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WO2005017129A1 - Phosphoramide et utilisations associees - Google Patents

Phosphoramide et utilisations associees Download PDF

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WO2005017129A1
WO2005017129A1 PCT/CA2004/001489 CA2004001489W WO2005017129A1 WO 2005017129 A1 WO2005017129 A1 WO 2005017129A1 CA 2004001489 W CA2004001489 W CA 2004001489W WO 2005017129 A1 WO2005017129 A1 WO 2005017129A1
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cps
phosphoramide
jejuni
genes
sugar
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WO2005017129B1 (fr
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Jean-Robert Brisson
Harold Jarrell
Christine M. Szymanski
Evgeny Vinogradov
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National Research Council of Canada
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/664—Amides of phosphorus acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/105—Delta proteobacteriales, e.g. Lawsonia; Epsilon proteobacteriales, e.g. campylobacter, helicobacter
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/28—Phosphorus compounds with one or more P—C bonds
    • C07F9/38—Phosphonic acids [RP(=O)(OH)2]; Thiophosphonic acids ; [RP(=X1)(X2H)2(X1, X2 are each independently O, S or Se)]
    • C07F9/44—Amides thereof
    • C07F9/4403—Amides thereof the acid moiety containing a substituent or a structure which is considered as characteristic
    • C07F9/4407—Amides of acyclic saturated acids which can have further substituents on alkyl
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/28—Phosphorus compounds with one or more P—C bonds
    • C07F9/38—Phosphonic acids [RP(=O)(OH)2]; Thiophosphonic acids ; [RP(=X1)(X2H)2(X1, X2 are each independently O, S or Se)]
    • C07F9/44—Amides thereof
    • C07F9/4434—Amides thereof the ester moiety containing a substituent or a structure which is considered as characteristic
    • C07F9/4438—Ester with hydroxyalkyl compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56922—Campylobacter
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to cell surface structures useful in the identification and targeting of C. jejuni.
  • BACKGROUND Campylobacter jejuni is the major bacterial cause of gastrointestinal disease in developed countries and infection can lead to the development of the neuropathy known as Guillain-Barre syndrome.
  • Carbohydrates are implicated in a variety of functions in all domains of life. There continues to be a growing demand for methodologies that can analyze carbohydrate structures with increasing levels of sensitivity and simplicity. Genome sequencing of C. jejuni NCTC11168 demonstrated that the strain contained four gene clusters necessary for carbohydrate biosynthesis.
  • the flagellar modification locus adjacent to the flagellin structural genes flaA and flaB, encodes enzymes involved in the biosynthesis of 0-linked pseudaminic acid and its derivatives.
  • the LOS and adjacent protein glycosylation loci encode enzymes involved in the formation of outer core ganglioside mimics and bacillosamine- containing N-linked heptasaccharide, respectively. While the capsular biosynthesis locus, containing a Kps transport system similar to that found in other encapsulated organisms, transfers a branched tetrasaccharide repeat to the outer membrane surface.
  • the CPS product of this locus has been demonstrated to be the major serodeterminant in the heat stabile typing scheme first described by Penner and Hennessy. However, an inconsistency in the literature developed when only a limited number of C.
  • jejuni serotypes were believed to produce capsules based on detection by immunoblotting yet, all strains examined contained ⁇ s genes necessary for capsule transport. It was then shown in C. jejuni 81-176 that the high molecular weight CPS was antigenically variable but it remained to be determined whether the loss in CPS reactivity was due to the lack of CPS production or changes in its structure. Since capsular polysaccharides are the outermost structure on the bacterial cell they play an important role in the interaction between the pathogen, host, and environment. In C. jejuni 81-176 the capsule is involved in I ⁇ T407 cell invasion, virulence in ferrets, serum resistance and maintenance of bacterial cell surface hydrophilicity. The C.
  • jejuni pgl locus encodes enzymes necessary for the glycosylation of multiple proteins and disruption of this pathway by mutagenesis results in multiple pleiotrophic effects.
  • the structure of the N-linked glycan is Gal ⁇ Ac- ⁇ l,4-Gal ⁇ Ac- ⁇ l,4-[Glc- ⁇ l,3-]GalNAc- ⁇ l,4-GalNAc- ⁇ l,4-GalNAc- ⁇ l,3-Bac.
  • C. jejuni LOS have received much attention due to their unique mimicry of human ganglioside structures and their potential involvement in the induction of the autoimmune polyneuropathies, Guillain-Barre (GBS) and Miller Fisher syndromes.
  • C. jejuni LOS have also recently been shown to be phase variable and important in virulence.
  • Capsular polysaccharides are found on the surface of a large number of bacterial species. CPSs are known to play an important role in bacterial survival and persistence in the environment and often contribute to pathogenesis.
  • bacterial CPSs play a role in evasion of host immune responses. Assembly of these surface polysaccharides is remarkably conserved in bacteria. Nucleotide diphosphate sugars are synthesized in the cytoplasm and sequentially added by glycosyltransferases to an undecaprenyl pyrophosphate carrier anchored in the membrane. Many Gram-negative bacteria flip the assembled polysaccharide across the membrane using an ABC transporter consisting of the transmembrane channel, KpsM, and the ATPase, KpsT.
  • jejuni 81-176 confirmed these findings and demonstrated a role for the capsule in serum resistance, epithelial cell invasion and diarrhoeal disease. Subsequent characterisation of the CPSs by Alcian blue staining led to the visualisation of capsule by electron microscopy. These experiments suggested that the previously described high molecular weight "lipopolysaccharides” (HMW LPSs) of C. jejuni are in fact CPSs.
  • HMW LPSs high molecular weight "lipopolysaccharides”
  • NCTC11168 was determined to contain 6-O- methyl-D- fycero- ⁇ -L-g/wco-heptose, ⁇ -D-glucuronic acid modified with 2-amino ⁇ 2- deoxyglycerol, ⁇ -D-Gal NAc and ⁇ -D-ribose.
  • NCTC11168 There are several notable features encoded by the cps locus of NCTC11168 that correlate well with the published structure: homologues of the GDP-D-gfycero-D-mannoheptose pathway (GrnhA2, HddA and HddC); hornologue of the UDP-glucose dehydrogenase, Udg, involved in the formation of UDP-glucuronic acid; and a hornologue of the UDP-pyranose mutase, Glf, predicted to catalyse the reversible conversion of pyranoses to furanoses and shown to cause loss of CPS when mutated in NCTC11168 ⁇ St Michael, 2002 #212 ⁇ .
  • CPS capsular polysaccharide
  • HR-MAS NMR High resolution magic angle spinning
  • jejuni NCTC11168 followed by phosphoramide filter analysis has allowed identification of multiple genes encoding enzymes involved in the biosynthesis of phosphoramide: cjl416c, cj 1417c, cj 1418c and cj 1421c (and potentially the duplicated gene, cj 1422c). All or most of these genes are missing in other Campylobacter species and genome sequenced strains belonging to the epsilon proteobacteria confirming their inability to synthesize phosphoramide. Preliminary examination of the C. jejuni mutants in human cell culture assays has demonstrated that the phosphoramide is required for efficient adherence but is not necessary for cell invasion. Furthermore, the expression of this modifications renders the bacteria more sensitive to human sera.
  • the phosphoramide further includes an alkyl group attached to the O which is attached to the P of the methyl amidophosphate group, wherein the alkyl group is a sugar producable in Campylobacter jejuni.
  • the sugzar may be a naturally occurring cam, an enantiomer, or other variant, or a non-natuarally- occurring sugar.
  • alkyl methyl amidophosphate or an immunologically active derivative thereof in the identification of Campylobacter jejuni, wherein the alkyl group is a sugar producable in Campylobacter jejuni.
  • the phosphoramides discussed above may be used as vaccines in mammal to compate or reduce the severity of C. jejuni infection.
  • a method of modulating the adhesion of C. jejuni cells to a surface comprising modulating the concentration of binders in surrounding fluid.
  • a substantially pure pharmaceutical composition comprising one or more the phosphoramide described herein and a physiologically acceptable carrier.
  • compositions of interest include those containing immunogenic conjugates and/or immunostimulants capable of enhancing immune response in a mammal (by way of non-limiting example, E. coli labile toxin).
  • immunogenic conjugates and/or immunostimulants capable of enhancing immune response in a mammal (by way of non-limiting example, E. coli labile toxin).
  • at least one of the phosphoramides described above which is linked through the O which is attached to the P of the methyl amidophosphate group, to an amino acid or alkyl group wherein the alkyl group is a sugar producable in Campylobacter jejuni and the amino acid is an amino acid producablein Campylobacter jejuni.
  • a kit comprising: a) the binder as described herein; and b) instructions for carrying out the method of claim 1 or 13.
  • nucleic acid encoding Campylobacter jejuni Cj 1421 c or Cj 1422c or a portion or variant thereof in producing a polypeptide sequence having wild-type transferase activity in producing an amino acid sequence useful in producing non-natually occurring antigenic compounds comprising the phosphoramide of claim 16 or 17.
  • nucleic acid sequence encodes an amino acid sequence at least 90% identical to the wild type Cj 1421c or Cj 1422c sequence.
  • an amino acid sequence encoding Campylobacter jejuni Cj 1421c or Cj 1422c or a portion or variant thereof having wild-type transferase activity in producing an amino acid sequence useful in producing non-natually occurring antigenic compounds comprising the phosphoramide described herein.
  • the amino acid sequence encodes a variant at least 90% identical to the wild type Cj 1421c or Cj 1422c sequence.
  • the wild type sequence of Cj 1421c or Cj 1422c can be readily determined by reference to published sequences.
  • FIGURE 1 Is a depiction of Proton NMR spectra of NCTC11168 and HS:2 serostrain.
  • HR-MAS proton NMR spectra with 10 ms CPMG filter of NCTC11168 (b) whole cells, (c) 1/100 dilution of whole cells, and (d) HS:2 serostrain.
  • the Asp (aspartic acid) resonances are labeled in (d).
  • the HOD resonance at 4.8 ppm was saturated and digitally filtered affecting the intensity of the anomeric resonance C in b) and c).
  • FIGURE 2 Is a depiction of comparison of individual colonies of NCTC11168.
  • FIGURE 3 Is a depiction of NMR experiments for the C jejuni NCTC11168 variant 2 CPS.
  • the structure of the CPS is shown above the spectra, (a) 1H spectrum of the purified CPS.
  • FIGURE 4 Is a depiction of MS analysis for the C jejuni NCTC11168 variant
  • FIGURE 5 Is a depiction of strategy for amplification of the cps regions.
  • the primers corresponding to the conserved cps genes (shown in open arrows) were used in combination with primers derived from conserved regions of kps genes (thick solid arrows) for long-range PCR as described in the section Experimental Procedures.
  • FIGURE 6 Is a depiction of graphical representation of the sequenced CPS biosynthethic regions.
  • the genes were given names of counterparts found in other bacteria. When no such similarity was found, the genes were assigned the names of respective genes from strain NCTC11168. The genes with no similarity to either NCTC11168 or other bacteria are given strain-specific systematic names.
  • the cps clusters of serostrains HS:23 and HS:36 are almost identical to that of strain 81-176 and are not shown (see text).
  • FIGURE 7 Is a depiction of summary of the C. jejuni capsular polysaccharide structures described in this study.
  • the CPS structures of the heat-stable (HS) Penner type strains HS:1, HS:19, HS:23 and HS:36 have been reviewed by Moran et al. ⁇ Moran, 2000 #217 ⁇ .
  • the structures of NCTC11168 CPS ⁇ St Michael, 2002 #212 ⁇ and HS:41 CPS ⁇ Hanniffy, 1999 #175 ⁇ have recently been described. Sugars are shown in pyranose configurations unless otherwise noted.
  • P phosphate
  • Gal galactose
  • Gro glycerol
  • Me methyl
  • Hep heptose
  • Rib ribose
  • GalNAc N- acetylgalactosamine
  • GlcA6 glucuronic acid
  • ⁇ Gro aminoglycerol
  • Glc ⁇ Ac N- acetylglucosamine
  • Ara arabinose
  • Alt altrose
  • Fuc fucose.
  • FIGURE 8 Is a depiction of proton ⁇ MR spectra of C. jejuni strains ⁇ CTC 12500 (HS: 1 serostrain) and Gl (HS: 1). a) HR-MAS spectrum of ⁇ CTC12500 (HS: 1) whole cells at 21°C. b) HR-MAS spectrum of Gl whole cells at 21°C. c) NMR spectrum of partially purified Gl CPS at 40°C with acetone as the internal reference. The CPS anomeric resonance corresponding to Gal and the methyl resonance from the common phosphoramide are labeled.
  • FIGURE 9 Is a depiction of proton NMR spectra of C. jejuni strains CCUG 10954 (HS:23 serostrain), ATCC 43456 (HS:36 serostrain) and 81-176 (HS:23/HS:36).
  • HR-MAS spectra were acquired at 21°C.
  • NMR spectra were acquired at 40°C.
  • acetone was used as the internal reference.
  • HMQC spectra the anomeric region is shown.
  • TOCSY the mixing time was 90ms. Crosspeaks between signals in the anomeric region (4.7 to 5.5 ppm) and the sugar ring region (3.4 to 4.4 ppm) are shown.
  • FIGURE 10 Is a depiction of comparisons of the cps clusters of 81-176 (top) with NCTC11168 (middle) and NCTC 12517 (bottom). Each of the genes are shown as boxes.
  • FIGURE 11 Is a depiction of regions of significant homology between the capsule locus (Cjl413c to Cjl448c) in C. jejuni NCTC11168 and C. jejuni RM1221, Helicobacter hepaticus ATCC51449, Helicobacter mustelae ATCC43772, and Wolinella succinogenes DMSZ 1740. No significant homology was seen with Helicobacter pylori strains 26695 or J99. Note that the genome sequences of C. jejuni RM1221 and H mustelae ATCC43772 are incomplete.
  • FIGURE 12 3 IP ⁇ MQC NMR analysis of purified capsule isolated from C. jejuni ⁇ S:1.
  • the signal at 3.81 ppm (arrow) is characteristic of a phosphoramide modification found on the capsule and originates from the CH 3 methyl group of the phosphoramide.
  • the signal at 4.84 ppm (arrow) is indicative of the capsular sugar to which the phosphoramide is attached. This sugar was not described in previous structural studies of HS:1 and is currently under investigation.
  • FIGURE 13 3 IP HMQC NMR analysis of purified capsule isolated from C. jejuni HS:19.
  • the signal at 3.76 ppm (arrow) is characteristic of a phosphoramide modification found on the capsule and originates from the CH 3 methyl group of the phosphoramide.
  • the signal at 4.26 ppm (arrow) is indicative of the capsular sugar to which the phosphoramide is attached (position 4 of ⁇ -GlcNAc).
  • FIGURE 14 Whole cell 31 P-1H filtered 1H HR-MAS NMR spectra of several strains of C. jejuni. The number of peaks represent the number of phosphoramide residues in different chemical( structural) environments. The amplitude of the peaks reflects the relative amounts of each residue.
  • FIGURE 15 Adherence and invasion results for phosphoramide mutants C. jejuni 1416-1, 1417-1, 1418-3 and their parent strain, NCTC11168H (UK-H) as well as the phosphoramide mutant C. jejuni 1421-3 and its parent strain, Variant 4 (V4).
  • Efficiency is defined as the number of bacteria that either adhered or invaded CaCo-2 cells divided by the total number of bacteria added and expressed as a percentage. Results are presented as the means of at least three experiments ⁇ the standard error of the mean.
  • FIGURE 16 Results of motility assays for phosphoramide mutants C. jejuni 1416-1, 1417-1, 1418-3 and their parent strain, NCTC11168H (UK-H) as well as the phosphoramide mutant C. jejuni 1421-3 and its parent strain, Variant 4 (V4). Results are presented as the mean of at least two experiments ⁇ the standard deviations.
  • FIGURE 17 Results of two serum sensitivity assays for the phosphoramide mutant C. jejuni 1416-1 and the parent strain, NCTC11168 (UK-H). Bars in grey represent colony counts without serum while bars in white represent counts in the presence of 100 ⁇ l of serum.
  • Bacterial strains and growth conditions - Campylobacter jejuni NCTC11168 (HS:2) was isolated from a case of human enteritis and later sequenced by Parkhill et al. C. jejuni serosrrains: HS:1 (ATCC 43429), HS:2 (ATCC 43430), HS:3 (ATCC 43431), HS:4 (ATCC 43432), HS:10 (ATCC 43438), HS:19 (ATCC 43446), HS:36 (ATCC 43456) and HS:41 (ATCC 43460) were obtained from ATCC; C.
  • jejuni HS:23 was obtained from Dr. Peggy Godschalk, Erasmus University Medical Center, Rotterdam; C. jejuni OH4382 and OH4384 were obtained from Health Canada; and C. coli HS:30 (NCTC 12532) was obtained from NCTC. All campylobacter strains were routinely grown on Mueller Hinton agar (Difco) under microaerophilic conditions at 37°C. C. jejuni NCTC11168 mutants were grown on Mueller Hinton agar with 30 ⁇ g/mL kanamycin. Spectroscopy - All CE-ESI-MS and CE-ESI-MS/MS experiments and structural analysis of the purified CPS by NMR were performed substantially as described (in St. Michael et al.
  • the total duration of the CPMG pulse (n*2 ⁇ ) was 10 ms with ⁇ set to (1/MAS spin rate).
  • One-dimensional selective TOCSY experiments with various spin-lock times from 30-150 ms and selective NOESY with mixing times from 100-400 ms were performed substantially as described in Uhrin and Brisson (2000) in NMR in Microbiology, p.165-210 Horizon Science Press, UK, and in Brisson et.al. (2002) in NMR spectroscopy of glycoconjugates ⁇ .59-93, Wiley-BCH, Weinheim.
  • the TOCSY sequences were modified so that the DIPSI-2 mixing sequence was replaced with the adiabatic WURST-2 pulses.
  • EXP selected spins, selective excitation bandwidth, mixing time
  • EXP is TOCSY or NOESY
  • proton spectra of bacterial cells could be obtained using 256 to 1024 transients (15 min to 1 hour).
  • the time for each TOCSY and ⁇ OESY varied from 1 to 8 hours.
  • Proteinase K treated whole cells of C. jejuni wild type and phase variants were prepared and analyzed by deoxycholate-PAGE substantially as described in St Michael (2002) above.
  • One portion of the gel was silver-stained while the other portion of the gel was transferred to a PVDF membrane (Roche Molecular Biochemicals) and immunodetected with HS:2 antiserum (1:500 dilution).
  • the immunoblot was then incubated with goat-anti-rabbit secondary antibody conjugated to alkaline phosphatase (1:2500 dilution, Sigma) and then developed with the nitro blue tetrazolium chloride / 5-bromo-4-chloro-3-indolyl phosphate detection system (Roche Molecular Biochemicals).
  • C. jejuni strains Bacterial strains and growth conditions - Sequences of cps regions from a wide range of C. jejuni strains have been investigated: ⁇ CTC11168 (HS:2, genome sequenced strain, enteritis isolate), 176.83 (HS:41 serostrain, enteritis isolate,
  • NCTC12517 (HS:19 serostrain, enteritis isolate), Gl (HS:1, GBS isolate), 81-176 (HS:23/36, enteritis isolate used in human challenge studies), CCUG 10954 (HS:23 serostrain, enteritis isolate) and ATCC 43456 (HS:36 serostrain, enteritis isolate).
  • NCTC11168, Gl and serostrain HS:19 Three of the strains examined in this study (NCTC11168, Gl and serostrain HS:19) and the additional strains used for comparative analysis of homopolymeric tracts in the cps region are listed in Table IV.
  • C. jejuni strains were grown in microaerophilic conditions at 37°C on 7% blood agar plates for 2 days. The E.
  • coli XL2 Blue MRF' strain (Stratagene), used in cloning experiments, was grown overnight at 37°C on LB agar plates supplemented with 100 ug/ml ampicillin when necessary. Sequencing of homopolymeric tracts in contingency genes - Genes cjl420 and cjl421 were amplified with primers akl49 (GAGTGCCACTGCTTACACGAGC S ⁇ Q. ID. NO.l) and akl50 (GCTCAACCCAAATTCAGCCATAGAAAG S ⁇ Q. ID. NO.2) and sequenced with primers akl52
  • Genes cjl426 and cjl429 were amplified using primers akl44 (CTCATTCGACCTTTGGAATTGCCTTTG S ⁇ Q. ID. NO.7) and akl45 (CTGTTTCATAATTTCTGTCCGATACTGC S ⁇ Q. ID. NO.8) and sequenced using 1 the same primers.
  • Gene cjl437 was amplified with primers akl54 (CTCCTTATTTATCTATTCCACAC S ⁇ Q. ID.
  • the corresponding primers could be used in combination with the I ⁇ sC and kpsF primers for long-range PCR.
  • Primer pairs specific to the biosynthetic cps genes of strain NCTC11168 are indicated in the supplementary material.
  • the primers designed for the genes found in internal cps regions of various strains were used in combination with the primers corresponding to the conserved flanking kpsC and kpsF genes to generate long PCR products (Fig. 5).
  • the long-range PCR resulted in overlapping products suitable for generation of complete sequences of the internal biosynthetic regions.
  • Long-range PCR was performed using the Expand 20 kb P US PCR System (Roche) using conditions described by the manufacturer.
  • KpsC specific primers akl88 (CCCCTAAAATCATCGAAGCATCATCTTCAACTTGAGC SEQ. ID. NO.16) and akl87 (CATGCTTTAAACCATTATACTTTGAAAAGCGGTTCTCAAG SEQ. ID. NO.17), for serostrain HS:19) and kpsF specific primer akl86 (GAAAAGGAAGCTTGTCCTTTGCAGCTTGC SEQ. ID. NO.18) were used in long-range PCR experiments. The long-range PCR products were treated with polynucleotide kinase, sonicated, and blunt-ended with T4 DNA polymerase.
  • the dried sample was then dissolved in water to a 1 % solution (w/v) and subjected to ultracentrifugation to yield a gel-like pellet containing LOS and supernatant containing the CPS.
  • Analytical methods - Sugars were determined by examining their alditol acetate derivatives by GLC-MS. Samples were hydrolyzed for 4 h using 4 M trifluoroacetic acid at 100°C. The sample was reduced in NaBD 4 overnight in H O and acetylated with acetic anhydride at 100°C for 2 h using residual sodium acetate as the catalyst.
  • the GLC-MS was equipped with a 30 M DB-17 capillary column (180°C to 260°C at 3.5°C/min) and MS was performed in the electron impact mode on a Varian Saturn II mass spectrometer.
  • HR-MAS NMR allows the screening of small amounts of bacterial cells directly without having to purify surface carbohydrates
  • HR-MAS experiments were performed on a Varian Inova 600 MHz spectrometer using a gradient 4 mm indirect detection high-resolution magic angle spinning nano-NMR probe (Varian) with a broadband decoupling coil as previously described ⁇ St Michael, 2002; Young, 2002 ⁇ .
  • Proton spectra of cells were acquired with the Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence ⁇ 90-( ⁇ -180- ⁇ ) n -acquisition ⁇ to remove broad lines arising from lipids and solid-like material.
  • the total duration of the CPMG pulse (n2 ⁇ ) was 10 ms with ⁇ set to (1/spin rate).
  • High-resolution NMR experiments on the partially purified CPS were acquired using a Varian Inova 500 MHz spectrometer equipped with a Z-gradient 3 mm triple resonance (1H, 13 C, 31 P) probe. The experiments were performed at 40°C with suppression of the water resonance.
  • MH Mueller-Hinton
  • strains from each well are diluted to 10 "2 and 10 "3 and plated on dry MH agar plates.
  • the plates are incubated under microaerophilic conditions for 2 days and colony counts are performed.
  • variant 1 predominantly exhibited a resonance at 3.2 ppm consistent with an N-ethanolamine modification on the glucuronic acid (GlcA) (Fig. 2d) in contrast to the major wild type form which exhibited GlcA modified with aminoglycerol (Fig. 2c).
  • Variant 2 showed extremely reduced levels of silver-staining and immunoblotting although the HR-MAS spectra clearly indicated that similar amounts of polysaccharides were present in both variant and wild type samples (Fig. 2e).
  • the HR-MAS spectrum of variant 2 revealed new resonances at 3.75 ppm (Fig. 2e) indicative of a novel modification, which had not been previously observed.
  • the sample also contained about 30% of the major wild-type CPS whose structure is shown in Fig. 1.
  • Proton chemical shifts for residue C for variant 2 were identified using a selective TOCSY experiment (Fig. 3b).
  • the 5D-4C and 3D-4C NOEs were also observed (Fig. 3c), as before for the wild-type CPS.
  • the proton spectrum for variant 2 (Fig.
  • Phosphoramides usually have 31 P signals between 10 and 20 ppm (41-47), which agrees with the position of the 31 P signal within the analyzed structure.
  • CE-MS analysis of C. jejuni NCTCl 1168 CPS phase variant 2 - In order to confirm the structure of the capsular glycan derived from NMR studies, the purified CPS sample was also analyzed by using CE-MS and CE-MS/MS techniques. All the CE-MS and CE-MS/MS experiments were acquired using high orifice voltage. With this experimental setup, the polysaccharide breaks up into shorter oligosaccharide units due to the front-end collision induced dissociation. In this study, a orifice voltage of 200 V was applied and the extracted mass spectrum is shown in Fig. 4a.
  • the ion [M + 1H] 1+ 884 corresponded to the mass of the one repeat unit minus H O.
  • the ions m/z 1181 and 1472 were assigned to one repeat unit plus CE, and to one repeat unit plus BD and A, respectively.
  • cps sequencing results is presented in Table II.
  • a schematic of all the cps loci compared in this study is shown in Fig. 6 with the genes involved in phosphoramide biosynthesis shown in bold (see below).
  • Some of the gene products are involved in the biosynthesis of activated sugars.
  • Such activated sugars contain energy-rich nucleotide-phosphate bonds and serve as substrates for glycosyltransferases involved in the biosynthesis of polysaccharides.
  • nucleotide sugars may be modified by enzymes such as epimerases, dehydratases and reductases before transfer of the final product.
  • Additional modifying enzymes can add groups such as O-methyl, phosphate, ethanolamine- and aminoglycerol- to further increase the complexity of the structures. Indeed, genes encoding these enzymes can be found in various C. jejuni cps regions.
  • the predicted function of cps genes from strain NCTCl 1168 (HS:2) based on the published genome sequence ⁇ Parkhill, 2000 ⁇ and the recently published CPS structure ⁇ St Michael, 2002 ⁇ are presented in Table III.
  • the CPS structures of NCTCl 1168 and the other strains used in this study are shown in Fig.7.
  • NCTCl 1168 There are three notable features encoded by the cps locus of NCTCl 1168: homologues of the GDP-D-g/ycero-D-m ⁇ wnoheptose pathway (HddC, GmhA2 and HddA), the presence of a UDP-glucose dehydrogenase homologue, Udg, responsible for the formation of UDP-glucuronic acid, and a UDP-pyranose mutase homologue, Glf, catalysing the reversible conversion of pyranoses to furanoses. NMR analysis of the HS:19 serostrain used in this study confirmed that the CPS structure was consistent with the published disaccharide repeat (Fig.7, results not shown).
  • the cps region of the HS:19 serostrain did not contain homologues of the heptose pathway but did have the udg homologue (Table IV) correlating well with the presence of ⁇ -D-glucuronic acid which is also amidated with 2-amino-2- deoxyglycerol.
  • NMR analysis also detected two acid-labile functional groups that were not reported previously. Both the phosphoramide modification recently described for NCTCl 1168 and an unknown labile group were observed during the analysis. In contrast to NCTCl 1168 (HS:2) and the HS:19 serostrain, the CPS locus of
  • Gl (HS:1) does not encode a homologue of UDP-glucose 6-dehydrogenase (Table V) and thus the strain should not have the ability to synthesise glucuronic acid.
  • this strain contains a potential tagD homologue encoding a glycerol-3-phosphate cytidylyltransferase necessary for the formation of CDP-glycerol (Table V, Gl.l l).
  • Gl also encodes a TagF homologue, which transfers glycerol-phosphate residues from CDP-glycerol. Therefore, the repeating unit of this CPS may contain glycerophosphate residues.
  • the HS:1 serostrain was reported to contain glycerol- 1 -phosphate residues alternating with galactose in the repeating unit (Fig. 7).
  • the NMR spectra of Gl revealed that the structure of this CPS is consistent with the HS:1 structure.
  • An additional anomeric resonance in the HR-MAS spectrum of Gl was not present in the partially purified CPS sample suggesting that this resonance probably came from the medium used.
  • Extensive NMR analysis by COSY, TOCSY, NOESY and HMQC indicated the presence of only one anomeric resonance consistent with the presence of one sugar in the repeating unit. 31 P NMR experiments indicated the presence of a phosphate diester linkage, also consistent with the reported structure.
  • the CPS loci of the HS:23 and HS:36 serostrains and of strain 81-176 all have exactly the same gene content ( Figure 6 and Table II).
  • the CPSs of HS:23 and HS:36 were found to contain repeating units of a-D-galactose, ⁇ -D-GlcNAc- and D-gfycero-D- ⁇ /tro-heptose or deoxy variants with and without methyl groups (Fig. 9). However, it was reported that the D-g ycero-D- ⁇ /tro-heptose variant was not detected in the HS:23 serostrain.
  • HMQC and TOCSY spectra for the HS:23 serostrain were the simplest with proton anomeric resonances at 5.06 ppm, 4.97 ppm and 4.77 ppm, corresponding to the Gal, Hep and GlcNAc anomeric resonances, respectively.
  • the C-6 crosspeaks of the 6-deoxy-heptose were observed at 34.8 ppm ( 13 C) and 2.06 and 1.71 ppm (1H).
  • HS:36 serostrain three anomeric resonances were also observed as detected by TOCSY and HMQC experiments on the CPS (Fig. 9b).
  • the 1H resonance at 4.92 ppm was confirmed to be a non-anomeric resonance using HMQC. While the anomeric carbon resonances had similar chemical shifts, the proton anomeric resonance of the heptose residue was different, probably due to different structural motifs on the heptose residue.
  • the anomeric resonances at 4.76 ppm and 5.06 ppm exhibited connectivities that were similar to those observed for the HS:23 serostrain, indicating the presence of similar sugars in both serostrains.
  • resonances characteristic of a 6-deoxy-heptose could not be observed, indicating that for this serostrain this modification was not predominant.
  • HMQC and TOCSY spectra for strain 81-176 showed correlation patterns similar to those observed for the HS:23 serostrain for the Gal, Hep and GlcNAc anomeric resonances, again indicating similar sugar structures to those of HS:23 and HS:36. This observation is in agreement with predictions derived from the 81-176 gene analysis. However, structural analysis of strain 81-176 also demonstrated the presence of additional resonances indicating the presence of a more complex repeating unit or the presence of another polysaccharide structure.
  • CPS isolated from the HS:41 serostrain were described to contain ⁇ -L-arabinose , 6-deoxy- ⁇ -D- ⁇ /tr ⁇ heptose , 6-deoxy- ⁇ -L- altrose and ⁇ -D-fucose all in the furanose form (Fig. 7). NMR analysis demonstrated that the CPS of the sequenced strain used in this study is consistent with the published structure.
  • genes cjl431 and cjl440 appear replaced with two genes (81176.16 and 81176.17 as well as the corresponding ORFs in the HS:23 and HS:36 serostrains) encoding glycosyltransferases.
  • the cps regions of the HS:23 and HS:36 serostrains and strain 81-176 are more similar to that of NCTCl 1168 than to serostrain HS:19 and strain Gl.
  • the cps region of serostrain HS:41 is interesting in that it lacks the cjl415- cjl420 genes conserved in the other strains.
  • heptose-related genes in the middle of the cps locus of serostrain HS:41 are almost identical to those in NCTCl 1168, although gmhA2 and hddA are separated via insertion of gene HS41.09 encoding a putative sugar transferase with low similarity to cj ' 1300 (Fig. 6, Table VII).
  • the mosaic patterns of similarity and divergence indicate that these cps regions have a diverse recent ancestry, suggesting that recombination between different cps clusters has occurred.
  • phase-variable genes could be responsible for the differential expression of deoxyheptose and phosphoramide observed in this study, ie HS:23 (Gal, GlcNAc, Hep, deoxyhep), HS:36 (Gal, GlcNAc, Hep, phosphoramide) and 81-176 (Gal, GlcNAc, Hep, deoxyhep, phosphoramide).
  • CE-MS/MS can be used to examine the structure and variability in C. jejuni LOS.
  • HR-MAS NMR has been used to investigate CPS structure, confirm serotype, demonstrate population variability, study the effect of mutagenesis, and detect N- linked glycoprotein sugars.
  • Campylobacter has a large repertoire of variable surface glycans in addition to a conserved N-linked glycan.
  • jejuni usually occur in the middle of this region with the exception of the heptose biosynthetic genes (Fig. 10).
  • the C-terminus of HS19.11 revealed no similarity to the corresponding region of CJ1440, and resembled instead that of the CJ1438 glycosyltransferase.
  • the finding supports the possibility of intra-cistron recombinations between the genes performing a similar function (e.g. encoding glycosyltransferases), which may result in altered substrate specificity and may contribute to antigenic variation of the CPS.
  • CPSs Structural variation of CPSs is not restricted to C. jejuni.
  • Other bacteria also developed various ways of changing cell surface properties through variation of CPSs.
  • S. pneumoniae where more than 90 different capsular serotypes have been described, the large majority of which are encoded by different cassettes at the same genomic locus.
  • Extensive variation in the cps regions of C. jejuni adds to its arsenal of antigenic variation mechanisms involving cell surface structures.
  • the requirement for CPS and variability of its structure may be dictated by changing host or environmental conditions. For example, colanic acid (exopolysaccharide) contributes to acid and heat tolerance in E. coli. Similarly, resistance of C.
  • jejuni to heat treatment during food preparation may be attributed to certain cell surface located structures, including CPS.
  • Variation in C. jejuni CPS structure may be a consequence of selective pressure in various environmental and in vivo conditions.
  • the discovery of the high conservation of some genes in the biosynthetic cps region along with the variation of others, serves as a basis for a PCR based typing procedure, which can provide a number of advantages over a classical Penner typing scheme.
  • the limited number of antisera available for serotyping (usually a panel of 66 antisera) used in the standard Penner typing protocol results in up to 20% of strains being untypeable.
  • PCR amplification of the cps loci can allow for differentiation of these strains based on their potential of CPS production.
  • PCR analysis allowed detection of CPS-related genes in the untypeable strain X, known to produce a CPS.
  • An advantage of a PCR-based typing scheme based on the sequences derived from the cps regions is that it is based on the presence of the genes, rather than on their expression, which may be affected by a number of factors, including growth conditions.
  • slight variation in the method of antigen preparation and conditions of passive hemagglutination may affect the results of typing using the classical Penner typing protocol. For example, the results of passive hemagglutination depend on the origin of erythrocytes. Therefore, a PCR-based approach based on genetic difference in the cps regions can produce a more reliable and comprehensive typing scheme.
  • Multi-strain comparison of C. jejuni CPS loci has revealed a high conservation in genes involved in heptose biosynthesis and those flanking the kps regions, particularly near kpsC.
  • the findings suggest that CPS clusters are exchanged between C. jejuni and other bacteria and may in part be responsible for the structural variation observed.
  • Other putative mechanisms of structural variation revealed here include gene duplication, deletion, recombination and contingency gene variation.
  • genes with as yet unknown function may be involved in the biosynthesis of CPSs with modified structures.
  • Analysis . of the polysaccharides using NMR has provided novel CPS structural information, including the demonstration that the recently identified phosphoramide modification is common to many C.
  • C. jejuni strains from different disease presentations and geographical locations were surveyed for the phosphoramide (Table Xa). Examination of the closely related Campylobacter coli demonstrated that this modification is absent from this species (Table Xb) and also absent in other species sampled with the exception of one C. fetus isolate from a human with bacterial septicemia. Multiple colonies from selected mutants were analysed to demonstrate their potential role in phosphoramide biosynthesis in NCTCl 1168.
  • the phosphoramide is added to capsules of strains with different serotypes and thus to different structures.
  • a more thorough analysis of the capsule structures of two serostrains known to produce phosphoramide, HS:1 and HS:19 was performed.
  • the HS:1 CPS structure consists of galactose and glycerol-phosphate.
  • an unusual sugar that has not previously been described was detected. It is to this unusual sugar that the phosphoramide is being attached (Fig. 12). More convincing results were obtained with HS:19 (Fig. 13).
  • the phosphoramide is attached at the 4-position of GlcNAc in contrast to NCTCl 1168 where the phosphoramide is attached at the 3-position of Gal/NAc.
  • the phosphoramide is detected in select mutants that lack the CPS. This suggests that the phosphoramide is added to alternate structures or that intermediate forms can be detected. Occasionally additional phosphoramide signals are observed during phosphate scans of capsulated C. jejuni isolates (Fig. 14). Thus, in addition to the possibilities mentioned, phosphoramides may be attached to a varying CPS backbone which could lead to additional signals.
  • tissue culture and serum sensitivity assays were performed. Preliminary adherence and invasion assays comparing wildtype to the phosphoramide mutants demonstrated that loss of phosphoramide caused decreased adherence to CaCo-2 cells while invasion appeared unaffected (Fig. 15). To ensure that the differences in the tissue culture assays were not due to differences in motility
  • motility assays were done comparing the wildtype strains with their respective mutants and demonstrated that all mutants had similar levels of motility compared to the parent (Fig. 16).
  • Preliminary serum sensitivity assays comparing wildtype to the 1416-1 mutant indicated that expression of the phosphoramide increases C. jejuni sensitivity to pooled human serum (Fig. 17).
  • Bac Bac
  • bacillosamine 2,4-diacetamido-2,4,6-trideoxy-D- glucopyranose
  • CE capillary electrophoresis
  • CPMG Carr-Purcell-Meiboom-Gill
  • CPS capsular polysaccharide
  • DIPSI-2 decoupling in the presence of scalar interactions
  • ESI-MS electrospray ionization mass spectrometry
  • GBS Guillain- Barre Syndrome
  • HR-MAS high resolution magic angle spinning
  • LOS lipooligosaccharides
  • LPS lipopolysaccharide
  • MAS magic angle spinning
  • NOESY nuclear Overhauser effect spectroscopy
  • PVDF polyvinylidene difluoride
  • TOCSY total correlation spectroscopy
  • WURST-2 wideband, uniform rate, and smooth truncation
  • HMQC heteronuclear multiple quantum correlation
  • HMBC heteronuclear multiple quantum correlation
  • Biners Molecules having a good binding affinity for this phosphoramide
  • Biners can be employed, either alone or as conjugates or on the surface of liposomes or other suitable cargo carriers or matricies to bind to C. jejuni cells.
  • Such molecules are functionally associated with a toxin or similar substance, they may be used to reduce C. jejuni viability or proliferation on a surface or in a solution, fluid, or semifluid of concern.
  • binders for this purpose are antibodies having specificity for the phosphoramide. Such antibodies may be single domain antibodies. In some cases only fragments of antibodies having the desired specificity will be employed. Such fragments may be expressed as part of a fusion protein with a "cargo" polypeptide of interest.
  • suitable binders are bacteriophages or portions thereof having a good affinity for the phosphoramide. In some instances the phage particles or portions will also be capable of lysing the C. jejuni cells.
  • Parts of interest can include any functional part.
  • phagetail sheaths and/or tail spike proteins may be employed.
  • Other binder molecules can be identified by screening of materials for specific binding to the phosphoramide.
  • the invention provides a use of the phosphoramide in identifying compounds, or materials useful in identifying or reducing the viability of C. jejuni.
  • binders which recognize the phosphoramide regardless of its sugar of attachment will be desired.
  • binders which recognize the phosphoramide in association with one or more particular sugars of attachment will be desired. Binders which specifically recognize the phosphoramide structure unique to
  • Campylobacter and no other phosphate compounds produced in nature are considered to be useful binders.
  • a method of modulating the adhesion of C. jejuni cells to a surface comprising modulating the concentration of binders in the surrounding fluid.
  • the surface may include a non-living material, cells, and/or cell-derived materials.
  • Table m Strain NCTCl 1168.
  • nrdb non-redundant database
  • contingency genes are shown in bold
  • the last three letters in the names of homologues are preceded by two letter codes for bacteria: Aa - Aquifex aeolicus, Af ' - Archaeoglobus fulgidus, At - Aneurinibacillus tliermoaerophilus, Au - Agrobacterium tumefaciens, Bb - Borrelia burgdorferi, Bf - B acter oides fragilis, Bh - Bacillus halodurans, Bj - Bradyrhizobiumjaponicum, Bs - Bacillus subt ⁇ lis, Ca - Clostridium acetobutylicum, Cd - Corynebacterium diphtheriae, Bc -E.
  • glycosyltransferases Function prediction of glycosyltransferases is based on either annotation by the Sanger Institute (for Cj 1421, Cj 1422, Cj 1432, Cj 1434, Cj 1438 and CJ1440) or similarity to other glycosyltransferases of C. jejuni (for Cj 1431 and CJ1442). These glycosyltransferases are labelled as conserved hypothetical without indicating E values. 2 According to ⁇ St Michael, 2002 #212 ⁇ .
  • UDP-galactose sugar epimerase UDP-glucuronate Udg
  • UDP-glucose 6-dehydrogenase UDP-galactose nucletidyl-sugar pyranose mutase sugar transferase sugar transferase sugar transferase sugar transferase sugar transferase sugar transferase sugar transferase sugar transferase sugar transferase sugar transferase sugar transferase sugar transferase sugar transferase sugar transferase sugar transferase sugar transferase sugar transfer
  • Table XI A summary of the distribution of the phosphoramide on the capsular polysaccharide of Campylobacter strains isolated from a variety of animal sources and geographical locations.
  • NCTC Enteritis (phage propagating)
  • NCTC Pigeon (phage propagating)
  • Campylobacter fetus venerealis (ATCC) 0/1 Campylobacter hominus (UK) 0/1
  • Table XLTI The commonality of genes involved in phosphoramide biosynthesis. The presence of Cjl416c, Cj 1417c, Cj 1418c, Cj 1421c and Cj 1422c homologues wa determined using sequencing and gene-specific polymerase chain reactions using primers found in Table XTV. The on off status of Cjl421c and Cjl422c, if known, i indicated as these genes are phase-variable, and was determined by sequencing. Empty boxes indicate that the experiment was not done.
  • Table XTV The sequences of primers used for the identification and characterization of Cj 1416c, Cj 1421c and Cj 1422c.
  • the chromosomal location represents the location of the rimer on the forward strand in the genome of NCTC 11168

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Abstract

L'invention concerne du phosphoramide OP=O(NH2)OMe et de l'alkyl méthyl amidophosphate ainsi que des utilisations associées dans l'identification, le traitement et le diagnostic de Campylobacter jejuni.
PCT/CA2004/001489 2003-08-18 2004-08-17 Phosphoramide et utilisations associees Ceased WO2005017129A1 (fr)

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US5200344A (en) * 1990-11-13 1993-04-06 Blaser Martin J Diagnostic testing for campylobacter jejuni or campylobacter coli infections using novel antigens

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US5200344A (en) * 1990-11-13 1993-04-06 Blaser Martin J Diagnostic testing for campylobacter jejuni or campylobacter coli infections using novel antigens

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SZYMANSKI C.M. ET AL.: "Detection of conserved N-linked glycans and phase-variable lipooligosaccharides and capsules from Campylobacter cells by mass spectrometry and high resolution magic angle spinning NMR spectroscopy", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 278, no. 27, 4 July 2003 (2003-07-04), pages 24509 - 24520 *

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EP1933865A4 (fr) * 2005-09-21 2010-12-22 Us Sectretary Of The Navy Composition d'une capsule immunogénique utilisée comme composant d'un vaccin contre campylobacter jejuni

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