MH) agar plates, supplemented with the appropriate antibiotic, if required. E. coli 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:

(2005); Poly, et al. J. Clin. Microbiol. 49: 1750 (2011);, Karlyshev, et al., Gene 522:

(2013)).

[0015] The CPS was extracted from cells by hot water-phenol extraction for 2 hours at °C. The aqueous layer was dialyzed (1000 Da) against water followed by ultracentrifugation to separate the CPS from the LOS. The supernatant material

2018204255 14 Jun 2018 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 ThermoFinnigan POLARIS™-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 Bruker cryo platform at 295 K with deuterated trimethylsilyl 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 HS1 CPS locus (McNally, et al., FEBS J. 272: 4407 (2005)). The DNA sequence of the capsule locus of the HS44 type strain 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 HS44 capsule biosynthesis locus is summarized in Table 1. All shared homologs were >96% identical, except for the putative MeOPN transferase (HS44.07) which showed only 47% identity to that of HS1.

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Table 1

Locus Tag	Putative function³	Relationship	Identity with HS1^b	Size (amino acid)
HS44.01	MeOPN biosynthesis	HS1.01	164/170 (96%)	170
HS44.02	MeOPN biosynthesis	HS1.02	252/253 (99%)	253
HS44.03	MeOPN biosynthesis	HS1.03	197/200 (98%)	200
HS44.04	MeOPN biosynthesis	HS1.04	775/779 (99%) 253/253	779
HS44.05	Methyl transferase	HS1.05	(100%)	253
HS44.06	Methyl transferase	HS1.06	255/257 (99%)	257
HS44.07	MeOPN transferase sugar-phosphate	HS1.07	308/642 (47%)	609
HS44.08	nucleotidyltransferase sedoheptulose 7-phosphate			224
HS44.09	isomerase D-glycero-D-manno-heptose 7-			201
HS44.10	phosphate kinase GDP-mannose 4,6-			360
HS44.11	dehydratase	-	-	343
HS44.12	GDP-fucose synthetase (fcl)	-	-	381
HS44.13	GDP-fucose synthetase (fcl)	-	-	385
HS44.14	Cj1429 like Nucleotide-sugar			310
HS44.15	epimerase/dehydratase Nucleotidyl-sugar pyranose			181
HS44.16	mutase	-	-	416
HS44.17	Heptosyl transferase CDP glycerol	-	1067/1095	1202
HS44.18	glycerophosphotransferase	HS1.09	(97%)	1100
HS44.19	Unknown Glycerol-3-phosphate	HS1.10	390/396 (98%)	397
HS44.20	cytidylyltransferase	HS1.11	128/129 (99%)	129

^aFunction attributed based on Blastp performed on non-redundant protein sequences database.

^bNumbers in parenthesis are the percentage of identity between the HS1 and HS44 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

2018204255 14 Jun 2018 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 reductases that have been recently demonstrated to be involved in 6-deoxyi///ra-heptosc biosynthesis. The CPS locus of HS44 also includes a gene (HS44.14) similar to CJ1429c coding for a protein of unknown function in NCTC 11168 (HS2), a nucleotidyl-sugar pyranose mutase (HS44.16) and a putative heptosyltansferase (HS44.17, 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 HS1. 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 HS 1/44 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): —>4)[MeOPN—»3)-P-D-Fru-(l—»]-a-Gal-(l—»2)-Gro-(l—»P—» (FIG. 2).

[0020] FIG. 3 shows the phosphorous-proton connections detected in HS1/44 CPS that emanate from the linkages of the teichoic-acid diester-phosphate (δρ 0.5 and 1.5) to position 1 of Gro and position 4 of Gal, and from the attachment of the MeOPN (δρ 14.3) to position 3 (διι 4.83) of Fru residues. The H-4 resonance of the 4-linked Gal carrying the Fru branches appeared at δ 4.68, whereas that of the defructosylated 4-linked Gal resonated at δ 4.49 (FIG. 3). A similar pattern was observed for the H-l resonances of Gro. Simultaneous analysis of the HS1 type strain and HS1/44 CPSs, suggested that the HS1/44 CPS contained a lower degree of fructosylation, as judged by the lower

2018204255 14 Jun 2018 intensities of the 2,3,4-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 HS1 and HS1/44 (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-deoxyga/acto-heptose (6d-ga/-Hep), 6-deoxy-a/tro-heptose (6d-a/tro-Hep) and, in lesser amounts, 6-deoxy-3-O-methyl-a/tro-heptose (6d-a/tro-3-(9-Me-Hep/). 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.

[0022] Accompanying NMR studies (FIG. 4B) confirmed the presence of deoxyheptoses (δ h 1.5- 2.0) and revealed that these units were present in the a anomeric configuration (δρ 5.15 - 5.42). A new MeOPN moiety (δρ 14.0), different from that expressed by HS1 and HS1/44 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-stoichiometric 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 wildtype in the complement as shown by gel; NMR analysis also confirmed complementation, but

2018204255 14 Jun 2018 the lower intensity of the MeOPN resonance in the ³¹P NMR (FIG. 5B) suggested that complementation in this case was partial. Thus, HS1.08 appears to encode a transferase that can transfer Fru to Gal.

[0024] Gene HS1.09 was annotated as a putative CDP glycerol transferase (Karlyshev et al., 2005). Mutation of this gene in HS1 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. 5 A), but restoration of CPS expression was confirmed by the ³¹P 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 HS1, HS1/HS44 and HS44 C. jejuni strains, comprises polysaccharide antigen, comprising the structure:

[—»4)-a-D-Gal/?-( 1 -*2)-Gro-( 1 P-*] _n

2 ΐ t 2 2 [MeOPN]—>3)-Fruf Fru/-(3<—[MeOPN], or a polymer comprising a repeating of the polysaccharide structure, wherein “n” is 1 to

100. The polysaccharide structure of HS44 comprises the above structure without “[MeOPN]—»3)-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 HS44 that comprises the structure:

[—>4)-a-D-Gal/?-(l—*2)-Gro-(l—>P—*]_n, wherein “n” is 1 to 100.

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Campylobacter jejuni strain PG 3588 (HS:1):

[0026] Upon treatment of Campylobacter jejuni strain PG 3588 (HS:1) capsule polysaccharide (CPS) with mild acetic acid (5%), the fructose (Fru/) side branches and their accompanying MeOPN units were removed. The ’H NMR of the defructosylated CPS showed the anomeric resonance at δ 5.21 that corresponds to the α-D-Gal residue (without the Fru/'substitutions). H5 δ 4.18 was assigned from the H6 δ 3.75 proton resonance, Gro resonances were found to be at Hl/1'δ 4.05/4.12, H2 δ 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 ^lH-¹³C HSQC are summarized in Table 2. A 2D ^!H-³¹P HMBC (Figure 4) showed a strong cross peak at (6h 4.54/ δρΐ.14), and (6h 4.05, 4.11/ δρΐ.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 'H-³ⁱP HMBC at δ 14.04 for a MeOPN moiety, and it showed a cross peak at δη 3.75/δρ 14.04, identifying the attachement of MeOPN at the C-6 of Gal.

Table 2: Ή, ¹³C chemical shifts of C.jejuni CPS PG 3588

Sugar residue	Hl/1' Cl	H2 C2	H3/3' C3	H4 C4	H5 C5	H6/6' C6
a-D-Gal	5.21 100.84	3.88 71.05	3.90 71.10	4.54 77.31	4.18 73.45	3.75 63.42
Gro	4.05/4.12 67.23	3.98 79.81	3.78/3.82 63.95

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Example 2: HS5 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:

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[MeOPN]⁺-	[MeOPN]⁺'-
1		1
κ 7		κ 7
a-Dideoxy-Hep		a-Dideoxy-Hep
1		1
A 6	Y	a 6 x
Ϊ -> 7)a-DD-Hep(l -»·	3)Glucitol(6 —>P —>	iii -> 7)a-DD-Hep(l -> 2)Glucitol(6 ->
7	2	2
ΐ	1	ΐ
1	1	1
a-Dideoxy-Hep	a-Dideoxv-Hep	a-Dideoxy-Hep
7 N	7 L	7 N
I	I	ΐ
[MeOPNp'-	[MeOPNfi-	[MeOPN]⁺-
c	X	B Z
11 -> 7)a-DD-Hep(l -> 2)Glucitol(6 ->	IV —> 7)a-DD-Hep(l —> 3)Glucitol(6 ——>
2		2 2
I		t I
1		1 1
a-Dideoxy-Hep	a-Dideoxy-Hep a-Dideoxy-Hep
7 N		7 x 7 l
I		ί ΐ
[MeOPNf-		[MeOPNR- [MeOPNR-

[0029] Results from monosaccharide composition analysis revealed that the capsule polysaccharide (CPS) of strain CG2995 (HS:5) contained 3,6-dideoxy-r;'/jo-heptose, glucitol, and D-glycero-D-mmzro-heptose (FIG. 6). Multiple linkages of each residue were observed; terminal 3,6-dideoxy-rz'/?o-heptose, 2,6-disubstituted Glucitol, 2,3,6trisubstituted Glucitol, 2-monosubstituted D-glycero-D-manzio-heptose, 2,6-disubstituted D-glycero-D-manno-heptose, 2,7-disubstituted D-giycero-D-manzio-heptose, and 2,6,7trisubstituted D-glycero-D-ma/ino-heptose.

[0030] The ID *H 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-r/ho-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 ³H-'H COSY, TOCSY, and NOESY

2018204255 14 Jun 2018 experiments. Linkages found through NMR experiments coincided with the linkages assigned by GC-MS.

[0031] With the aid of 2D *H-¹³C HSQC and HMBC the Glucitol residues (Χ,Υ,Ζ) could are assigned, along with the ring region resonances from the six heptose residues. As expected carbons involved in the glycosidie linkages, C2 (δ 78.1) of the D-glycero-Dmzzzzzzo-heptose A, B and C, C6 (δ 76.8) of D-glycero-D-mazzzzo-heptose A, C2 (δ 81.6) of Glucitol Y and Z, C2 (δ 82.5) of Glucitol X, and C3 (δ 78.8) of Glucitol Y and Z, were found to be down-field resonances (LIG. 8 (A)). The deoxy resonances associated with the 3,6-dideoxy-rz'ho-heptose were easily observed at δ 37.1 (C3) and δ 36.1 (C6). Selective ID nOe experiments (LIG. 8(B)) also showed the presence of the linkages aforementioned.

[0032] The ID ³¹P and 2D *H-³¹P HMBC NMR revealed resonances at 0.96 and 1.30 ppm, indicating that the capsular polysaccharide repeats were linked with a phosphate bridge (PIG. 9). This bridge links through the 6-position of the Glucitol and the 7position of the D-glycero-D-muzzzzo-heptose (Pig. 9). The ID ³¹P spectra also gave rise to a peak δ 14.5 indicating MeOPN, and through the 2D *H-³¹P HMBC the MeOPN could be linked to being a 7-subsituted 3,6-dideoxy-rz'ho-heptose (PIG. 10).

[0033] One main capsular polysaccharide was observed with a backbone of [-7)-a-Dglycero-D-mazzzzo-heptose-(l-3)-Glucitol-(6-)-P-] with 2,6-disubstitution of the D-glyceroD-muzzzzo-heptose, and 2-monosubstitution of the Glucitol with a-3,6-dideoxy-rz'hoheptose (PIG. lli). Three other variations of the capsular polysaccharide repeat were also noted; variation 1 with 2-monosubstituted D-glycero-D-muzzzzo-heptose and Glucitol linked through the 2-position instead of 3 to D-glycero-D-muzzzzo-heptose (PIG. 1 lii),

2018204255 14 Jun 2018 variation 2 with 2,6-disubstitution of the D-glycero-D-manno-heptose and Glucitol linked through the 2-position instead of 3 to D-glycero-D-manno-heptose (FIG. 1 liii), and variation 3 with 2-monosubstituted D-glycero-D-manno-heptose and 2-monosubstituted

Glucitol (FIG. lliv).

Example 3: Conjugation of CPS polysaccharide to protein carrier [0034] One or more polysaccharides or polysaccharide polymers can be conjugated to a carrier molecule to improve immunity. 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 CG2995 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 CRM 197 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

2018204255 14 Jun 2018 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-OH 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 HS1 polysaccharide [0037] A glycoconjugate composed of HS1 teichoic acid CPS and the protein carrier CRM 197 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 CRM197 by carbodiimide-type coupling of the newly created carboxylic acid functionalities in the

CPS and exposed CRM197 lysine units. Importantly, analysis of the HS1 CPS-CRM197 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 HS1 CPS indicated that half of the backbone Gal residues were branched by the Fru-cntaining

MeOPN units.

Example 4: Polysaccharides in HS complexes [0038] Polysaccharide structures were identified in C. jejuni Penner serotype complexes. For example, anti-HS4 serum results in cross-reaction to other strains strains

2018204255 14 Jun 2018 of the HS4 complex, including HS13, HS4/13/64 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 /Strain	Structure
HS4 type strain	A)-l-Ji-l)-zi/zz-llep-(l>4)-jM)-(,lc\Ac-(!> (non-stoichiometric MeOPN at C-4 of LD-zf/o-Hep)
HS13	-*3)-6d-p-D-z<fo-Hep-(l—>4)-p-D-Glc-(l—+ (50%) (non-stoichiometric MeOPN at C-2 and/or C-7 of 6d-zt/o-Hep); and/or >3)-l-[!-l)-z’i/z>-llep-(!>4)-ji-l)-Glc-(l> (50%)
HS4,13,64 (e.g., strain CG8486)	>3)-6d-p-l)-zi/z>-llep-( !H)-p-l)-Glc\Ac-(l> (80%) (non-stoichiometric MeOPN at C-2 and/or C-7 of 6d-zt/o-Hep); and/or A)-l-P-l)-zi/zz-lle\|)-(l>4)-p-l)-(,lc\Ac-(l> (20%) (non-stoichiometric MeOPN at C-2 of LD-z't/o-Hep)
HS50	>3)-l-[!-l)-z’i/z>-llep-(!>4)-\|i-l)-Glc-(l> (85%) (non-stoichiometric MeOPN at C-4 of LD-z't/o-Hep); and/or >3)-6d-p-l)-z’<P>-l lep-( l>4)-[S-l)-Glc-{ 1> (15%) (non-stoichiometric MeOPN at C-7 of 6d-zt/o-Hep)

[0039] 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. jejuni strain CG8486 (HS: 4:13:64)

2018204255 14 Jun 2018 consisted mainly of a disaccharide repeating unit [—>3)-6ά-β-Ο-ζ'ί/ο-Ηερ-(1->4)-βGlcNAc-(l—>], with non-stoichiometric O-methyl phosphoramidate substituent attached to C-2 and C-7 positions of zt/o-heptose. A minor component of L-g/ycerz»-D-zz/z»-heptose (LD-z'z/o-Hep) was detected by GLC-MS, using alditol acetate derivatives for compositional analysis and permethylated alditol acetate derivates for lingage analysis of, and was newly found in this strain. The sugar ring configuration of 6-deoxy-heptose and L-g/yce/O-D-heptose were assigned as idose. The traces of l,7-anhydro-L-g/ycerz>-D-z'z/z>heptose (1,7-anhydro-LD-z't/o-Hep) and 1,6-anhydro-L-g/ycero-D-z't/o-heptose (1,6anhydro-LD-z'z/o-Hep) originated from LD-z'z/o-Hep during acid hydrolysis.

[0041] In addition to previously reported linkage types in C. jejuni CG8486 CPS (3substistuted 6d-z'z/z»-heptose [—»3)-6d-z'i/o-Hep-(l—»] and 2,3-di-substistuted 6d-idoheptose [-»2,3)-6d-z'z/z>-Hep-(l—>], 3,7-di-substistuted 6d-z'z/z»-heptose [-»3,7)-6d-z'z/z»Hep-(1—»], 4-substituted N-acetyl-glucosamine [->4)-GlcNAc-(l->]), the GLC profile of

GLC-MS of permethylated alditol acetate derivatives of C. jejuni HS:4:13:64 CPS showed two additional linkage types from LD-zz/o-Hep which were not detected in previously reported structure, including 3-substistuted L-g/ycerz»-D-z'z/z»-heptose [—>3)-LDz'z/z>-Hep-(l—»] and 2,3-di-substistuted L-g/ycerz»-D-z'z/z»-heptose [-»2,3)-LD-z'z/z>-Hep(!->]· [0042] The ID *H NMR of C. jejuni CG8486 CPS showed two resonances of two β[0043] glycosides at δ 4.94 and δ 4.66 which were 6d-z'z/o-Hep/LD-z'z/z>-Hep and GlcNAc, respectively. The presence of two anomeric proton resonances for three monosaccharide residues (6d-z'z/z»-Hep, LD-z'z/o-Hep, and GlcNAc) suggested that both 6d-z'z/z»-Hep and LDzz/z»-Hep may contain the same chemical shifts through the sugar ring system except the

2018204255 14 Jun 2018

H-6 position since the only difference between them was at the C-6 position with or without a hydroxyl group. The ¹H NMR spectrum also revealed one methyl singlet at δ 2.07 which was characteristic of the A-acetyl moiety from GlcNAc and methylene signals at δ 1.77 and δ 2.03 which were 6-deoxy-moiety from 6d-zt/o-Hep. In addition, ID ³¹P NMR detected a characteristic MeOPN signal at δρ 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 LDido-Hep within its CPS:

[—»3)-6d-P-z'i/o-Hep-(l—»4)-P-GlcNAc-(l—»] (with non-stoichiometric MeOPN at C-2 and/or C-7 of 6d-ido-Hep) as a major repeat; and [—>3)-LD-[i-z<7o-Hep-(l —>4)-β-Ο1εΝΑε-(Ί ^-] (with non-stoichiometric MeOPN at C2 of LD-ido-Hep) as a minor repeat.

CPS determination of C. jejuni HS:4 Type Strain (Strain MK7) [0044] The CPS isolated from C. jejuni strain MK7 (HS4) was composed of L-glycero-Ozf/o-hcptosc (LD-zf/o-Hep) and A-acctyl-glucosamine (GlcNAc) by GC-MS profde 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:4,13,64; strain CG8486), which contains mostly 6-deoxy-zf/o-hcptose (6d-z2/o-Hep) instead of LD-z't/o-Hep. GC-MS of permethylated alditol acetate derivatives showed the following linkage types of each monosaccharide: 3-substituted L-g/ycero-D-zf/o-heptose [—»3)-LD-z'i/o-Hep-(l—»] and 4-substituted A-acetyl glucosamine [-»4)-GlcNAc-(l—»]. [0045] The ’H NMR spectrum of the C. jejuni strain MK7 (type strain HS:4) CPS

2018204255 14 Jun 2018 showed two β-anomeric proton resonances at δ 4.70 and δ 4.94 for GlcNAc and LD-zt/oHep, respectively. The *H NMR spectrum also revealed one methyl singlet at δ 2.07 which was characteristic of the A-acetyl moiety from GlcNAc and a broad range of overlapping sugar ring proton resonances between δ 3.50 and δ 4.55. In addition, ID ³¹P NMR detected a weak trace of MeOPN signals at δρ 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 C. jejuni C. jejuni Serotype HS:13 (Strain MK16) [0046] C. jejuni HS4 type-strain (MK7) contains a CPS composed of the following disaccharide repeat: [-»3)-L-P-D-z't/o-Hep-(l—>4)-P-GlcNAc-(l->]. The monosaccharide composition analysis, using GC-MS of alditol acetate derivatives of C. jejuni strain MK16 (serotype HS:13) revealed the presence of glucose (Glc), 6-deoxy-z'i/o-heptose (6d-z't/o-Hep), and L-g/ycero-D-z't/o-heptose (LD-z't/o-Hep) by GS-MS determinationof alditol acetate derivative profiles. Linkage analysis of profiles of permethylated alditol acetate derivatives showed that these units were present as 4substituted glucose [—>4)-Glc-(l—>], 3-substituted 6-deoxy-z't/o-heptose [-»3)-6d-z't/oHep-(l-->], 2,3-di-substituted 6-deoxy-z't/o-heptose [—»2,3)-6d-z'i/o-Hep-(l-»], 3substituted L-g/ycero-D-zi/o-heptose [->3)-LD-z't/o-Hep-(l->], and 3,7-di-substituted 6deoxy-z't/o-heptose [-»3,7)-6d-z'i/o-Hep-(l—»]. 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 Glc as backbone instead of 4-substituted GlcNAc (seen in serotypes HS: 4:13:64 and HS:4).

2018204255 14 Jun 2018 [0047] The *H NMR spectrum of C. jejuni serotype HS:13 CPS showed two β-anomeric proton resonances at δ 4.63 and δ 4.92 which assigned as Glc and 6d-zz/o-Hep/ LD-idoHep, respectively (Fig. 15A). The *H NMR spectrum also revealed the methylene signals (multiplet) at δ 1.86 and δ 2.00 which were characteristic of the 6-deoxy moiety from 6dzz/zz-Hep and a broad range of overlapping sugar ring proton resonances between δ 3.30 and δ 4.55. ID ³¹P NMR detected two resonances at δρ 14.1 and δρ 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 nonstoichiometrically attached to C-2 and C-7 of 6ά-β-ζζ/ο-Ηερ):

[->3)-6d-P-D-zifo-Hep-( 1 ->4)-β-ϋ1ο-( 1 ->]; and [-»3)-L-P-D-z'z/o-Hep-(l—»4)-β-01ο-(1—»].

C. jejuni serotype HS3/13/50 [0049] HS: 3:13:50 complex has been identified predicated on a quantitatively low level immune-crossreactivity. C. jejuni strain BH-01-0142 (serotype HS: 3:13:50) was composed of galactose (Gal), 6-deoxy-z'z/o-heptose (6d-z'z/o-Hep), and L-glycero-D-idoheptose (LD-zz/o-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 [-»4)-Gal-(l—>], 3-substituted 6deoxy-heptose [-»3)-6d-Hep-(l—>] and 3-substituted L-g/ycero-D-z'z/o-heptose [—»3)-ldz'z/o-Hep-(l->] were found to make up the CPS of serotype HS:3:13:50, using GC-MS

2018204255 14 Jun 2018 profile analysis of permethylated alditol acetate derivatives of C. jejuni BH-01-0142 CPS (serotype (HS: 3:13:50)). In addition, minor components, 3,4-di-substituted galactose [—»3,4)-Gal-(l—»], 2,3-di-substituted 6-deoxy-heptose [—»2,3)-6d-Hep-(l—»], and2,3-disubstituted L-g/ycero-D-z'i/o-heptose [->2,3)-LD-z'i/o-Hep-(l—>] were also characterized.

The above results suggested that the backbone units of C. jejuni serotype HS: 3,13,50

CPS were [-»4)-Gal-(l—»], [->3)-6d-Hep-(l->], and [->3)-LD-z'i/o-Hep-(l->], with three other non-sugar components were non-stoichiometrically attached to the C-3 of Gal, and

C-2 of 6d-z'i/o-Hep and LD-zf/o-Hep. Also, a terminal Gal [Gal-(1—>] was also determined and was suggested as a non-reducing end.

[0051] The *H NMR spectrum of the C. jejuni serotype HS:3:13:50 CPS showed broad overlapping peaks between δ 5.00 ppm and δ 5.30 ppm representing the anomeric proton signals. These overlapping peaks suggested the presence of α-anomeric sugars. In addition, the 'HNMR spectrum also revealed a methylene signal at δ 1.80 and δ 2.02 which are characteristic of 6-deoxy moiety from 6d-zf/o-Hep. Another proton resonance at δ 2.72 was later confirmed as a methylene signal which also revealed in the *H NMR 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 δρ 15.3 revealed the presence of an O-methyl phosphoramidate groups (MeOPN) or CH3OP(O)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

2018204255 14 Jun 2018 analysis revealed the presence of minor component of 1,3,4-linked Gal, 1,2,3-linked 6dz't/o-Hep and 1,2,3-linked LD-z'z/o-Hep.

[0053] 2D ‘H-³¹P HMBC 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 δρ 15.3/5h 3.78 represented the correlation between the phosphorus and the methyl group of the

MeOPN. A strong proton-phosphorus correlation between δρ 15.3 and 6h 4.56 suggested the linkage site of the MeOPN group, with also a weak proton-phosphorus correlation between δρ 15.3 and the anomeric proton at 6h 5.10. Thus, the combination of the results from monosaccharide linkage type analysis and 2D *Η-³¹Ρ HMBC NMR showed that the O-methyl-phosphoramidate group (residue C) was attached to the C-2 position of 6d-idoHep and LD-z'z/o-Hep (residue A').

[0054] A 2D *H-¹³C HMBC NMR experiment (FIG. 17) showed that a second nonsugar moiety was that of 3-hydroxypropanoyl. The cross-peaks at 6h 3.89/δο 173.0 and διι 2.72/δο 173.0 showed three-bond and two-bond connectivities of the carbonyl ester ΟΙ with H-3 and H-2 of 3-hydroxypropanoyl group (residue D), respectively. The 3hydroxypropanoyl group was observed to be connected to the C-3 of Gal, by interpreting the cross-peak at διι 5.20/6c 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 O-methyl phosphoramidate at C-2 of 6d-a-/z/o-Hep/L-a-D-/z/o-Hep and 3-hydroxypropanoyl ester at C-3 of a-Gal):

[-»3)L-a-D-z'i/o-Hep-( 1 —>4)-a-Gal-( 1 —»]; and

2018204255 14 Jun 2018 [-»3)- 6d-a-z'i/o-Hep-( 1 —>4)-a-Gal-( 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-Barre 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, HS2, HS3, HS4, HS4/13/64, HS50 and HS13.

[0057] In one embodiment, the composition comprises one or more polysaccharide structures selected from the group consisting of:

[MeOPN]”· a-Dideoxy-Hep

-* 7)a-DD-Hep( 1 —> 3)Glucitol(6 —* P —» a-Dideoxy-Hep a-Dideoxy-Hep [MeOPN]”· [MeOPN]”·

2018204255 14 Jun 2018 [MeOPN]

7 α-Did^oxy-Hep • 7)a-DD-Hep(l2 T 1 a-Dideoxy-Hep

T [MeOPN] ” >2)Glucitol(6—>P—* 7)a-DD-Hep( 1 —* 2)Glucitol(6 ->P->

T a-Dideoxy-Hep

T [MeOPN]’

7)a-DD-Hep(l —

T a-Dideoxy-Hep

T [MeOPN]·

3)Glucitoi(6 -+P —ι 2 T 1 a-Dideoxy-Hep

T [MeOPN]' [^>4)-a-D-Gal/>-(l—>2)-Gro-(l—>P—>]_n, derived from C. jejuni strain HS44; and [—>4)-a-D-Gal/?-(l —>2)-Gro-( 1 —>P—+]_n3 2

T T 1 1 [MeOPN]—>3)-Fru/ Fru/-(3<—[MeOPN], derived from C. jejuni strain HS1 and/or

HS1/44;

[—>3)-P-3-D-ido-Hep-(l->4)-P-D-GlcNAc-(l—>]_n, derived from C. jejuni strain

2018204255 14 Jun 2018

HS4/HS13/HS64, with non-stoichiometric substitution of O-methyl-phosphoramidate at position 2 of L-D-ido-heptose;

[—>3)-6d-P-D-ido-Hep-(l—>4)-p-D-GlcNAc-(l—>]_n, derived from C. jejuni strain HS4/13/64, with non-stoichiometric MeOPN at C-2 and/or C-7 of 6d-ido-Hep;

[—>3)-L-P-D-ido-Hep-(l—»4)-P-D-GlcNAc-(l-^]_n, derived from C. jejuni strain HS4, with non-stoichiometric MeOPN at C-4 of LD-ido-Hep;

[—>3-6d-p-D-ido-Hep-(l—>4)-P-D-Glc-(l—>]_n, derived from C. jejuni strain HS13, without MeOPN or with non-stoichiometric MeOPN at C-2 and/or C-7 of 6d-ido-Hep;

[MeOPN] (3,6,-O-Me)-D-glycero-a-L-glc-Hep/?

[->2)-P-D-Rib/-( 1 ->5)-P-D-Gal/NAc-( 1 ^4-a-D-GlcpA6-( 1 ->]_n

MeOPN] [NGro/Etn], whererin NGro = aminoglycerol; Etn = ethanolamine, derived from HS2;

[—>3)-L-alpha-D-ido-Hep-(l->4)-alpha-Gal-(l “»]n, derived from C. jejuni strain HS3, with non-stoichiomoetric substitution O-methyl-phosphoramidate at position 2 of 6deoxy-alpha-D-ido-heptose with or without a 3-hdroxypropanoyl ester at C-3 of a-Gal;

2018204255 14 Jun 2018 [-*3)-L-p-D-ido-Hep-(l—>4)-p-D-Glc-(l—>]_n, derived from HS50, with nonstoichiometric MeOPN at C-4 of LD-ido-Hep;

and [—>3-6d-P-D-ido-Hep-(l—»4)-P-D-Glc-(l-->]n, 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 to 100 polysaccharides linked together (i.e., “n” greater than or equal to 1).

[0058] 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 CRM 197.

Example 6: Induction of immune response by CPS conjugates.

Induction of immune response against HS1, HS1/HS44 and HS44 [0059] In one embodiment an immunogenic composition, useful for inclusion in a vaccine composition against HS1, HS1/HS44 and HS44 C. jejuni strains, comprises a polysaccharide, comprising the structure:

[—»4)-a-D-Gal/?-( 1 -*2)-Gro-( 1 -> P-^]_n3 2 ΐ ί 1 1 [MeOPN]->3)-Fru/ Fru/-(3«—[MeOPN],

2018204255 14 Jun 2018 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 “[MeOPN]—>3)-Fruf‘‘ unit. [0060] Surprisingly, the above structure found in HS1 and HS1/HS44 strains induces an immune response against HS44 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 pg of vaccine (by weight) per dose had a significantly higher endpoint titer (P<0.05) than mice receiving 10 pg per dose. These results illustrate that HS1 is capable of generating high levels of anti-CPS antibodies in mice. The results of these studies is illustrated in FIG.

18. Also, shown in FIG. 19, a dot blot demonstrating immunogenicity of an HS1CRM197 vaccine. Purified capsules (1 mg/ml) were dot blotted in triplicate (2 ul each) to nitrocellulose and immunodetected with rabbit polyclonal antiserum to an HS1-CRM197 vaccine. HS1, wildtype HS1 capsule; HS1.08, capsule from a fructose transferase mutant of HS1 that lacks the fructose branch and the MeOPN; HS23/36, capsule from 81-176 which expresses a heterologous capsule (HS23/36).

Induction of an immune response using an HS-5 polysaccharide composition [0061] The ability of isolated HS5 polysacccharide to induce an immune response was evaluated. It is contemplated that isolated HS5 polysaccharide could be used conjugated

2018204255 14 Jun 2018 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 CRM197 predicated on the method in Example 3. BALB/c mice were given three doses each of 10 pg or 50 pg of HS5 polysaccharide-conjugate at 4 week intervals, with 200 pg of ALHYDROGEL® (Brenntag AG, Germany). The mice received a total of three 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 BALB/c mice were immunized via subcutaneous injection with conjugate vaccine (HS3 from BH0142 conjugated to CRM197) in aluminum hydroxide 3 times at 4-week intervals.

Vaccine was given by weight. A dose of 5 pg corresponded to approximately 0.5 pg of conjugated polysaccharide and a dose of 25 pg 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 immunoblotted with rabbit polyclonal antisera made against formalin killed whole cells of members of the HS4 complex. HS4 anti-serum was found to cross react to HS13 and

HS4. Anti-HS4/13/64 serum was found to crossreact with HS64 and HS4 and to a small extent to HS50.

2018204255 14 Jun 2018 [0065] In similar studies, rabbit anti-HS13 serum was found to cross react with HS4 and

HS13 and anti-HS64 serum was found to cross react with HS4, HS13, HS4/13/64 and

HS50. Similarly, rabbit polyclonal antiserum made to conjugate vaccine composed of the capsule of HS4/13/64 strain conjugated to CRM197 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 crossreacted to HS4 and HS64, 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 1 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 HS1 and HS1 complex (HS1, HS1/HS44 or HS44), HS2, HS3, HS4, HS5, HS13, 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

2018204255 14 Jun 2018 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 pg 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 HS1, HS1/HS44 or HS44. In the inventive method, the composition is used to induce an immune response against HS1, 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 autoimmune responses such as Guillain-Barre syndrome, such as derived from HS1, are used to induce an immune response against HS1, HS1/HS44 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 HS4, HS13, HS4/HS13/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, HS4/HS13/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 CRM 197.

[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: HS1 and HS1 complex (HS1, HS1/HS44 or

HS44), HS2, HS3, HS4, HS5, HS13, HS4/13/64, and HS50, wherein capsule

2018204255 14 Jun 2018 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 pg 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:

[MeOPN]”· a-Did|oxy-Hep

6

-»7)a-DD-Hep( 1 —»3)Glucitol(6 —* P —»

2

1 a-Dideoxy-Hep a-Dideoxy-Hep

7

T T [MeOPN]”· [MeOPN]”[MeOPN]”^- !

a-Did^oxy-Hep ► 7)a-DD-Hep(l -> 2)Glucitol(6 2 T 1 a-Dideoxy-Hep

T [MeOPN]”^{1- 2}

2018204255 14 Jun 2018 —* 7)a-DD-Hep( 1 —»2)Glucitol(6 —t-P -+

T a-Dideoxy-Hep

T [MeOPN]’ 7)a-DD-Hep(l — 3)Glucitoi(6 ->P a-Dideoxy-Hep a-Dideoxy-Hep

T [MeOPN]’

T [MeOPN]' [—»4)-a-D-Gal/?-(l -^2)-Gro-( 1 jejuni strain HS44;

>P—>]_n, derived from the derived from the C.

[—>4)-a-D-Gal/?-(l —>2)-Gro-(l -+ P^] _n

2

T T 1 1 [MeOPN]—>3)-Fru/~ Fru/-(3<—[MeOPN], derived from the C. jejuni strain HS1 and/or

HS1/44;

[—>3)-L-P-D-ido-Hep-(l->4)-P-D-GlcNAc-(l—>]_n, derived from

HS4/HS13/HS64, with non-stoichiometric substitution of O-methyl-phosphoramidate at position 2 of F-Dido-heptose;

[—>3)-6d-3-D-ido-Hep-(l—>4)-3-D-GlcNAc-(l^>]n, derived from HS4/13/64, with non-stoichiometric MeOPN at C-2 and/or C-7 of 6d-ido-Hep;

2018204255 14 Jun 2018 [-G)-L-p-D-ido-Hep-(l—>4)-P-D-GlcNAc-(l—>]_n, derived from HS4, with nonstoichiometric MeOPN at C-4 of LD-ido-Hep;

[—»3-6d-P-D-ido-Hep-(l—>4)-P-D-Glc-(l—»]_n, derived from HS13, without MeOPN or with non-stoichiometric MeOPN at C-2 and/or C-7 of 6d-ido-Hep;

[MeOPN] (3,6,-O-Me)-D-glycero-a-L-glc-Hep/?

[->2)-P-D-Rib/-( I -»5)-P-D-Gal/NAc-( 1 -^4-a-D-Glc/?A6-( 1 -+]_n3 5

T ΐ [MeOPN] [NGroNEtn], derived from HS2, whererin NGro = aminoglycerol; Etn = ethanolamine;

[—>3)-L-alpha-D-ido-Hep-(l->4)-alpha-Gal-(l-^]_n, derived from HS3, with non-stoichiomoetric substitution O-methyl-phosphoramidate at position 2 of 6deoxy-alpha-D-ido-heptose with or without a 3-hdroxypropanoyl ester at C-3 of ot-Gal;

[—»3)-L-P-D-ido-Hep-(l—>4)-P-D-Glc-(l-->]n, derived from HS50, with nonstoichiometric MeOPN at C-4 of LD-ido-Hep;

and

2018204255 14 Jun 2018 [—>3-6d-P-D-ido-Hep-(l—>4)-P-D-Glc-(l—+]_n, derived from HS50, with nonstoichiometric MeOPN at C-7 of 6d-ido-Hep, wherein the same polysaccharide is linked to form a polysaccharide polymer comprising 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 0.1 pg to 10 mg per dose.

[0072] Another embodiment comprises a method of immunizing against Campylobacter jejuni strains HS1; HS1/HS44 and/or HS44 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, HS1/HS44, 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 pg 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:

2018204255 14 Jun 2018 [—>4)-a-D-Gal/7-(l—>2)-Gro-(l—»P—*]n, derived from the derived from the C. jejuni strain HS44; or [—>4)-a-D-Gal/?-(1 -»2)-Gro-( 1 -*Ρ-*]η 3 2 ΐ ΐ 1 1 [MeOPN]—Gj-Fru/’ Fru/-(3<—[MeOPN], derived from the C. jejuni strain HS1 and/or

HS1/44;

wherein the same polysaccharide is linked to form a polysaccharide polymer comprising to 100 polysaccharides linked together (i.e., “n” greater than or equal to 1);

[0073] Another embodiment comprises a method of immunizing against Campylobacter jejuni strains HS4, 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, HS4/HS13/H64 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 pg to 10 mg per dose

2018204255 14 Jun 2018 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-Hep-(l->4)-p-D-GlcNAc-(l—>]_n, derived from HS4/HS13/HS64, with non-stoichiometric substitution of O-methyl-phosphoramidate at position 2 of L-D-ido-heptose;

[—>3)-6d-P-D-ido-Hep-(l—>4)-P-D-GlcNAc-(l—>]_n, derived from HS4/13/64, with non-stoichiometric MeOPN at C-2 and/or C-7 of 6d-ido-Hep;

[—>3)-L-P-D-ido-Hep-(l—>4)-P-D-GlcNAc-(l—>]_n, derived from HS4, with nonstoichiometric MeOPN at C-4 of LD-ido-Hep;

[—»3-6d-P-D-ido-Hep-(l—>4)-P-D-Glc-(l—*]_n, derived from HS13, without MeOPN or with non-stoichiometric MeOPN at C-2 and/or C-7 of 6d-ido-Hep;

[—>3)-L-P-D-ido-Hep-(l—->4)-P-D-Glc-(l —>]_n, derived from HS50, with nonstoichiometric MeOPN at C-4 of LD-ido-Hep; and [—>3-6d-P-D-ido-Hep-(l—+4)-P-D-Glc-(l—>]_n, derived from HS50, with nonstoichiometric MeOPN at C-7 of 6d-ido-Hep,

2018204255 14 Jun 2018 wherein the same polysaccharide is linked to form a polysaccharide polymer comprising to 100 polysaccharides linked together (i.e., “n” greater than or equal to 1);

[0074] The polysaccharide polymers can be conjugated or unconjugated to a carrier molecule and the composition. In the above method, immunogenic composition can be administered orally, nasally, subcutaneously, intradermally, transdermally, transcutaneously, intramuscularly or rectally. Also, the carrier molecule can be a protein, for example CRM197, 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, olgodeoxynucleotides (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.

2018204255 14 Jun 2018

Claims

Claims:

1. A method of inducing an immune response in a mammal against a Campylobacter jejuni strain of an HS4 complex comprising administering to said mammal an immunogenic composition comprising one or more isolated capsule polysaccharides or polysaccharide polymers derived from Campylobacter jejuni strain HS:50; wherein said immunogenic composition does not contain Campylobacter jejuni lipooligosaccharide structures associated with GuillainBarre Syndrome.
2. A method of inducing an immune response in a mammal against Campylobacter jejuni strain HS:50 comprising administering to said mammal an immunogenic composition comprising one or more isolated capsule polysaccharides or polysaccharide polymers derived from one or more Campylobacter jejuni strains of an HS4 complex; wherein said immunogenic composition does not contain Campylobacter jejuni lipooligosaccharide structures associated with GuillainBarre Syndrome.
3. A method of inducing an immune response in a mammal against a Campylobacter jejuni strain comprising administering to said mammal an immunogenic composition comprising one or more isolated capsule polysaccharides or polysaccharide polymers derived from one or more Campylobacter jejuni strains, wherein said Campylobacter jejuni strain is selected from the group consisting of HS4, HS4/13/64, HS5, and HS50, wherein said immunogenic composition does not contain Campylobacter jejuni lipooligosaccharide structures associated with Guillain-Barre Syndrome, wherein the structure of HS4 is [—>3)-L-P-D-ido-Hep(1—>4)-P-D-GlcNAc-(l—»]_n, with non-stoichiometric MeOPN at C-4 of LD-idoHep, wherein the structure of HS4/13/64 is [-^3)-6d-P-D-ido-Hep-(l—»4)-β-ϋGlcNAc-(l—*]_n, with non-stoichiometric MeOPN at C-2 and/or C-7 of 6d-idoHep, and wherein “n” is greater than or equal to 1.
4. The method of claim 1 wherein the isolated capsule polysaccharides or polysaccharide polymers derived from Campylobacter jejuni strain HS:50 is

2018204255 14 Jun 2018 selected from the group consisting of [—>3)-L-p-D-ido-Hep-(l—>4)-P-D-Glc(I—>]_n, with non-stoichiometric MeOPN at C-4 of LD-ido-Hep, and [—>3-(κΙ-β-ϋido-Hep-(l—>4)-P-D-Glc-(l—»]n, with non-stoichiometric MeOPN at C-7 of 6dido-Hep, and wherein “n” is greater than or equal to 1.
5. The method of any one of claims 1 to 3 wherein the isolated capsule polysaccharides or polysaccharide polymers are linked to a carrier molecule to improve immunity.
6. The method of claim 5 wherein the carrier molecule is a protein carrier.
7. The method of claim 6 wherein the protein carrier is CRM197.
8. The method of any one of claims 1 to 3 wherein said method comprises

a. administering the immunogenic composition to said mammal at a dose range of 0.1 pg to 10 mg per dose; and

b. administering a boosting dose of said immunogenic composition to said mammal at a dose range of 0.1 pg to 10 mg per dose.
9. The method of any one of claims 1 to 3 wherein said method comprises administering 3 doses of the immunogenic composition to said mammal at a dose range of 0.1 pg to 10 mg per dose.
10. The method of any one of claims 1 to 3 wherein the immunogenic composition comprises an adjuvant.
11. The method of claim 10, wherein the adjuvant is selected from the group consisting of LTR192G, aluminum hydroxide, RC529, QS21, oligodeoxynucleotides (ODN), and aluminum phosphate.
12. The method of claim 11 wherein the oligodeoxynucleotides (ODN) is a CpGcontaining oligodeoxynucleotide.
13. The method of any one of claims 1 to 3 wherein the immunogenic composition is administered by a route selected from the group consisting of orally, nasally,

2018204255 14 Jun 2018 subcutaneously, intradermally, transdermally, transcutaneously, intramuscularly and rectally.
14. Use of one or more isolated capsule polysaccharides or polysaccharide polymers derived from Campylobacter jejuni strain HS:50 for the manufacture of a medicament for inducing an immune response against a Campylobacter jejuni strain of an HS4 complex in a mammal in need thereof, wherein said medicament does not contain Campylobacter jejuni lipooligosaccharide structures associated with Guillain-Barre Syndrome.
15. Use of one or more isolated capsule polysaccharides or polysaccharide polymers derived from one or more Campylobacter jejuni strains of an HS4 complex for the manufacture of a medicament for inducing an immune response against Campylobacter jejuni strain HS:50 in a mammal in need thereof, wherein said medicament does not contain Campylobacter jejuni lipooligosaccharide structures associated with Guillain-Barre Syndrome.
16. Use of one or more isolated capsule polysaccharides or polysaccharide polymers derived from one or more Campylobacter jejuni strains for the manufacture of a medicament for inducing an immune response against a Campylobacter jejuni strain in a mammal in need thereof, wherein said Campylobacter jejuni strain is selected from the group consisting of HS4, HS4/13/64, HS5, and HS50, wherein said immunogenic composition does not contain Campylobacter jejuni lipooligosaccharide structures associated with Guillain-Barre Syndrome, wherein the structure of HS4 is [—>3)-L-P-D-ido-Hep-(l—->4)-P-D-GlcNAc-(l—>]_n, with non-stoichiometric MeOPN at C-4 of LD-ido-Hep, wherein the structure of HS4/13/64 is [—>3)-6d-P-D-ido-Hep-(l —>4)-P-D-GlcNAc-(l—>]_n, with nonstoichiometric MeOPN at C-2 and/or C-7 of 6d-ido-Hep, and wherein “n” is greater than or equal to 1.
17. The use of claim 14 wherein the isolated capsule polysaccharides or polysaccharide polymers derived from Campylobacter jejuni strain HS:50 is selected from the group consisting of [—>3)-L-P-D-ido-Hep-(l—>4)-P-D-Glc47

2018204255 14 Jun 2018 (1—*]_π, with non-stoichiometric MeOPN at C-4 of LD-ido-Hep, and [—+3-6ά-β-ϋido-Hep-(l-^4)-P-D-Glc-(l—>]_n, with non-stoichiometric MeOPN at C-7 of 6dido-Hep, and wherein “n” is greater than or equal to 1.
18. The use of any one of claims 14 to 16 wherein the isolated capsule polysaccharides or polysaccharide polymers are linked to a carrier molecule to improve immunity.
19. The use of claim 18 wherein the carrier molecule is a protein carrier.

20. The use of claim 19 wherein the protein carrier is CRM197.

The United States of America as represented by the Secretary of the Navy Patent Attorneys for the Applicant/Nominated Person SPRUSON & FERGUSON

2018204255 14 Jun 2018

FIG. 1

1/21

2018204255 14 Jun 2018

J-CXFIG.

2/21

2018204255 14 Jun 2018

FIG. 3

3/21

2018204255 14 Jun 2018

FIG, 4

4/21

2018204255 14 Jun 2018

FIG. 5

5/21

2018204255 14 Jun 2018

100 90 80-y 8 © ά 70 ] 60 fcos ; ·ί o : ss © Φ 3»? 8 T3 ; S u ; ;i 3 ](5 © ε 5 Ω O .Ω < ds S 1 50 © Ίδ? > T Ω Tb 40 © 30 9 20/] 10;:; |

30 35 40 45 50

Time (min)

FIG. 6

6/21

2018204255 14 Jun 2018

FIG. 7

7/21

2018204255 14 Jun 2018

FIG. 8

8/21

2018204255 14 Jun 2018

FIG. 9

9/21

2018204255 14 Jun 2018

FRI 10

10/21

2018204255 14 Jun 2018

I [MeOPN] [MeOPN)’- j 1 x 7 i a-Dsd^oxy-Hep κ 7 a-Did^oxy-Hep i A δ V i i -» 7)a-DD-Hepil --- 3)Glucitol{6— P — ! ' 7 ' 2 5 4 Λ 5 ! : s 6 x iii ····» 7)a-DD-Hep( 1. — 2)Ghicitol(S — * P --<> 2 -X ί ! ί ί ί ct-Dsdeoxy-He» a-ESdeoxy-He» ί 7 x ' 7 ί. ' «-Didsoxy-Hep 7 X ί [MeOPN]''· [MeOPN]- pvleOPKT' ί U — 7)a~DD«Hep(l — 2)Glucs»oi(6 -»2» — ! 7 iv 7)a-DD~Hepa -> 3)Gl»ciloS(6 ~».P -» 2 2 T * ί ί a-Dideoxy-Hep ί 7 χ 1 i a-Dideoxy-Hep a-Dideoxy-Hep 7 x 7 l i [MeOPN]' (MeOPN} [MeOPNj- -

FIG. 11

11/21

2018204255 14 Jun 2018

100-.

95/

80-/

80/

75/

707 c 80 .:

CO ·!

c 55/ I 50 3

1 453 co ··:

ffl 40-3-: & .-:

30/

25-:/

20/

15/

10/

5-/

G32

38 38 40

42 44 46

Tims (min)

FIG, 12

50 52 54

12/21

2018204255 14 Jun 2018 fid 13

13/21

2018204255 14 Jun 2018

25010075-

28 15 10 S 0-5 ppm

FIG. 14

14/21

2018204255 14 Jun 2018

5,0 4,5 4,«3 3,5 3.S 2.5

24 23 22 23 20 39 18 37 38 35 U 33 « 33 30 8 8 ? ppm

FIG, 15

15/21

2018204255 14 Jun 2018

FIG. 16

16/21

2018204255 14 Jun 2018

Ή

FIG. 17

17/21

2018204255 14 Jun 2018 p <0.05

10 yg 50yg

2»wk 3rd immuns&tbn

HS1 HS1 kpsM HS44 HS1 1,08

FIG. 18

18/21

2018204255 14 Jun 2018

HSl

HS1.08

HS23/36

FTC. 19

19/21

2018204255 14 Jun 2018 ·&««:

2wk post 3rd imffl orto&o

FIG. 20

20/21

2018204255 14 Jun 2018

PBS 5 ug 25 μ

2wfe post 3rd

FIG, 21

21/21