WO2013078172A1 - Glycoconjugaison médiée par tempo d'une composition immunogène contre campylobacter jejuni - Google Patents

Glycoconjugaison médiée par tempo d'une composition immunogène contre campylobacter jejuni Download PDF

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Publication number
WO2013078172A1
WO2013078172A1 PCT/US2012/066007 US2012066007W WO2013078172A1 WO 2013078172 A1 WO2013078172 A1 WO 2013078172A1 US 2012066007 W US2012066007 W US 2012066007W WO 2013078172 A1 WO2013078172 A1 WO 2013078172A1
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WIPO (PCT)
Prior art keywords
polysaccharide
immunogenic composition
hep
tempo
protein
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PCT/US2012/066007
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English (en)
Inventor
Patricia Guerry
Mario Artur Monteiro
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The United States Of America As Represented By The Secretary Of The Navy
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Application filed by The United States Of America As Represented By The Secretary Of The Navy filed Critical The United States Of America As Represented By The Secretary Of The Navy
Publication of WO2013078172A1 publication Critical patent/WO2013078172A1/fr

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    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
    • 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
    • A61K2039/62—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier
    • A61K2039/627—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier characterised by the linker
    • 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 inventive subject matter relates to a Campylobacter polysaccharide- protein conjugate with improved structural integrity and immunochemical properties and a method of producing said polysaccharide protein conjugate and use to stimulate anti-Ca pylobacter immunity.
  • C. jejuni is a leading cause of diarrheal disease worldwide and a documented threat to US military personnel (Taylor, Current status and future trends. Amer. Soc. Micro., (1992); Tauxe, Current status and future trends. Amer. Soc. Micro.(1992).
  • the symptoms of Campylobacter mediated enteritis include diarrhea, abdominal pain, and fever and often accompanied by vomiting. Stools usually contain mucus, fecal leukocytes, and blood, although watery diarrhea is also observed (Cover and Blaser, Ann. Rev. Med., 40: 269-285 (1999)).
  • 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: 665-668 (2000); arlyshev et al., Mol. Microbiol., 35: 529-541 (2000)).
  • Conjugate vaccines containing bacterial polysaccharides may be an effective tool in controlling bacterial infections (Jennings, Adv.
  • An object of this invention is an m ⁇ . ⁇ -C. jejuni immunogenic composition, comprising a polysaccharide conjugate with improved immunochemical properties. Induction of immune responses against Campylobacter by administration of polysaccharide polymers is advantageous due to the low likelihood of inducing Guillain-Barre syndrome (GBS).
  • GBS Guillain-Barre syndrome
  • Another object of the invention is a method of producing a polysaccharide- protein conjugate that retains structural and immunochemical integrity utilizing a method wherein 2,2,6,6-tetramethylpiperidin-l-oxyl (TEMPO) is used to stoichiometrically oxidize primary alcohols of polysaccharides.
  • TEMPO 2,2,6,6-tetramethylpiperidin-l-oxyl
  • another object of the invention is a method of administering the carrier conjugated or unconjugated anti- C. jejuni capsule polysaccharide composition in order to induce an immune response.
  • FIG. 1 Schematic representation of controlled oxidation scheme
  • oxidation occurs on the C-7 of the heptose unit, followed by coupling of the CPS to carrier protein through the newly oxidized carboxylic acid at C7 of the heptose residue.
  • FIG. 2 Schematic representation of selective oxidation at C-7 of 6d- cfo-Hep via TEMPO-mediated oxidation at pH 10.0 followed by EDC-mediate coupling.
  • coupling is of BH-01-0142 capsule polysaccharide is conjugated to BSA.
  • FIG. 3 ID ⁇ NMR spectrum of C. jejuni BH-01-0142 of the activated CPS by TEMPO oxidation at pH 10.0.
  • FIG. 4 Gel electrophoresis of C. jejuni BH-01-0142 conjugate.
  • FIG. 5 GLX profile of GLC-MS of alditol acetate derivatives of (A) intact
  • FIG. 6 (A) ID ⁇ NMR spectrum: and (B) ID 31 P NMR spectrum of C. jejuni
  • FIG. 7 SDS-PAGE electrophoresis and immunoblot of C. jejuni BH-01-0142 capsular conjugate.
  • FIG. 8 IgG endpoint titer of mice immunized with BH-01-0142-CRM] 9 7.
  • oxidation of polysaccharides is achieved utilizing 2,2,6,6-tetramethylpiperidin-l-oxyI (TEMPO) mediated oxidation (Zuchao, et al., Carbohydrate Research 346: 343-347 (201 1)).
  • TEMP selectively oxidizes primary alcohols to carboxylic acids. Therefore, in this embodiment, stoichiometric oxidation by TEMP of the capsule polysaccharides produced by Campylobacter jejuni is followed by the conjugation to protein carrier. As an example, as illustrated in FIG. 1, the C.
  • jejuni capsule trisaccharide composed of galactose (Gal), 3-0-methyl-6- deoxy-altro-heptose (6dHep) and N-acetyl-glucosamine (GlcNAc), non- stoichiometrically substituted at 0-2 of Gal by O-methyl-phosphoramidate (MeOPN) is oxidized followed by conjugation to protein carrier.
  • Gal galactose
  • 6dHep 6- deoxy-altro-heptose
  • GlcNAc N-acetyl-glucosamine
  • MeOPN O-methyl-phosphoramidate
  • the embodied method contemplates the conjugation of other Campylobacter jejuni polysaccharides to other protein carriers.
  • the Campylobacter jejuni polysaccharides that are envisioned to be coupled to protein include:
  • An additional embodiment is a method of conjugating polysaccharides to carrier proteins comprising limiting the number of monosaccharides oxidized per polysaccharide chain. Typically, only 2-3 monosaccharide units in each chain are oxidized.
  • polysaccharide capsule structures from other bacterial species are oxidized and conjugated using TEMPO-mediated glycoconjugation method.
  • TEMPO-mediated glycoconjugation method As examples, the stoichiometric oxidation by TEMPO of nigeran [(1 ⁇ 3) - a-glucan] and amylase [(l ⁇ 4)-a-glucan], and the capsule polysaccharide produced by the Actinobacillus suis (serotype 0:1; CPSAcimobacuius), followed by their conjugation to BSA.
  • capsule polysaccharide is conjugated to a protein by:
  • step (b) exposing TEMPO reacted polysaccharide of step (a) to TEMPO and oxidant; c. exposing oxidized polysaccharide of step (b) to a protein carrier in the presence of a coupling agent.
  • the oxidant can be any number of compound, however in one embodiment NaBr and/or NaOCl is utilized. Oxidant is typically exposed to limiting amounts of NaOCl. Any number of potential coupling agents can be utilized, however, as illustrated in FIG. 1, in one embodiment, l-ethyl-3-(3-dimethylaminopropyl) carbodiimide can be utilized. Reacting to TEMPO is conducted at a pH range of 8.0 to 10.0 at a temperature of 23° C to 37° C. In one embodiment, creation of the carboxylic acid residue is on the C-7 of 6d- i/o-Hep.
  • Example 1 Methods of oxidation and conjugation of amylose and nigeran
  • An embodiment of the current invention is a method of producing an immunogenic composition comprising a polysaccharide conjugate to Campylobacter with improved immunochemical properties due to improved retention of structural integrity.
  • the embodied method comprises the TEMPO oxidation of the polysaccharide, using stochiometric amounts of TEMPO. The oxidized
  • polysaccharide is then directly conjugated to a carrier protein using the newly created carboxylic acid units as functional groups.
  • the activated PS, or the conjugate may be reduced with NaBH 4 .
  • the oxidized polysaccharides were conjugated to protein carrier (i.e., BSA) by first dissolving the oxidized polysaccharides in MES buffer (pH 5.5, 2 mL/1.2 mg of polysaccharide) to which l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (10 ⁇ 171.2 mg polysaccharide) was added, and followed by the addition of BSA (0.3- 14.4 mg/1.2 mg sugar). The pH value of the reaction mixture was adjusted to 5.5 by adding 0.5 M HC1.
  • BSA protein carrier
  • the mixture was stirred at 23 °C or 37 °C for 1-3 days, and then dialyzed against de-ionized water for 1-3 days to remove unreacted polysaccharide, EDC and buffer ions.
  • the dialyzed preparation was lyophilized to afford the conjugates.
  • the conjugation conditions are summarized in Table 2.
  • the first bacterial polysaccharide oxidized was CP ActinobacHim, a P-(l ⁇ 6)-gIucan
  • CPScampy bacter (approximately 6000Da) from
  • Campylobacter jejuni strain 81 176 was oxidized under relatively milder conditions than for Actinobacter.
  • the estimated percentages of oxidized monosaccharide units are illustrated in Table 3.
  • Table 3 When compared with the 2D ⁇ - ⁇ NMR HMBC spectrum of the intact C campyioba er, a new correlation was observed in the spectrum of the oxidized CPScampyiobaaer, between a proton at 3.60 ppm and a carboxyl carbon at 175 ppm after oxidation.
  • FIG. 1 illustrates the selective oxidation followed by EDC coupling to protein (i.e., BSA). The results are illustrated in Table 4. Corroborating the GC-MS results, the ⁇ NMR and 3, P NMR spectra of the oxidized CPScampyiobaaer also showed that the polysaccharide remained structurally intact.
  • the final conjugate was purified by dialysis and size exclusion column to remove any unconjugated polysaccharides.
  • ⁇ NMR experiments on the conjugates showed strong polysaccharide resonances along with some weak BSA signals.
  • Gel electrophoresis revealed the presence of high and low molecular weight conjugates. It is possible that the low molecular weight bands may also contain unconjugated BSA units. More significantly, an immunoblot experiment, using anti-sera against C.
  • a method for inducing an anti-Campylobacter jejuni immune response comprising: comprising the steps:
  • polysaccharide is conjugated to a protein carrier by the method described above.
  • immunogenic composition is covalently linked to said protein by reacting said polysaccharide to 2,2,6,6-tetramethylpiperidin-l-oxyl (TEMPO); exposing said TEMPO reacted polysaccharide to an oxidant and exposing said oxidant exposed polypeptide to said protein in the presence of a coupling agent.
  • TEMPO 2,2,6,6-tetramethylpiperidin-l-oxyl
  • the oxidant can be any number of compounds, however in one embodiment, NaBr and NaOCI is used, wherein the reaction to The TEMPO is conducted at a pH range of 8.0 to 10.0. In this method the composition is administered at 0.1 ⁇ g to 10 mg per dose with or without adjuvant;
  • any protein conjugate can be used, including CRM197.
  • the terminal monosaccharide can be other monosaccharrides. However, in a preferred embodiment, the terminal monosaccharide is 6d- i/o-Hep and the created carboxylic acid is on residue is C-7. Furthermore, in one embodiment, the polysaccharide contains only 1 to 3 monosaccharide units linked to the protein carrier.
  • the polysaccharide polymer is selected from the group consisting of ⁇ 4)-[P ⁇ 3 ]-alpha-D-GaI-( 1 ⁇ 3)-[P ⁇ 2/7] -6d-alpha-D-ido-Hep-( 1 - ; - )-[P ⁇ 3]-alpha-D-Gal-(l ⁇ 3)-[P ⁇ 2]-L-glycero-alpha-D-ido-Hep-(l ⁇ ; ⁇ 3)-6-d-p- D-ido-Hep-( 1 - ⁇ 4)-p-D-GlcNAc-( 1 ⁇ ; and a-D-Gal-( 1 ⁇ [-2)-6d-3-0-Me-a-D-altro- Hep-(l ⁇ 3)-P-D-GlcNAc-(l ⁇ 3)-a-D-Gal-]n, wherein is polysaccharide polymer also contains O-methyl-phosphoramide on gal
  • HS3 capsule polysaccharide contains a heptose monosaccharide it its polysaccharide repeating chain (Aspinall, et al., Eur J. Biochem., 231 : 570-578 (1995)). Therefore, attachment to CRM197 to the isolated capsule polysaccharide, via the C-7 of 6d-ido-Hep, was undertaken via TEMPO-mediated oxidation followed by EDC-mediated coupling. Illustration of the overall scheme of oxidation and coupling is illustrated in FIG. 2. TEMPO-mediated oxidation was used to avoid disruption of potential immunogenic epitopes of the HS3 CPS.
  • SDS-Polyacrylamide gel electrophoresis (SDS-PAGE) analysis of CPS B H-OI- 0142-BSA conjugate showed a significant amount of a lower molecular weight band correlating with the presence of CPSBH-OI-042-BSA conjugate as a single-ended conjugated. This is shown in FIG. 4.
  • lane 1 represents molecular weight markers
  • lane 2 is CPSBH- O I- O I42-BSA conjugate
  • lane 3 is BSA.
  • the molecular weight of BSA was determined to be approximately 67 kDa. However, some degraded BSA was also observed. Additionally, some higher molecular weight conjugate was observed that likely represents cross-linked glycoconjugate.
  • Anti-sera raised against whole cells of C. jejuni BH-01-0142 (HS:3,13,50) were reacted to immunoblots of CPSBH- O I- O I42-BSA conjugate or whole cells of C. jejuni TGH9011 (HS:3 type strain).
  • immune serum reacted to both CPSBH-OI-OI42-BSA conjugate, as well as to C. jejuni whole cell lysates.
  • panels (A) and (B) represent intact CPS and activated CPS of C. jejuni BH-01-0142 by TEMPO oxidation at pH 8.0, respectively.
  • panels (A) and (B) represent the results of ID ⁇ NMR spectrum and 31 P NMR spectrum, respectfully, of C. jejuni BH-01- 0142 of the activated CPS by TEMPO-mediated oxidation at pH 8.0.
  • FIG. 7 (B) shows an immunoblot using antisera raised against whole cell C. jejuni BH-01 -0142 (HS:3, 13, 50).
  • the antisera recognized the TEMPO-derived CPS B H-OI-OI4 2 - CRM197 glycoconjuate, in which two substitutions of MeOPN and 3-hydroxypropanoyl remained intact.

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
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  • Medicinal Chemistry (AREA)
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  • Mycology (AREA)
  • Pharmacology & Pharmacy (AREA)
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  • Polysaccharides And Polysaccharide Derivatives (AREA)

Abstract

L'invention concerne des procédés de production d'une composition de polymère polysaccharidique de capsule immunogène ayant une intégrité structurale améliorée et de meilleures propriétés immunogènes, et son utilisation pour induire une réponse immunitaire contre Campylobacter jejuni.
PCT/US2012/066007 2011-11-23 2012-11-20 Glycoconjugaison médiée par tempo d'une composition immunogène contre campylobacter jejuni WO2013078172A1 (fr)

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US201161629823P 2011-11-23 2011-11-23
US61/629,823 2011-11-23

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999052849A1 (fr) * 1998-04-09 1999-10-21 Merck & Co., Inc. Procede d'oxydation au moyen de tempo
US7700578B2 (en) * 2005-09-21 2010-04-20 The United States Of America As Represented By The Secretary Of The Navy Capsule composition for use as immunogen against Campylobacter jejuni
US20100239604A1 (en) * 2007-06-26 2010-09-23 Glaxosmithkline Biologicals. S.A. Vaccine comprising streptococcus pneumoniae capsular polysaccharide conjugates

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999052849A1 (fr) * 1998-04-09 1999-10-21 Merck & Co., Inc. Procede d'oxydation au moyen de tempo
US7700578B2 (en) * 2005-09-21 2010-04-20 The United States Of America As Represented By The Secretary Of The Navy Capsule composition for use as immunogen against Campylobacter jejuni
US20100239604A1 (en) * 2007-06-26 2010-09-23 Glaxosmithkline Biologicals. S.A. Vaccine comprising streptococcus pneumoniae capsular polysaccharide conjugates

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