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WO2025021704A1 - Vaccin - Google Patents

Vaccin Download PDF

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Publication number
WO2025021704A1
WO2025021704A1 PCT/EP2024/070606 EP2024070606W WO2025021704A1 WO 2025021704 A1 WO2025021704 A1 WO 2025021704A1 EP 2024070606 W EP2024070606 W EP 2024070606W WO 2025021704 A1 WO2025021704 A1 WO 2025021704A1
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WIPO (PCT)
Prior art keywords
polysaccharide
antigen
conjugate
carrier protein
fvi
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/EP2024/070606
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English (en)
Inventor
Santosh Renukuntla
Dharmesh Patel
Vikram Madhusudan Paradkar
Carlo GIANNELLI
Roberta DI BENEDETTO
Renzo ALFINI
Francesca Micoli
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
GlaxoSmithKline Biologicals SA
Biological E Ltd
Biological E Ltd
Original Assignee
GlaxoSmithKline Biologicals SA
Biological E Ltd
Biological E Ltd
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Publication date
Priority claimed from GBGB2311231.1A external-priority patent/GB202311231D0/en
Priority claimed from GBGB2311233.7A external-priority patent/GB202311233D0/en
Priority claimed from GBGB2318238.9A external-priority patent/GB202318238D0/en
Application filed by GlaxoSmithKline Biologicals SA, Biological E Ltd, Biological E Ltd filed Critical GlaxoSmithKline Biologicals SA
Publication of WO2025021704A1 publication Critical patent/WO2025021704A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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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
    • A61K39/025—Enterobacteriales, e.g. Enterobacter
    • A61K39/0275—Salmonella
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/6415—Toxins or lectins, e.g. clostridial toxins or Pseudomonas exotoxins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
    • 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
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
    • C07K16/1228—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K16/1235—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia from Salmonella (G)
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • 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
    • A61K2039/70—Multivalent vaccine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • 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

  • Vaccine Field of the invention relates to conjugates comprising polysaccharides comprising 3- deoxy-D-manno-actulosonic acid (KDO) moieties, particularly conjugates produced using random conjugation methods, methods for preparing such conjugates, immunogenic compositions and vaccines comprising the conjugates, and methods of treatment or medical uses using the compositions and vaccines.
  • KDO deoxy-D-manno-actulosonic acid
  • Background to the invention Typhoid fever is a bacterial disease caused by Salmonella enterica subspecies enterica serovar Typhi (Salmonella Typhi), a human host–restricted organism [Crump, 2019].
  • typhoid fever may require hospitalization and potentially fatal complications such as Typhoid Intestinal Perforation (TIP).
  • TIP Typhoid Intestinal Perforation
  • MDR multidrug- resistant
  • Salmonella first identified in 1980 and defined as strains resistant to ampicillin, chloramphenicol and trimethoprim sulfamethoxazole.
  • Paratyphi A is ranked second as a causative agent of enteric fever, preceded only by Salmonella enterica serovar Typhi (S. Typhi). Enteric fever caused by S. Paratyphi A, or Paratyphoid fever was thought to be responsible for a comparatively smaller proportion of enteric fever cases. However, since the 1980s both the incidence and relative frequency of Paratyphoid fever have risen in Nepal, Pakistan, and Thailand. Moreover, the populous nations of India and China have reported substantial numbers of S. Paratyphi A cases.
  • polysaccharides are T-independent antigens, however, they are poorly immunogenic. Conjugation to a carrier can convert T-independent antigens into T- dependent antigens, thereby enhancing memory responses and allowing protective immunity to develop.
  • the most effective polysaccharide vaccines are therefore based on glycoconjugates, and the prototype conjugate vaccine was against Haemophilus influenzae type b ('Hib') [e.g. see chapter 14 of Vaccines (2004) eds. Plotkin & Orenstein. ISBN 0-7216-9688-0].
  • Gram-negative bacteria are surrounded by an outer membrane that contains lipopolysaccharide.
  • Lipopolysaccharides are a diverse group of molecules that act as endotoxins and elicit strong immune responses in mammals.
  • Each lipopolysaccharide comprises three parts: an O-antigen (also referred to as the O-specific polysaccharide or O-polysaccharide), a core domain, and a lipid A domain.
  • O-antigen also referred to as the O-specific polysaccharide or O-polysaccharide
  • Vaccines have therefore been envisaged that contain O- antigens conjugated to carrier proteins.
  • O-antigen-based conjugate vaccines have been proposed for various Salmonellae (e.g.
  • the O-antigen is linked to the core domain from the full- length lipopolysaccharide (i.e. the conjugated polysaccharide is a lipopolysaccharide without its lipid A domain).
  • the polysaccharide is conjugated to the carrier via its core domain.
  • Various methods for conjugating O-antigens from bacteria such as Salmonella Paratyphi A (S. Paratyphi A) have been considered.
  • WO 2013/038375 proposes using a selective method in which the KDO moiety is linked to a linker or a carrier protein using direct (selective) reductive amination.
  • Summary of the invention The present Examples demonstrate that conjugates produced by the direct (selective) reductive amination method of WO2013/038375 are not very stable.
  • the Examples demonstrate that, when a S. Paratyphi A O-antigen-CRM 197 conjugate was formulated into a bivalent vaccine further comprising a Salmonella Typhi (S. Typhi) Vi- CRM 197 conjugate, the S.
  • Paratyphi A O-antigen-CRM 197 conjugate made using the selective reductive amination method of WO2013/038375 was not stable. This is particularly concerning as infections by S. Paratyphi A and S. Typhi are particularly prevalent in developing countries, and in such countries it is particularly important for the supply chain that a vaccine be stable.
  • the present Examples demonstrate, however, that if the S. Paratyphi A O-antigen is conjugated using a random conjugation method introducing multiple activated sites, the conjugate is significantly more stable and therefore much more suitable for use in a vaccine.
  • a first aspect provides a conjugate comprising a polysaccharide comprising a 3-deoxy-D-manno-octulosonic acid (KDO) moiety conjugated to a carrier protein by a random conjugation method, wherein the polysaccharide comprises more than one activated site.
  • KDO 3-deoxy-D-manno-octulosonic acid
  • a conjugate comprising a polysaccharide comprising a KDO moiety conjugated to a carrier protein using a conjugation method comprising a step of: (i) activating the polysaccharide by 1-cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP) chemistry to provide an activated polysaccharide; or (ii) oxidising the polysaccharide to provide an oxidised polysaccharide.
  • CDAP 1-cyano-4-dimethylaminopyridine tetrafluoroborate
  • a conjugate comprising an O-antigen conjugated to a carrier protein using a conjugation method comprising a step of: (i) activating the O-antigen by CDAP chemistry to provide an activated O- antigen; or (ii) oxidising the O-antigen to provide an oxidised O-antigen.
  • a method for producing a conjugate comprising a polysaccharide comprising KDO moiety conjugated to a carrier protein comprising a step of introducing multiple activated sites into the polysaccharide.
  • a method for producing a conjugate comprising an O-antigen conjugated to a carrier protein comprising a step of introducing multiple activated sites into the polysaccharide.
  • a method for producing a conjugate comprising a polysaccharide comprising a KDO moiety conjugated to a carrier protein comprising a step of: (i) activating the polysaccharide by CDAP chemistry to provide an activated polysaccharide; or (ii) oxidising the polysaccharide to provide an oxidised polysaccharide.
  • a method for producing a conjugate comprising an O-antigen conjugated to a carrier protein comprising a step of: (i) activating the O-antigen by CDAP chemistry to provide an activated O- antigen; or (ii) oxidising the O-antigen to provide an oxidised O-antigen.
  • a conjugate obtainable by the method of the invention.
  • a conjugate obtained by the method of the invention there is provided an immunogenic composition comprising the conjugate of the invention.
  • a vaccine comprising the immunogenic composition of the invention.
  • a method of preventing an infection comprising administering an effective amount of the immunogenic composition or vaccine of the invention to a subject.
  • a use of the immunogenic composition or vaccine of the invention for the manufacture of a medicament for use in a method of preventing an infection Brief description of the Figures
  • Figure 1 Reaction scheme for the selective reductive amination method of WO2013/038375.
  • Figure 2 Reaction scheme for conjugation of S. Paratyphi A O-antigen to CRM 197 - ADH by NHS-EDAC chemistry.
  • Figure 3 Reaction scheme for conjugation of S.
  • Paratyphi A O-antigen to CRM 197 by reductive amination with a step of random oxidation using periodate.
  • Figure 4 Reaction scheme for conjugation of S. Paratyphi A O-antigen to CRM 197 by a random CDAP chemistry approach.
  • Figure 5 Graphs showing the immunogenicity of various S. Paratyphi O-antigen conjugate vaccines. Mice were immunised as described in Example 8, and the immunogenicity of the vaccines measured by ELISA. Bars with hatching represent the antibody level 42 days after first immunisation and bars with no fill represent the antibody levels 28 days after first immunisation.
  • Figure 6 Published structure of the O-antigen (including the core domain) from S. Paratyphi A.
  • FIG 7 Structure of the Vi monomeric repeating unit.
  • Figure 8 Sequence listing.
  • Figure 9 Graph showing geometric mean concentrations (GMCs) of anti-Vi antibodies as measured by ELISA.
  • the vaccines administered (and doses used) are as described in Example 9.
  • Figure 10 Graph showing geometric mean concentrations (GMCs) of anti-O:2 (S. Paratyphi O-antigen) antibodies as measured by ELISA.
  • the vaccines administered (and doses used) are as described in Example 9.
  • Figure 11 - Graph showing geometric mean titres (GMTs) of anti- S. Paratyphi O- antigen bactericidal antibodies as measured by SBA.
  • the vaccines administered (and doses used) are as described in Example 9.
  • Figure 12 A) HPLC SEC of O:2 [16kDa + 100kDa] , O:2 [16kDa] , O:2 [100kDa] ; B) analytical characterization of the O:2 populations used for the conjugation with CRM 197 .
  • Figure 13 Summary graphs of anti-O:2 IgG geometric mean units (bars) and individual antibody levels (dots) are reported. Left-hand bars represent Day 27, and right-hand bars represent Day 42.
  • O:2 size O:2-CRM 197 conjugates synthesized using O:2 with different sizes (16 kDa, 100 kDa and 16kDa + 100kDa) and with a similar O:2/CRM 197 w/w ratio were compared.
  • the term “consisting of” is intended to be limiting.
  • the phrase “An immunogenic composition consisting of a conjugate” should be understood to mean that the immunogenic composition has a conjugate and no further components.
  • the word “comprising” is replaced with the phrase “consisting essentially of”.
  • the term “consisting essentially of” means that specific further components can be present, namely those not materially affecting the essential characteristics of the subject matter.
  • the term “about” or “around” when referring to a value refers to that value but within a reasonable degree of scientific error.
  • a value is “about x” or “around x” if it is within 10%, within 5%, or within 1% of x.
  • conjugates refers to a molecule formed by a covalent linkage between an antigen and a carrier.
  • the conjugates of the present invention comprise a polysaccharide.
  • polysaccharide refers to any linear or branched polymer consisting of monosaccharide residues, usually linked by glycosidic linkages, and thus includes oligosaccharides.
  • the carrier is generally a carrier protein.
  • protein we mean or include any linear or branched molecule consisting of amino acid residues.
  • Polysaccharide comprising a KDO moiety and/or an O-antigen The conjugates of the invention comprise a polysaccharide comprising a KDO moiety and/or an O-antigen.
  • the methods of the invention are methods for producing conjugates comprising a polysaccharide comprising a KDO moiety and/or an O-antigen.
  • a 3 KDO moiety has the following structure:
  • the polysaccharides comprised within the conjugates comprise a KDO moiety at the reducing terminus.
  • the KDO moiety may be used for direct selective coupling of the polysaccharide to a carrier protein.
  • the conjugates may comprise an O-antigen (also referred to as OAg and O:2).
  • the polysaccharide comprising a KDO moiety may be an O-antigen.
  • the outer membrane of gram-negative bacteria comprises a lipopolysaccharide.
  • This lipopolysaccharide comprises an O-antigen, which is linked via to the core domain to a lipid A domain.
  • O-antigen O-antigen
  • O:2 refer to a polysaccharide made up of the O- antigen alone, or the O-antigen linked to the core domain of the lipopolysaccharide. Purification of these O-antigen-cores may be carried out using a method based on the phenol-water method of Westphal and Jann, first described in the 1960s (Westphal and Jann (1965) Methods Carbohydr. Chem. 5:83-91), followed by detoxification of the lipopolysaccharide with acetic acid or anhydrous hydrazine.
  • the O-antigen is modified to remove the lipid A.
  • extraction and purification of polysaccharide can be performed by acetic acid hydrolysis as described in for example Watson et al., (1992) Infect Immun. 60(11):4679-86; Konadu et al. (1996) Infect Immun. (7):2709-l5; Konadu et al. (1994) Infect Immun. 62(11):5048-54; Ahmed et al. (2006) J Infect Dis. 193(4):515-21; Cox et al. (2011) Glycoconj J 28 :165-182; Chu et al. (1991) Infect Immun.
  • the O-antigen is from the lipopolysaccharide of a Salmonella bacterium, e g. from Salmonella serogroups A, B or D, and particularly from Salmonella Paratyphi A.
  • Salmonella serogroups A, B and D have been described and are thought to share a common backbone: ⁇ 2- ⁇ -D-Manp-(l ⁇ 4)- ⁇ -L-Rhap-(l ⁇ 3)- ⁇ -D- Galp-(1 ⁇ .
  • the serogroup specificity of Salmonella Paratyphi A is conferred by an ⁇ - 3,6-dideoxyglucose ( ⁇ -D-paratose) linked (1 ⁇ 3) to the mannose of the backbone.
  • the ⁇ -L-rhamnose of the backbone is partially O-acetylated at C-3 (Konadu et al. (1996) Infect Immun. (7):2709-l5).
  • the published structure of the O-antigen and core domain from S. Paratyphi A is shown in Figure 6, including the KDO subunit and primary amine group (within a pyrophosphoethanolamine group) in the core domain.
  • the O- antigen may also be from S. Typhimurium.
  • the O-antigen may also be from S. Enteriditis.
  • Naturally-derived O-antigens may contain structural variations compared to the published structures for these O-antigens.
  • the O-antigen may also be from Shigella species, e.g. from S. flexneri. Other Shigella species that may provide the O-antigen used in the invention are S. sonnei, S. dysenteriae and S. boydii [Knirel et al. (2011) Glycobiology. (10): 1362-72].
  • the O- antigen and core domain may also be from E. coli, such as E. coli 0157.
  • Other lipopolysaccharide-containing Gram-negative bacteria that may provide the O-antigen used in the invention are Klebsiella pneumonia [Chhibber et al.
  • the polysaccharide and/or O-antigen may be chemically modified relative to the polysaccharide and/or O-antigen as found in nature.
  • the polysaccharide and/or O-antigen may be de-O-acetylated (partially or fully), but it is preferred for O- antigens not to be de-O-acetylated.
  • de-acetylation may occur before, during or after other processing steps, but typically occurs before any coupling step.
  • the effect of de-acetylation etc. can be assessed by routine assays.
  • the relevance of O-acetylation on S. Paratyphi A O-antigen is discussed in Konadu et al. (1996) Infect Immun. (7):2709-l5.
  • the native O-antigen of S. Paratyphi A is said in this document to have about 80% O-acetylation.
  • Conjugated de-O-acetylated O-antigen did not elicit anti-lipopolysaccharide antibodies with bactericidal activity. Accordingly, when the O-antigen used in the present invention is S.
  • the O- antigen may have between 0 and 100% O-acetylation, but it is preferred for the O- antigen to be O-acetylated.
  • the level of O-acetylation may depend on the bacterial strain that provided the O-antigen.
  • the degree of O-acetylation of the S. Paratyphi A O-antigen may be 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 55- 95%, 55-85%, 60-80% or 65-75%.
  • the degree of O-acetylation of the S. Paratyphi A O-antigen is 55-85%, particularly 65-75%, or 45-80%, particularly 40-50% or 60-70%.
  • the degree of O-acetylation of the S. Paratyphi A O-antigen may also be 60-100%, 70-100%, 80-100%, or 90-100%.
  • the degree of O-acetylation is based on the amount of Rhamnose (Rha).
  • the degree of O-acetylation of the polysaccharide can be determined by any method known in the art, for example, by proton NMR ⁇ e.g. as described in Ravenscroft et al. Carbohydr Res. 2015 ;404:108-16, the Hestrin method [24] or HPAEC-CD [25].
  • O- acetyl groups may be removed by hydrolysis, for example by treatment with a base such as sodium hydroxide or anhydrous hydrazine [Konadu et al. (1996) Infect Immun. (7):2709-l5].
  • a base such as sodium hydroxide or anhydrous hydrazine [Konadu et al. (1996) Infect Immun. (7):2709-l5].
  • treatments that lead to hydrolysis of the O-acetyl groups are minimised, e.g. treatments at extremes of pH.
  • the O-antigen used in the present invention is S.
  • the average molecular weight of the O-antigen is between 5 kDa and 120 kDa, between 10 kDa and 100 kDa, between 10 kDa and 80 kDa, between 10 kDa and 70 kDa, between 10 kDa and 60 kDa, between 10 kDa and 50, between 10 kDa and 40 kDa, between 10 kDa and 30 kDa, between 10 and 25 kDa, between 10 and 20 kDa, 11 kDa and 19 kDa, 12 kDa and 19 kDa, 13 kDa and 19 kDa, 14 kDa and 18 kDa, 15 and 18 kDa, or between 16 kDa and 18 kDa .
  • the O-antigen has a target molecular weight of between 16 and 18 kDa (e.g. it has been made by a method that typically generates O-antigen having a molecular weight within this range).
  • the molecular weight of the O-antigen may be determined by HPLC-SEC.
  • Conjugated to a carrier protein The conjugates of the invention comprise a carrier protein.
  • the methods of the invention are methods for producing conjugates comprising a carrier protein. In general, conjugation of polysaccharides to carrier proteins enhances the immunogenicity of the polysaccharides as it converts them from T-independent antigens to T-dependent antigens, thus allowing priming for immunological memory.
  • Carrier proteins include bacterial toxins, such as diphtheria or tetanus toxins, or toxoids or mutants thereof.
  • the carrier protein is CRM 197 .
  • the sequence of CRM 197 is provided in SEQ ID NO: 1.
  • the polysaccharide of the invention is S. Paratyphi A O-antigen and the carrier protein is CRM 197, and the O:2/ CRM 197 w/w ratio may be between 0.1 and 3.5, between 0.2 and 3.0, between 0.25 and 3.0, between 0.25 and 0.9, between 0.25 and 0.8, between 0.35 and 0.8, between 0.4 and 0.8, between 0.5 and 0.8, between 0.6 and 0.7, between 0.61 and 0.67 or between 0.62 and 0.66.
  • the ratio may also be between 0.5 and 3.0, 0.7 and 3.0, 1.0 and 3.0, 1.5 and 3.0, 2.0 and 3.0, or 2.5 and 3.0. Random conjugation and activated sites
  • the conjugates of the invention are conjugates comprising a polysaccharide and/or an O-antigen conjugated to a carrier protein by a random conjugation method.
  • the methods of the invention are methods for producing a conjugate by a random conjugation method.
  • the term “random conjugation method” refers to a method of conjugating a polysaccharide to a carrier protein which is not selective, i.e. a conjugation method in which one or both of (i) the number of linkages (i.e.
  • the number of positions on the polysaccharide to which a carrier protein is linked) and (ii) the position of the linkages (i.e. where on the polysaccharide the linkages occur) is not predetermined by the chemistry used and may differ between conjugates produced using the same conjugation chemistry.
  • some of the conjugates may comprise 2 linkages (i.e. two linkages between the polysaccharide and the carrier protein or two carrier protein molecules being linked to the same polysaccharide) and other conjugates may comprise only 1.
  • some of the conjugates are conjugated at a first reaction site on the polysaccharide, but some of the conjugates are conjugated at a second reaction site on the polysaccharide.
  • Suitable random conjugation methods include conjugation using 1- cyano-4-dimethylaminopyridine (CDAP) chemistry and using reductive amination using a step of random oxidation, for example using an agent such as sodium periodate.
  • the polysaccharide or O-antigen may comprise more than one activated site or the method may comprise a step of introducing multiple activated sites into the polysaccharide or O-antigen.
  • activated site is intended to refer to a site or functional group on the polysaccharide that has been activated by a step in a conjugation chemistry method such that it is primed to be conjugated to a carrier protein.
  • the polysaccharide is “activated” by the addition of CDAP if the addition of CDAP introduces cyanoester groups.
  • the CDAP activation introduces cyanoester groups at one or more sites, and the positions of these introduced cyanoester groups would be considered to be “activated sites”.
  • the polysaccharide may be linked (conjugated) to a carrier protein at one or more (in some cases all) of the activated sites.
  • the term “activated sites” includes sites that have been activated and not linked to carrier protein and also sites that have been activated and are linked to a carrier protein.
  • the random conjugation method comprising a step of: (i) activating the polysaccharide or the O-antigen by CDAP chemistry to provide an activated polysaccharide or O-antigen; or (ii) oxidising the polysaccharide or the O-antigen to provide an oxidised polysaccharide or O-antigen. It is possible to determine the average number of activated sites of a polysaccharide. If the activated sites are introduced using CDAP chemistry, the number of activated sites may be measured using the ADH quenching/TNBS colorimetric method as reported in Lees A, Vaccines (Basel). 2020;8(4):777.
  • the number of activated sites may be measured using microBCA reagents to measure the formed aldehydes or by HPAEC-PAD to detect the oxidized sugar ring; both options are reported in Stefanetti et al. Vaccine. 2014;32(46):6122-9.
  • the polysaccharide or the O-antigen comprises 1.5 or more, 2.0 or more, or 2.5 or more activated sites.
  • the polysaccharide or the O-antigen is part of a composition comprising multiple polysaccharide or O-antigen saccharides
  • the polysaccharide or O-antigen will comprise 1.5 or more activated sites if the average number of activated sites on each polysaccharide or O-antigen molecule is 1.5 or more.
  • the conjugates of the invention are part of a composition comprising multiple conjugates, and/or the methods of the invention produce a composition comprising multiple conjugates, and the average number of activated sites on the conjugates in the composition is greater than 1, optionally 1.5 or more, 2 or more, or 2.5 or more activated sites. In such cases different O-antigen molecules within the composition may have different numbers of activated sites.
  • Conjugate further comprises a linker
  • the conjugate may further comprise a linker.
  • the methods of the invention may comprise conjugating an activated or oxidised polysaccharide to a linker and/or a carrier protein-linker compound.
  • a linker is a compound that can be used to link a protein and a polysaccharide. Any suitable linker may be used in the conjugates and methods of the invention. Suitable linkers include an adipic acid dihydrazide (ADH) linker, which is a compound having the following structure: .
  • ADH adipic acid dihydrazide
  • linkers include adipic acid, glutaric acid, carbonyl, ⁇ -propionamido (WO00/10599), adipic acid bis(N-hydroxysuccinimmide), dihydrazides analogous to ADH but with different chain lengths, hexamethylenediamine (or analogous diamines with different chain lengths), nitrophenyl-ethylamine (Gever et al. (1979) Med. Microbiol. Immunol. 165, 171-288), haloacyl halides (U.S. Pat. No. 4,057,685), glycosidic linkages (U.S. Pat. Nos.
  • the conjugate comprises a polysaccharide comprising a KDO moiety and/or an O-antigen that was conjugated to a carrier protein using a conjugation method comprising a step of activating the polysaccharide and/or the O-antigen by CDAP chemistry to provide an activated polysaccharide.
  • the methods comprise a step of activating the polysaccharide by CDAP chemistry to provide an activated polysaccharide.
  • Activating the polysaccharide or O-antigen by CDAP chemistry comprises mixing the polysaccharide and/or the O-antigen with CDAP such that cyanoester groups are introduced into the polysaccharide or O-antigen.
  • Example 7 discloses a suitable method of activating an O-antigen by CDAP chemistry.
  • Activating the polysaccharide and/or the O-antigen by CDAP chemistry results in an activated polysaccharide or O-antigen.
  • Activating the polysaccharide and/or the O-antigen by CDAP chemistry introduces cyanoester groups, and so an activated polysaccharide and/or O-antigen comprises cyanoester groups that were introduced using CDAP.
  • a method comprises a step of activating a polysaccharide and/or an O-antigen by CDAP chemistry if the method comprises mixing the polysaccharide and/or the O- antigen with CDAP and the number of cyanoester groups present on the polysaccharide and/or the O-antigen present after the step of mixing with CDAP is higher than the number of cyanoester groups present on the polysaccharide and/or the O-antigen prior to that step.
  • the number of cyanoester groups present may be measured using the ADH quenching/TNBS colourimetric method as reported in Lees A., Vaccines (Basel), 2020; 8(4):777.
  • activating the O-antigen and/or the polysaccharide by CDAP chemistry comprises mixing the polysaccharide or the O-antigen with CDAP at a w/w ratio of between 0.05:1 and 5:1, between 0.1:1 and 5:1, between 0.2:1 and 2:1, or around 0.3:1 (CDAP to polysaccharide or O-antigen).
  • the step of activating the O-antigen and/or the polysaccharide by CDAP chemistry comprising mixing the polysaccharide or the O-antigen with CDAP takes place in a salt solution, such as a solution of NaCl or KCl.
  • the step of activating the O-antigen and/or the polysaccharide by CDAP chemistry comprising mixing the polysaccharide or the O-antigen with CDAP takes place in a solution of NaCl or KCl at a concentration between 50 mM and 1 M, between 100 mM and 250 mM, between 125 mM and 200 mM, or around 150 mM.
  • the pH is adjusted, optionally to a pH between 6 and 10, between 7 and 9, or between 9 and 10.
  • the pH is adjusted by adding a base, such as triethylamine, sodium hydroxide, or pyridine.
  • the pH is adjusted by adding between 5 % and 15%, between 8% and 12%, or around 10% (v/v) triethylamine.
  • the mixture is incubated at a temperature between 18oC and 30oC, between 20oC and 28oC, room temperature, or around 25oC.
  • the solution is incubated with stirring prior to conjugation of the activated polysaccharide or O-antigen to the carrier protein.
  • a suitable method for activating the polysaccharide by CDAP chemistry is described in Example 7.
  • polysaccharides and/or O-antigens that have been activated using CDAP chemistry comprise cyanoester groups (at activated sites), and these cyanoester groups may be covalently linked to hydrazide or amino groups.
  • polysaccharides and/or O-antigens that have been activated using CDAP chemistry may be linked directly to carrier proteins (via amino groups), or may be conjugated to a carrier protein via a linker comprising a hydrazide or amino group.
  • Suitable linkers include the ADH linker described above.
  • the methods may comprise reacting the activated polysaccharide or O-antigen with hydrazide/amino groups on a carrier protein or a carrier-protein linker compound.
  • the methods may comprise a step of reacting the activated polysaccharide or O-antigen with hydrazide/amino groups on a carrier protein-linker compound.
  • the method may further comprise steps to prepare the carrier protein-linker compound.
  • the linker is an ADH linker
  • the method may comprise a step of preparing an ADH-carrier protein compound (such as an ADH-CRM 197 compound), for example as reported in Micoli et al. Vaccine 2011, 29, (4), 712-20.
  • Reacting the activated polysaccharide or O-antigen with hydrazide/amino groups on a carrier protein or a carrier-protein linker compound may comprise mixing the carrier protein or the carrier-protein linker compound with the activated polysaccharide or O- antigen under conditions suitable for a covalent bond to be formed between the cyanoester groups (activated sites) on the activated polysaccharide or O-antigen and the hydrazide/amino groups on the carrier protein or the carrier protein-linker compound. For example, it may be simply a case of mixing the activated polysaccharide or O- antigen with the carrier protein or the carrier protein-linker compound.
  • Reacting the activated polysaccharide or O-antigen with hydrazide/amino groups on the carrier protein or a carrier protein-linker compound may comprises mixing the activated polysaccharide or O-antigen with the carrier protein or the carrier protein-linker compound at a w/w ratio of between 0.1:1 and 5:1, between 0.2:1 and 3:1, between 0.5:1 and 2:1, or around 1:1, (polysaccharide or O-antigen to carrier protein or carrier protein-linker).
  • the step of mixing the activated polysaccharide or O- antigen with the carrier protein or the carrier protein-linker provides a conjugation mixture.
  • mixing the activated polysaccharide or O-antigen with the carrier protein or the carrier protein-linker compound takes place at a pH between 8 and 11, between 9 and 10, or around 9.5.
  • the pH is maintained at between 8 and 11, between 9 and 10, or around 9.5 for at least 1 hour, at least 2 hours, between 30 minutes and 10 hours, between 1 hour and 5 hours, or between 2 hours and 3 hours.
  • the pH is maintained using a base, such as triethylamine, sodium hydroxide, or pyridine.
  • the pH is maintained using triethylamine.
  • the method may further comprise a step of adding glycine solution (to quench the cyanoester groups).
  • the glycine solution is added in a concentration of 0.5M to 5M, 0.5M to 2M, or around 1M.
  • the glycine solution is added to a volume of the conjugation mixture with is substantially equal to the volume of the glycine solution. A volume is substantially equal to another volume if it is within 10%.
  • a further step of adjusting the pH using a base such as triethylamine, sodium hydroxide, or pyridine.
  • this further step occurs after a step of adding a glycine solution.
  • the pH is adjusted to a pH between 7 and 9, or around 8.
  • the incubation step comprises incubating at a temperature below 15oC, between 12oC, below 10oC, between 0oC and 10oC, or between 2oC and 8oC.
  • the incubation step takes place for between 10 and 30 hours, or between 10 and 20 hours.
  • the method may further comprise a chromatography step to remove any unconjugated polysaccharide and/or O-antigen.
  • the chromatography step comprises hydrophobic interaction chromatography or anion exchange chromatography.
  • the conjugate comprises a polysaccharide comprising a KDO moiety and/or an O-antigen that was conjugated to a carrier protein using a conjugation method comprising a step of oxidising the polysaccharide and/or the O-antigen to provide an oxidised polysaccharide.
  • the methods comprise a step of oxidising the polysaccharide to provide an oxidised polysaccharide.
  • Oxidising the polysaccharide and/or O-antigen may be used as part of a random conjugation method, as an oxidation step may be used to introduce one or more activated (oxidised) sites at a variety of positions on the polysaccharide chain.
  • the step of oxidising the polysaccharide or O-antigen comprises mixing the polysaccharide or O-antigen with an oxidising agent.
  • Suitable oxidising agents include periodate, for example sodium periodate.
  • mixing the polysaccharide or O-antigen with an oxidising agent comprises mixing the polysaccharide or O-antigen with an oxidising agent at a ratio of between 1 mg/ml: 10 mM and 100 mg/mL: 10 mM, between 1 mg/mL :10 mM and 50 mg/mL: 10 mM, between 1 mg/mL: 10 mM and 25 mg/mL: 10 mM, or around 10mg/mL: 10 mM (polysaccharide or O-antigen to oxidising agent).
  • mixing the polysaccharide or O-antigen with an oxidising agent occurs at a pH between 3 and 7, between 4 and 6, or around 5.
  • mixing the polysaccharide or O-antigen with an oxidising agent occurs in a buffer such as an acetate buffer.
  • the buffer is sodium acetate.
  • the step of oxidising the polysaccharide or O-antigen comprises incubating a mixture of the polysaccharide or O-antigen and the oxidising agent.
  • incubating the mixture of the polysaccharide or O-antigen and the oxidising agent takes place in the dark.
  • incubating the mixture of the polysaccharide or O-antigen and the oxidising agent takes place for between 1 and 10 hours, between 1 and 5 hours, between 1 and 3 hours, or around 2 hours.
  • incubating the mixture of the polysaccharide or O-antigen and the oxidising agent takes place at a temperature between 20oC and 30oC, between 22oC and 28oC, or around 25oC, or at room temperature.
  • the step of oxidising the polysaccharide or O-antigen may also comprise a step of quenching excess oxidising agent.
  • the step of quenching excess oxidising agent may take place after the step of mixing the polysaccharide or O-antigen with an oxidising agent and/or the step of incubating the mixture of the polysaccharide or O-antigen and the oxidising agent, optionally in water.
  • the step of quenching excess oxidising agent may comprise adding a quenching agent.
  • a suitable quenching agent is sodium sulphite (Na 2 SO 3 ).
  • quenching excess oxidising agent comprises adding sodium sulphite to the mixture of the polysaccharide or O- antigen and oxidising agent and incubating the resulting mixture for at least 10 minutes, between 5 minutes and 30 minutes, or around 15 minutes at a temperature between 15oC and 30oC, between 20oC and 28oC, room temperature, or around 25oC.
  • the step of oxidising the polysaccharide or O-antigen may also comprise a step of purifying the oxidised polysaccharide or O-antigen.
  • the step of purifying the oxidised polysaccharide or O-antigen may comprise desalting the mixture of the polysaccharide or O-antigen and the oxidising agent, for example using a PD10 column.
  • the methods may also comprise a step of lyophilising the oxidised polysaccharide or O- antigen to provide a lyophilised oxidised polysaccharide or O-antigen.
  • a suitable method for oxidising the polysaccharide is described in Example 6. Conjugating an oxidised polysaccharide to a carrier protein with or without a linker
  • the methods may comprise a step of reacting the oxidised polysaccharide or O-antigen with the carrier protein or a carrier protein-linker compound comprising the carrier protein.
  • the oxidised polysaccharide or O-antigen may be reacted with the carrier protein or the carrier protein-linker compound using reductive amination.
  • the step of reacting the oxidised polysaccharide or O-antigen with the carrier protein comprises mixing the oxidised polysaccharide or O-antigen and the carrier protein with a reducing agent.
  • Suitable reducing agents include cyanoborohydrides such as sodium cyanoborohydride, borohydrides such as sodium borohydride, or boranes such as amino borane, pyridine borane, dimethylborane or trimethylamine borane.
  • the reducing agent is a cyanoborohydride.
  • the reducing agent is sodium cyanoborohydride.
  • the step of reacting the oxidised polysaccharide or O-antigen with the carrier protein or carrier protein-linker compound comprises mixing the oxidised polysaccharide or O-antigen and the carrier protein or carrier protein-linker compound at a w/w ratio of between 0.5: 1 and 20: 1, between 1:1 and 10: 1, between 1.5:1 and 5:1, or around 2:1 (polysaccharide or O-antigen to carrier protein).
  • the step of reacting the oxidised polysaccharide or O-antigen with the carrier protein or carrier protein-linker compound comprises mixing the carrier protein or the carrier protein- linker compound and the reducing agent at a w/w ratio of between 0.25:1 and 20: 1, between 0.5:1 and 10: 1, between 0.75:1 and 5:1, or around 1:1 (carrier protein or carrier protein-linker compound to reducing agent).
  • the step of mixing the oxidised polysaccharide or O-antigen with the carrier protein or carrier protein-linker compound results in a reaction mixture comprising the oxidised polysaccharide or O- antigen and the carrier protein or carrier protein-linker compound.
  • the method may comprise incubating the reaction mixture for between 4 and 20 hours, between 6 and 15 hours, between 7 and 10 hours, or overnight.
  • the method may comprise incubating the reaction mixture at a temperature between 30oC and 42oC, between 33oC and 40oC, between 35oC and 39oC, or around 37oC.
  • the method may further comprise quenching.
  • the quenching quenches residual oxidised sites.
  • the quenching comprises adding a quenching agent. Suitable quenching agents include sodium borohydride and lithium aluminium hydride.
  • the quenching agent is sodium borohydride and the w/w ratio of oxidised polysaccharide or O-antigen to sodium borohydride is between 10:1 and 0.1: 1, between 5:1 and 0.5: 1, or around 1:1 (w/w polysaccharide or O-antigen to sodium borohydride).
  • the conjugate is purified by chromatography such as hydrophobic interaction chromatography. A suitable method for conjugating an oxidised polysaccharide to a carrier protein or a linker is described in Example 6. Conjugates obtained/obtainable by the methods In some aspects of the invention there is provided a conjugate obtained by or obtainable by the methods of the invention. Such conjugates may comprise any of the features of the conjugates described here.
  • conjugates may comprise S. Paratyphi A O-antigen and CRM 197 as the carrier protein.
  • Immunogenic compositions The conjugates of the invention may be comprised within an immunogenic composition.
  • the immunogenic composition may comprise a conjugate of the invention and additional components, such as a pharmaceutically acceptable excipient, an adjuvant, and/or further antigens.
  • the immunogenic composition may further comprise a pharmaceutically acceptable excipient.
  • Typical ‘pharmaceutically acceptable excipients’ include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition.
  • Suitable carriers are typically large, slowly metabolised macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, sucrose, trehalose, lactose, and lipid aggregates (such as oil droplets or liposomes). Such carriers are well known to those of ordinary skill in the art. Pharmaceutically acceptable excipients may also contain diluents, such as water, saline, glycerol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present.
  • the immunogenic composition comprises phosphate buffered saline (and optionally an aluminium adjuvant as described further below).
  • the immunogenic composition comprises phosphate buffered saline at a pH between 6 and 7, for example pH 6.5.
  • Immunogenic compositions may be prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared (e.g.
  • the immunogenic composition may be prepared for topical administration e.g. as an ointment, cream or powder.
  • the immunogenic composition may be prepared for oral administration e.g. as a tablet or capsule, as a spray, or as a syrup (optionally flavoured).
  • the immunogenic composition may be prepared for pulmonary administration e.g. as an inhaler, using a fine powder or a spray.
  • the composition may be prepared as a suppository or pessary.
  • the immunogenic composition may be prepared for nasal, aural or ocular administration e.g. as drops.
  • the immunogenic composition may be in kit form, designed such that a combined composition is reconstituted just prior to administration to a mammal.
  • kits may comprise one or more antigens in liquid form and one or more lyophilised antigens.
  • Immunogenic compositions may be presented in vials, or they may be presented in pre-filled syringes. The syringes may be supplied with or without needles. A syringe will include a single dose of the composition, whereas a vial may include a single dose or multiple doses. Immunogenic compositions of the invention may be packaged in unit dose form or in multiple dose form. For multiple dose forms, vials are preferred to pre-filled syringes. Effective dosage volumes can be routinely established, but a typical human dose of the composition has a volume of 0.5ml e.g. for intramuscular injection. The composition will be sterile.
  • Immunogenic compositions of the invention may be isotonic with respect to humans. Thus, immunogenic compositions of the invention may be useful as vaccines. Vaccines according to the invention may either be prophylactic (i.e. to prevent infection) or therapeutic (i.e. to treat infection), but will typically be prophylactic. Immunogenic compositions used as vaccines comprise an effective amount of antigen(s), as well as any other components, as needed.
  • effective amount i.e. an immunologically effective amount
  • Immunogenic compositions of the invention may include an antimicrobial, particularly when packaged in multiple dose formats.
  • the immunogenic composition comprises a conjugate of the invention at a dose of 1 ⁇ g to 50 ⁇ g, 10 ⁇ g to 50 ⁇ g, 20 ⁇ g to 30 ⁇ g, or around 25 ⁇ g (polysaccharide or O-antigen).
  • the immunogenic composition comprises an fVi conjugate at a dose of 1 ⁇ g to 50 ⁇ g, 10 ⁇ g to 50 ⁇ g, 20 ⁇ g to 30 ⁇ g, or around 25 ⁇ g (fVi).
  • Adjuvants The immunogenic compositions of the invention may comprise an adjuvant. Any suitable adjuvant may be used. However, in some embodiments the adjuvant is a mineral salt, such as an aluminium salt or a calcium salt. Suitable mineral salts include hydroxides (e.g. oxyhydroxides), phosphates (e.g. hydroxyphosphates, orthophosphates), sulphates, etc. or mixtures of different mineral compounds, with the compounds taking any suitable form (e.g.
  • the mineral containing compositions may also be formulated as a particle of metal salt.
  • the adjuvants known as “aluminium hydroxide” are typically aluminium oxyhydroxide salts, which are usually at least partially crystalline.
  • Aluminium oxyhydroxide which can be represented by the formula AlO(OH)
  • AlO(OH) 3 can be distinguished from other aluminium compounds, such as aluminium hydroxide Al(OH) 3 , by infrared (IR) spectroscopy, in particular by the presence of an adsorption band at 1070cm -1 and a strong shoulder at 3090-3100cm -1 [chapter 9 of ref. Vaccine Design: The Subunit and Adjuvant Approach (eds.
  • the aluminium salt comprises or consists of aluminium phosphate and/or aluminium hydroxide.
  • the aluminium salt comprises or consists of aluminium hydroxide.
  • the immunogenic compositions comprise aluminium hydroxide at a dose of 0.05 mg to 1.00 mg, 0.05 mg to 0.50 mg, 0.30 mg to 0.40 mg, or around 0.375 mg (Al 3+ ).
  • Salmonella Typhi antigen The immunogenic composition may further comprise an antigen from Salmonella typhi (S. typhi).
  • the antigen from S. Typhi is a Vi polysaccharide.
  • Vi or “Vi polysaccharide” relates to the capsular polysaccharide of Salmonella enterica serovar Typhi purified from Citrobacter (Rondini et al., J. Infect. Dev. Ctries, 2012).
  • the Vi polysaccharide is a fragmented Vi polysaccharide (fVi).
  • fragmented in reference to the Vi polysaccharide refers to the Vi polysaccharide having undergone size reduction thus reducing the number of repeating units in the polysaccharide. Fragmented Vi therefore has a lower average molecular weight compared to native Vi.
  • fragmented Vi may comprise 30 to 300 repeating units, compared to over 600 repeating units for native Vi.
  • a structure of Vi monomeric repeating unit is shown in Figure 7.
  • the fragmented Vi preferably no changes in the structure of the repeating unit is observed compared to native Vi. This can be confirmed by 1H NMR analysis (see WO2015/068129).
  • the percentage of O-acetyl groups in the fragmented Vi is preferably the same as the native Vi (i.e. about 95% O-acetylation) but may vary and decrease to about 65% O-acetylation.
  • O- acetylation can be determined by standard measurements such as 1H NMR or the Hestrin colorimetric method.
  • the Vi polysaccharide In its native size, the Vi polysaccharide has an average molecular weight measured by HPLC size exclusion chromatography (HPLC-SEC) of about 165kDa.
  • HPLC-SEC HPLC size exclusion chromatography
  • the fVi polysaccharide has an average molecular weight of between 10 kDa and 90 kDa, between 25 kDa and 70 kDa, between 40 kDa and 55 kDa, between 41 kDa and 49 kDa, or between 51 kDa and 55 kDa.
  • the fVi polysaccharide has a target molecular weight of between 51 kDa and 55 kDa (e.g.
  • the molecular weight of the Vi polysaccharide may be determined by HPLC-SEC. Typically, the average molecular weight is calculated by running the sample on a TSK gel 3000 PWXL column, (30 cm x 7.8 mm; particle size 7 ⁇ m; cod. 808021) with a TSK gel PWXL guard column (4.0 cm x 6.0 mm; particle size 12 ⁇ m; cod.
  • the fVi polysaccharide used in the present invention have certain average molecular weight (avMW) range distributions which can be further characterized in terms of polydispersity index (PDI).
  • avMW average molecular weight
  • PDI polydispersity index
  • the fVi polysaccharide may have an avMW distribution characterised in that at least 80% of the pool has an avMW between 25 kDa and 70 kDa.
  • Fragmentation of the Vi polysaccharide may be carried out by a number of methods known in the art such as chemical hydrolysis of the native polysaccharide, enzymatic fragmentation of the native polysaccharide, gamma irradiation of the native polysaccharide, or mechanical methods such as sonication, or high pressure homogenizer/microfluidizer/HPCDS (High pressure cell disruption system) of the native polysaccharide.
  • the fragmentation method used in the present invention is selected such that it can yield fVi polysaccharide having an avMW of less than 90 kDa, less than 80 kDa, less than 60 kDa, or between 40 and 55 kDa.
  • the method may also be selected such that there are no alterations to the repeating units' structure.
  • fragmentation is not by mechanical methods.
  • fragmentation is not by alkaline hydrolysis.
  • the fVi polysaccharide may be obtained by chemical hydrolysis with hydrogen peroxide. Using this method, it was found that the Vi polysaccharide could be reduced in size without altering the repeating units' structure.
  • the fVi polysaccharide may be part of an fVi conjugate comprising fVi and a carrier protein.
  • the carrier protein in the fVi conjugate is tetanus toxoid, CRM 197 , or diphtheria toxoid.
  • the carrier protein is CRM 197 .
  • the fVi polysaccharide may be conjugated to the carrier protein via any suitable conjugation chemistry.
  • Conjugation of the fVi polysaccharide to the carrier protein may be via a -NH2 group, e.g., through the side chain(s) of a lysine residue(s) or arginine residue(s) in the carrier polypeptide.
  • this group can react with an amine in the protein to form a conjugate by reductive amination.
  • Conjugation to the carrier may also be via a -SH group, e.g., through the side chain(s) of a cysteine residue(s) in the carrier polypeptide.
  • the fVi polysaccharide may be conjugated to the carrier protein via a linker molecule.
  • the fVi polysaccharide will typically be activated or functionalised prior to conjugation. Activation may involve, for example, cyanylating reagents such as CDAP (l-cyano-4-dimethylamino pyridinium tetrafluoro borate).
  • cyanylating reagents such as CDAP (l-cyano-4-dimethylamino pyridinium tetrafluoro borate).
  • Other suitable techniques use carbodiimides, hydrazides, active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU (see, e.g., the introduction to WO 98/42721).
  • Direct conjugation to the carrier protein may comprise oxidation of the fVi polysaccharide followed by reductive amination with the protein, as described in, for example, U.S. Pat No. 4,761,283 and U.S. Pat No. 4,356,170.
  • Conjugation via a linker group may be made using any known procedure, for example, the procedures described in U.S. Pat No. 4,882,317 and U.S. Pat No. 4,695,624.
  • the linker is attached via an anomeric carbon of the polysaccharide.
  • a preferred type of linker is an adipic acid linker, which may be formed by coupling a free -NH2 group (e.g., introduced to a polysaccharide by amination) with adipic acid (using, for example, diimide activation), and then coupling a protein to the resulting saccharide-adipic acid intermediate (see, e.g., EP-B-0477508, Mol. Immunol, (1985) 22, 907-919, and EP-A-0208375).
  • a similar preferred type of linker is a glutaric acid linker, which may be formed by coupling a free -NH group with glutaric acid in the same way.
  • Adipic and glutaric acid linkers may also be formed by direct coupling to the polysaccharide, i.e., without prior introduction of a free group, e.g., a free -NH group, to the polysaccharide, followed by coupling a protein to the resulting saccharide-adipic/glutaric acid intermediate.
  • a free group e.g., a free -NH group
  • Another preferred type of linker is a carbonyl linker, which may be formed by reaction of a free hydroxyl group of a modified polysaccharide with CDI (Bethell G.S. et al. (1979) J. Biol. Chem. 254, 2572-4 and Hearn M.T.W. (1981) J. Chromatogr.
  • linkers include ⁇ -propionamido (WO00/10599), nitrophenyl-ethylamine (Gever et al. (1979) Med. Microbiol. Immunol. 165, 171-288), haloacyl halides (U.S. Pat. No. 4,057,685), glycosidic linkages (U.S. Pat. Nos. 4,673,574; 4,761,283; and 4,808,700), 6- aminocaproic acid (U.S. Pat. No.
  • a bifunctional linker may be used to provide a first group for coupling to an amine group in the polysaccharide (e.g., introduced to the polysaccharide by amination) and a second group for coupling to the carrier (typically for coupling to an amine in the carrier).
  • the first group is capable of direct coupling to the polysaccharide, i.e., without prior introduction of a group, e.g., an amine group, to the polysaccharide.
  • the fVi conjugate is obtained by or obtainable by a method (i.e. a method for preparing an fVi conjugate) comprising the steps of: a.
  • fragmenting Vi polysaccharide to obtain a fragmented Vi (fVi) polysaccharide having an average molecular weight of between 10 kDa and 90 kDa, between 25 kDa and 70 kDa, between 40 kDa and 55 kDa, between 41 kDa and 49 kDa, or between 51 kDa and 55 kDa; b. reacting the fVi polysaccharide obtained in step a. with a carbodiimide and N- hydroxysuccinimide at a pH of 5 to 6 to form an N-hydroxysuccinimide ester fVi derivative; and c.
  • fVi fragmented Vi
  • the carrier protein may be derivatised by reacting it with a carbodiimide and a linker.
  • the carbodiimide is 1-ethyl-3-(3-Dimethylaminopropyl) carbodiimide (EDAC).
  • EDAC 1-ethyl-3-(3-Dimethylaminopropyl) carbodiimide
  • Any suitable linker (such as those discussed above) may be used.
  • the linker is an ADH linker.
  • derivatising the carrier protein produces a derivatised carrier protein.
  • the carrier protein is CRM 197 and derivatising the carrier protein comprises one or more of the following steps: (i) providing CRM 197 is an appropriate buffer, optionally MES buffer; (ii) mixing the CRM 197 with EDAC at a ratio between 1:0.05 and 1:0.5, between 1:0.1 and 1:0.3, or around 1:0.15 (w/w CRM 197 to EDAC); (iii) mixing the CRM 197 with ADH at a ratio of between 1:1 and 1:6, between 1:2 and 1:4, or around 1:3.5 (w/w CRM 197 to ADH); (iv) incubating a mixture of CRM 197 and EDAC and optionally ADH for at least 30 minutes, or between 30 minutes and 2 hours, optionally with stirring; and (v) purifying derivatised CRM 197 , optionally by tangential flow filtration.
  • the carrier protein is derivatised by a method that comprises steps (i), (ii), and (iv). In some embodiments, the carrier protein is derivatised by a method that comprises steps (i), (ii), (iii), and (iv). In some embodiments, the carrier protein is derivatised by a method that comprises steps (i), (ii), (iv), and (v). In some embodiments, the carrier protein is derivatised by a method that comprises all of steps (i) to (v) above. In some embodiments, steps (i) to (v) above are performed in the order set out above, except that steps (ii) and (iii) may be performed simultaneously.
  • the fVi conjugate may be obtainable or obtained by a method comprising a step of reacting the fVi polysaccharide with a carbodiimide and N-hydroxysuccinimide at a pH of 5 to 6 to form an N-hydroxysuccinimide ester fVi derivative.
  • the carbodiimide is EDC (N-3-dimethylamino propyl(-N-ethyl carbodiimide).
  • reacting the fVi polysaccharide with a carbodiimide and N-hydroxysuccinimide comprises mixing the fVi with a carbodiimide such as EDC in the presence of N- hydroxysuccinimide (NHS).
  • reacting the fVi polysaccharide with a carbodiimide and N-hydroxysuccinimide comprises mixing the fVi polysaccharide with NHS.
  • reacting the fVi polysaccharide with a carbodiimide and N- hydroxysuccinimide comprises mixing the fVi polysaccharide with NHS such that the NHS concentration is between 0.1 M and 0.5M, or around 0.33M, and the fVi polysaccharide concentration is between 1 mg/mL and 100 mg/ml, or around 50 mg/ml.
  • reacting the fVi polysaccharide with a carbodiimide and N- hydroxysuccinimide comprises mixing the fVi polysaccharide with EDC to have a molar ratio of EDC to fVi repeating unit or between 1:1 and 20:1, between 1:1 and 10:0, between 2:1 and 7:1, or around 5:1.
  • mixing the fVi polysaccharide with EDC is carried out after mixing the fVi polysaccharide with NHS.
  • reacting the fVi polysaccharide with a carbodiimide and N-hydroxysuccinimide comprises a step of incubating a mixture of fVi polysaccharide, NHS and EDC for at least 30 minutes, or around 1 hour at room temperature.
  • reacting the N-hydroxysuccinimide ester fVi derivative with the carrier protein comprises mixing the N- hydroxysuccinimide ester fVi derivative with the carrier protein (or carrier protein derivative).
  • reacting the N-hydroxysuccinimide ester fVi derivative with the carrier protein comprises mixing the N- hydroxysuccinimide ester fVi derivative with the carrier protein (or carrier protein derivative) at a ratio of between (w/w) 1:0.1 and 1:10, between 1:0.5 and 1:5, between 1:0.75 and 1:2, or around 1:1.
  • mixing the the N-hydroxysuccinimide ester fVi derivative with the carrier protein (or carrier protein derivative) is carried out in a buffer at a pH between 5 and 7, or around 6.
  • mixing the N- hydroxysuccinimide ester fVi derivative with the carrier protein (or carrier protein derivative) is carried out in MES buffer.
  • mixing the N-hydroxysuccinimide ester fVi derivative with the carrier protein (or carrier protein derivative) is carried out at a temperature between 20oC and 30oC or around room temperature, optionally with mixing.
  • the method for preparing an fVi conjugate may comprise one or more of the following additional steps, after the step of reacting the N-hydroxysuccinimide ester fVi derivative with the carrier protein (or carrier protein derivative): (i) quenching by addition of a quencher such as Phenyl HP buffer; (ii) filtering the fVi conjugate; (iii) purifying the fVi conjugate, optionally using hydrophobic interaction chromatography; (iv) concentrating the fVi conjugate, optionally by tangential flow filtration; and (v) filtering the conjugate, optionally using one or more 0.2 ⁇ m filters.
  • a quencher such as Phenyl HP buffer
  • filtering the fVi conjugate optionally using hydrophobic interaction chromat
  • the method for preparing an fVi conjugate comprises 2 or more, 3 or more, 4 or more, or all 5 of steps (i) to (v) above.
  • the method comprises step (i) above.
  • the method comprises steps (i) to (iii) above.
  • the method comprises steps (i) to (v) above.
  • the method comprises steps (i) to (iii) above in the order recited above.
  • the method comprises steps (i) to (v) above in the order recited above.
  • a suitable method for conjugating the fVi polysaccharide to CRM 197 using EDAC chemistry via an ADH linker is set out in Example 2.
  • the immunogenic composition comprises (i) a conjugate comprising O- antigen from S. Paratyphi A and CRM 197 and (ii) a conjugate comprising fVi polysaccharide and CRM 197 at a (w/w) ratio of between 1:0.1 and 1:10, between 1:0.5 and 1:5, between 1:0.75 and 1:3, or around 1:1 (O-antigen to fVi).
  • Stability assay The stability of the conjugates of the invention (optionally within an immunogenic composition of the invention) (e.g.
  • the amount of polysaccharide or O-antigen released from the conjugate after 4 weeks) may be determined using the following stability assay: (i) incubate the immunogenic composition for 4 weeks at 37oC; (ii) prepare a post-incubation sample of the incubated immunogenic composition and remove the conjugated polysaccharide or O-antigen by deoxycholate precipitation; and (iii) determine the amount of free polysaccharide or O-antigen in the post- incubation sample as a percentage of the amount of total polysaccharide in the post-incubation sample.
  • Incubating the immunogenic composition for 4 weeks at 37oC is a simple step in which the immunogenic composition is simply maintained at 37oC for 4 weeks.
  • the immunogenic composition comprises a conjugate comprising O-antigen conjugated to CRM 197 and an fVi conjugate
  • the user will transfer a sample of that immunogenic composition to an appropriate vessel (such as a syringe with a single dosage) and store it at 37oC.
  • the post-incubation sample(s) may be a sample of any suitable size (i.e. any size that allows the user to accurately detect the amount of free polysaccharide or O-antigen in the immunogenic composition).
  • the amount of free polysaccharide or O-antigen in the post-incubation sample may be determined by removing the intact conjugate and measuring the amount of polysaccharide or O-antigen remaining. This involves deoxycholate precipitation.
  • the deoxycholate will precipitate out the carrier protein, and any polysaccharide or O- antigen that is still part of a conjugate will be precipitated out with the carrier protein.
  • the precipitated protein may be removed by centrifugation.
  • the conjugate carrier protein, and any polysaccharide or O-antigen that is still part of a conjugate
  • the user should then examine the amount of polysaccharide or O-antigen that is remaining in the sample (post-incubation) once the polysaccharide or O-antigen that is still part of a conjugate has been removed.
  • This may be achieved using a Phenol- sulphuric assay or preferably high performance anion exchange chromatography with pulsed amperometric detection (HPAED-PAD).
  • HPAED-PAD pulsed amperometric detection
  • the Phenol-Sulphuric Assay quantifies the total amount of detectable sugar in the sample as hexose weight equivalent (glucose calibration curve).
  • the assay uses concentrated sulphuric acid and phenol.
  • hexose monosaccharides (glucose standard or the ones coming from O:2 sample hydrolysis) form hydroxymethyl furfurals that react with phenol resulting in the production of chromophores (Molisch reaction).
  • Sample and glucose standard solutions absorbance at 490 nm is read using a spectrophotometer.
  • Glucose standard solution at several dilutions (0, 25, 50, 75, 100 ⁇ g/mL) is treated as the sample in the assay and used as a calibration curve.
  • HPAED-PAD can be used to detect the amount of detectable sugars, such as the amounts of the rhamnose (rha), galactose (gal), glucose (glc) and mannose (man) of the S. Paratyphi A O-antigen repeating unit. Commercial monomer sugars may be used to build calibration curves.
  • a suitable HPAED-PAD method is described in more detail in PLoS One. (2012): 7(11):e47039. For example, the following method may be used to detect amounts of the S. Paratyphi O-antigen. For detecting amounts of Rha, Gal, Glc in the S.
  • the column After washing for 20 min with 100 mM AcONa in 28 mM NaOH, the column may be re-equilibrated with 18 mM NaOH for 20 min.
  • the effluent may be monitored using an electrochemical detector in the pulse amperometric mode with a gold working electrode and an Ag/AgCl reference electrode.
  • the Dionex standard quadruple-potential waveform for carbohydrates may be used.
  • the resulting chromatographic data may be processed using Chromeleon software 6.8. Calibration curves may be built for each sugar monomer (0.5–10 ⁇ g/mL). The standards may be hydrolysed and analysed in the same way as samples.
  • the user can then calculate the amount of free polysaccharide or O-antigen in the sample as a percentage of the total amount of polysaccharide in the sample.
  • concentration of conjugate in the immunogenic composition and can readily calculate the amount of conjugate in the sample by determining the volume of the conjugate in the sample and multiplying the volume by the concentration.
  • Stability The conjugates of the invention have increased stability compared to conjugates of the invention prepared using non-random conjugation methods.
  • the conjugate may be stable in the immunogenic composition for at least 4 weeks (at 37oC).
  • an immunogenic composition of the invention may comprise a conjugate of the invention (comprising a polysaccharide comprising a KDO moiety or an O-antigen) and an fVi conjugate, and in these embodiments the conjugate of the invention will be stable in the immunogenic composition in the presence of the fVi conjugate.
  • the immunogenic composition may comprise a conjugate comprising an S. Paratyphi A O-antigen and CRM 197 and an fVi conjugate, and the conjugate comprising an S. Paratyphi O-antigen and CRM 197 is stable in the composition for at least 4 weeks.
  • the conjugate (comprising the polysaccharide comprising a KDO moiety or the O-antigen) is stable in the immunogenic composition for at least 4 weeks if less than 20%, less than 18%, less than 17%, less than 15%, between 0% and 20%, or between 1% and 15% of the polysaccharide or O-antigen is released from the conjugate in 4 weeks.
  • the amount of polysaccharide or O-antigen released from the conjugate in 4 weeks may be determined using a stability assay as set out above.
  • the conjugate (comprising the polysaccharide comprising a KDO moiety or the O-antigen) is stable in the immunogenic composition for at least 4 weeks, if the amount of polysaccharide or O-antigen released from the conjugate in 4 weeks is substantially less than an equivalent immunogenic composition in which the conjugate comprises an S. Paratyphi O-antigen conjugated to CRM197 using direct reductive amination via an ADH-SIDEA linker.
  • equivalent immunogenic composition should contain the same excipients etc. as the immunogenic composition, and the only difference should be the nature of the conjugate in the immunogenic composition.
  • the conjugate is stable in the immunogenic composition for at least 4 weeks, if the amount of polysaccharide or O-antigen released from the conjugate in 4 weeks is similar to the amount released in an equivalent reference immunogenic composition in which the conjugate is an S. Paratyphi O-antigen conjugated to CRM 197 using CDAP chemistry.
  • similar to is within 25%, within 15%, or within 10% of.
  • Immunogenicity The Examples demonstrate that the conjugates of the invention are not only stable, but also have good immunogenicity.
  • the immunogenic composition induces a similar level of anti-polysaccharide or anti-O-antigen antibodies compared to an equivalent immunogenic composition in which the conjugate comprises a polysaccharide or O-antigen conjugated to a carrier protein using a selective (non- random) conjugation method.
  • the immunogenic composition induces a similar level of anti-polysaccharide or anti-O-antigen antibodies compared to an equivalent immunogenic composition in which the conjugate comprises the polysaccharide or O-antigen conjugated to a carrier protein using selective reductive amination, such as the reductive amination method described in Example 1.
  • the immunogenic composition induces a similar level of anti-S.
  • Paratyphi O-antigen antibodies compared to an equivalent immunogenic composition in which the conjugate comprises an S. Paratyphi O-antigen conjugated to CRM 197 using direct reductive amination via an ADH-SIDEA linker.
  • An immunogenic composition should be considered to be one that “induces” antibodies if it is capable of inducing antibodies, i.e. if the immunogenic composition has the structural features of an immunogenic composition that can induce antibodies if it is administered to a mammal such as a mouse. Whether an immunogenic composition “induces” antibodies may be determined by administering a sample of the immunogenic composition to a mouse and determining whether the relevant antibodies are raised. Optionally, a similar level is a level within 25%, within 15%, or within 10%.
  • a user may determine whether the immunogenic composition is one that induces anti-polysaccharide or anti-O-antigen antibodies by measuring the level of anti- polysaccharide or anti-O-antigen antibodies induced using the following immunogenic assay: (i) immunise mice at days 0 and 28 with the conjugate subcutaneously at a dose of 25 ⁇ g (polysaccharide); (ii) measure the anti-polysaccharide antibody level by ELISA at day 42. If the immunogenic composition comprises an S.
  • the level of anti-O-antigen antibodies induced may be measured using the following immunogenic assay: (i) immunise mice at days 0 and 28 with the conjugate subcutaneously at a dose of 25 ⁇ g (O-antigen); (ii) measure the anti-O-antigen antibody level by ELISA at day 42.
  • a suitable ELISA assay involves: - coating ELISA plates with the polysaccharide or O-antigen; - taking serum samples from the immunised mice at day 42 (42 days after first immunisation), and applying them to the plates; and - measuring the antibody titers by adding an anti-mouse Fc antibody conjugated with an enzyme and the color substrate.
  • the immunogenic composition may comprise further antigens.
  • the further antigens comprise Salmonella antigens.
  • the further antigens comprise a Salmonella Typhimurium antigen, and/or a Salmonella Enteritidis antigen.
  • the Salmonella Typhimurium antigen, and/or the Salmonella Enteritidis antigen are outer membrane vesicles.
  • the immunogenic composition comprises S. Typhimurium GMMA and/or S. Enteritidis GMMA.
  • the S. Typhimurium GMMA and/or the S. Enteritidis GMMA comprise detoxified lipid A.
  • Typhimurium GMMA and/or the S. Enteritidis GMMA are derived from bacteria that do not comprise a gene encoding a functional MsbB and/or PagP protein.
  • the S. Typhimurium GMMA and/or the S. Enteritidis GMMA are derived from bacteria that are modified to at least partially delete the msbB and/or pagP gene.
  • the S. Typhimurium GMMA and/or the S. Enteritidis GMMA are ⁇ msbB and/or ⁇ pagP.
  • the S. Typhimurium GMMA and/or the S. Enteritidis GMMA are derived from bacteria that are hyperblebbing.
  • Typhimurium GMMA and/or the S. Enteritidis GMMA are derived from bacteria that do not comprise a gene encoding a functional TolR protein.
  • the S. Typhimurium GMMA and/or the S. Enteritidis GMMA are derived from bacteria that are modified to at least partially delete the tolR gene.
  • the S. Typhimurium GMMA and/or the S. Enteritidis GMMA are ⁇ tolR. Medical uses and methods of treatment
  • an immunogenic composition of the invention for use in a method of preventing an infection.
  • a method of preventing an infection comprising administering an effective amount of the immunogenic composition or vaccine of the invention to a subject.
  • the method of preventing an infection may comprise administering an effective amount of the immunogenic composition or vaccine of the invention to a subject.
  • the method of preventing an infection may be a method of preventing Salmonella infection, optionally an invasive non-typeable Salmonella infection.
  • the method of preventing an infection is a method of preventing infection by S. Typhimurium, S. Enteritidis, S. Typhi and/or S.
  • the term “preventing Salmonella infection” in the method/immunogenic composition for use/use of the immunogenic composition in the manufacture of a medicament of the invention comprises raising an immune response in a subject.
  • the immune response may be protective and may raises antibodies, such as IgG antibodies.
  • the subject of the invention is a mammal, optionally a human.
  • the human may be an adult i.e. subject is 18 years old or above 18 years old.
  • the vaccine is for prophylactic use
  • the human may be a child i.e. below 18 years old.
  • the vaccine is for prophylactic use, the child may be between 12 to 72 months, preferably between 24 to 59 months, more preferably between 6 to 12 months.
  • the child may be around 9 months.
  • the human is preferably a child.
  • a vaccine intended for children may also be administered to adults e.g. to assess safety, dosage, or immunogenicity.
  • Examples Example 1 - Production of S. Paratyphi OAg – CRM 197 conjugates via an ADH-SIDEA linker through reductive amination Conjugates of the S. Paratyphi A O-antigen (also referred to as O:2) and CRM 197 (via an ADH-SIDEA linker) were produced using the following protocol. The method used for producing S.
  • OAg O antigen-CRM197 conjugates via an ADH-SIDEA linker through reductive amination
  • S. Paratyphi O-antigen (OAg) is solubilized in 100 mM AcONa pH 4.5 at a concentration of 20–40 mg/mL, ADH and then NaBH 3 CN are added in sequence as solids to have a ratio of OAg/ADH/ NaBH 3 CN 1 : 2 : 2 w/w/w. After mixing the solution at 30 °C for 1 hour (h), the reaction mixture is desalted against water using a G- 25 column.
  • OAg-ADH is dried, solubilized in water/DMSO 1:9 (v/v) at a concentration of about 50 mg/mL (O-Ag), triethylamine (TEA) and SIDEA (see Figure 1 for the SIDEA structure) are added to have an NH 2 (OAg) : TEA : SIDEA molar ratio of 1 : 5 :12, and the reaction is kept at room temperature for 3h. Adding twice the reaction volume of 100 mM citrate buffer pH 3, unreacted SIDEA precipitates and is discarded after centrifugation.
  • OAg-ADH-SIDEA is isolated from the reaction mixture by precipitation adding ethanol up to 80% (v/v) and is recovered after centrifugation.
  • OAg-ADH- SIDEA is solubilized in phosphate buffer pH 7.2 with CRM 197 to give a protein concentration of 20 mg/mL and a molar ratio of active ester groups on OAg-ADH- SIDEA to CRM 197 of 30 to 1.
  • the reaction mixture is mixed at room teemperature for 2h and the conjugate is purified by Size Exclusion Chromatography (SEC) or by Hydrophobic Interaction Chromatography (HIC).
  • SEC Size Exclusion Chromatography
  • HIC Hydrophobic Interaction Chromatography
  • OAg-CRM 197 conjugate made as described above and a conjugate of fragmented Vi polysaccharide from S. Typhi (fVi) conjugated to CRM 197 .
  • 1) Fragmentation of Vi Polysaccharide Step 1: Fragmentation Reaction and Quenching: Vi-Polysaccharide fragmentation is achieved by an Oxidation-reaction using hydrogen peroxide in the presence of iron sulphate. The reaction is quenched with EDTA (Ethylenediaminetetraacetic acid). Native Vi polysaccharide is diluted with WFI.
  • EDTA Ethylenediaminetetraacetic acid
  • Step 2 Buffer Exchange: Buffer exchange is performed by Tangential Flow Filtration (TFF) with 100mM Sodium phosphate (pH: 7.2 ⁇ 0.2) using 30 kDa cassettes to remove residual H 2 O 2 . Fragmented Vi (fVi) polysaccharide is concentrated.
  • Step 3 Stabilization of fVi polysaccharide: Post fragmentation, 30 kDa retentate is stabilized by incubating at 80 ⁇ 5° C for 120 +/- 15 min.
  • Step 4 fVi Purification by Anion Exchange (Resin: Capto-Q): AA chromatography step is used to separate the desired molecular size of fVi polysaccharide (25-70 KDa). This is performed using a linear gradient elution, with Capto-Q Buffer A and Capto-Q Buffer B using a Capto-Q Resin which has the binding capacity of 13 mg of fVi/mL.
  • the eluted fractions are collected based on the conductivity for every 1 mS/cm; i.e., from 35 to 50 mS/cm and estimating the Molecular size distribution by SEC/HPLC.
  • the Capto- Q fractions are pooled based on the Molecular Size (kDa) distribution.
  • Step 5 Desalting: Pooled Capto-Q fractions are concentrated by Tangential Flow Filtraion (TFF) using a 10 kDa Cut-off cassette and then dia-filtered using WFI until permeate conductivity reaches ⁇ 30 ⁇ S/cm.
  • Step 6 0.2 ⁇ m Filtration of fVi polysaccharide: The fVi polysaccharide is filtered through a 0.22 ⁇ m filter. The purified fVi polysaccharide is stored in PETG bottles. 2) CRM197 Derivatization: Step1: Thawing of CRM197: Purified CRM197 is thawed at 2-8°C prior to buffer exchange with 100 mM MES (Morpholino Ethanesulfonic acid) buffer. Post thawing, CRM 197 is filtered using 0.5 ⁇ m filter.
  • MES Magneto Ethanesulfonic acid
  • Step 2 Buffer Exchange with 100 mM MES Buffer: Buffer exchange is performed by TFF with 100 mM MES buffer (pH 6.0 ⁇ 0.2) using 10 KDa cassette after CRM 197 thawing.
  • Step 3 CRM 197 Derivatization: The required concentration of CRM 197 is diluted with 100 mM MES buffer followed by addition of calculated quantity of ADH (Adipic Acid Dihydrazide) and EDAC (1-Ethyl-3-(3-Dimethylaminopropyl) carbodiimide) to make a CRM : ADH : EDAC 1 : 3.5 : 0.15 w/w/w ratio.
  • ADH Adipic Acid Dihydrazide
  • EDAC 1-Ethyl-3-(3-Dimethylaminopropyl) carbodiimide
  • Step 4 Purification of CRM 197 : Post reaction, CRM 197 is purified by TFF using a 10 KDa cassette with 5 mM MES buffer. Step 5: Filtration of Dia-Filtered CRM 197 : 0.2-micron filtration is performed for dia- filtered CRM 197 solution followed by storage at 2-8°C in glass bottle.
  • Step 1 fVi polysaccharide drying by Rota Vapor: fVi is further concentrated by drying at 30°C using rotavapor. Concentrated fVi polysaccharide is reconstituted by using 100 mM MES buffer (pH: 6.0) in order to get a 50 mg/mL concentration.
  • Step 2 Activation of fVi polysaccharide with NHS: fVi carboxylates (-COOH) are activated with EDC (N-3-Dimethylamino propyl-N Ethyl Carbodimide) in the presence of N-hydroxysuccinimide (NHS), by forming an active ester intermediate, to increase the efficiency of conjugation with CRM 197 previously activated with ADH.
  • EDC N-3-Dimethylamino propyl-N Ethyl Carbodimide
  • NHS N-hydroxysuccinimide
  • the dried fVi polysaccharide is re-constituted to a desired concentration (50 mg/mL) with 100 mM MES buffer (pH: 6.2 ⁇ 0.2); and activated in the presence of NHS (concentration of 0.33 M) followed by EDAC addition to have an EDAC /fVi RU molar ratio of 5 :1.
  • Step 1 Conjugation of fVi with CRM 197 -ADH: The conjugation reaction creates a covalent bond between the activated fVi and CRM 197 -ADH.
  • the activated and derivatized reaction mixture is diluted with 100mM MES pH: 6.0 and CRM 197 -ADH is added in a w/w ratio of 1 : 1 (fVi : CRM 197 ) to reach the final fVi concentration of the activated fVi and CRM 197 -ADH of 5 mg/mL.
  • the conjugation reaction is performed at room temperature with slow mixing until protein consumption is ⁇ 70% measured by HPLC-SEC at 280 nm absorbance.
  • Step 2 Quenching and conditioning of Conjugation Reaction: The conjugation reaction is quenched by adding equal volume of Phenyl HP Buffer-B Tris 50 mM pH: 8.0. NaCl as powder is added to reach a final salt concentration of 3 M.
  • Step 3 Conjugation Mixture Filtration: The fVi-CRM 197 crude conjugate is 0.65 filtered.
  • Step 4 Purification of fVi-CRM 197 Crude Conjugate: Purification of the conjugate from the conditioned reaction mixture is performed through a HIC Phenyl Sepharose High Performance (HP) column.
  • Step 5 Concentration and Buffer Exchange using PBS: Purified conjugate is concentrated by TFF using a 50 kDa Cut-off cassette and then buffer exchanged using PBS buffer until the permeate conductivity meets PBS buffer conductivity.
  • Step 6 Pre Filtration of fVi-CRM197 Conjugate using 0.2 ⁇ m filter: fVi-CRM197 conjugate is filtered through a 0.2 ⁇ m filter for bioburden reduction.
  • Step 7 Sterile Filtration of fVi-CRM 197 Conjugate using 0.2 ⁇ cellulose acetate filter: The fVi-CRM 197 conjugate is filtered through a 0.2 ⁇ m cellulose acetate filter.
  • bivalent formulations with or without an aluminium adjuvant were prepared according to the following protocols. Not adsorbed formulation: (containing 25 ⁇ g fVi, 25 ⁇ g O:2 (O-antigen from S. Paratyphi A, 4 mg 2-PE per dose) Saline solution containing 2-PE is dispensed aseptically into the formulation vessel, the appropriate volume of fVi conjugate antigen (as prepared in Example 2) is added (depending on the number of doses required – see header for dosage) and the solution is stirred for 15-20 minutes. A calculated volume of S.
  • Paratyphi O-Antigen (as prepared in Example 1) is added (depending on the number of doses required – see header for dosage) and the solution is stirred for 15-20 minutes. pH is checked and adjusted to 6.5 ⁇ 0.2. Stirring is continued for 15-20 minutes.
  • the formulated solution is filtered using sterilized 0.22 ⁇ m filter into another sterilized glass bottle aseptically.
  • Adsorbed formulation (containing 25 ⁇ g fVi, 25 ⁇ g O:2, 0.375 mg Al 3+ , 4 mg 2-PE per dose) Saline solution with 2-PE is dispensed aseptically into the formulation vessel, the volume of Vi Antigen is added and the solution is stirred for 15-20 minutes. Calculated volume of O:2 Antigen is added and the solution is stirred for 15-20 minutes.
  • Example 3 Stability study on the bivalent composition produced prepared in Example 2 Samples of a bivalent not adsorbed formulation, composed of fVi-CRM and O:2-CRM (O:2ADH-SIDEA-CRM as per example 1) of the bivalent composition were stored in a fridge at 2-8oC for 12 months, and the presence of free Paratyphi OAg was tested at 0 months, 3 months, 6 months, 9 months and 12 months using the following assay.
  • a phenol sulfuric colorimetric assay is used, performed after protein (conjugate) precipitation with Deoxycholate in acidic conditions.
  • the phenol-Sulfuric Assay quantifies the total amount of detectable sugar in the sample as a hexose weight equivalent (glucose calibration curve).
  • the assay uses concentrated sulfuric acid and phenol. With the addition of sulfuric acid the temperature raises due to acid hydration and the polysaccharide is hydrolized to corresponding sugar monomers. In these conditions hexose monosaccharides (glucose standard or the ones coming from O:2 sample hydrolysis) form hydroxymethyl furfurals that react with phenol resulting in chromophors (Molisch reaction).
  • the stability of the Paratyphi A OAg was assessed using an accelerated stability study performed at 37oC over 4 weeks of time. 1 H NMR and HPLC-SEC data indicated that Paratyphi A OAg sugar composition and chain length did not change over time. 2.
  • the purity of the sodium cyanoborohydride reagent as well as its decomposition kinetic in reaction conditions were assessed using NMR. The NMR spectra indicated that the sodium cyanoborohydride reagent did not contain any significant impurities and a suitable residual quantity is still present after 5h in reaction conditions, and so it seems that impurity of the sodium cyanoborohydride reagent and its decomposition was not the source of the instability (due to incomplete reduction of imine bond in the intermediate of reaction). 3.
  • KDO alpha-keto-acid
  • ADH hydrazide
  • the ADH peak in the sample is quantified by comparison of the ABS 214 nm with a ADH standard calibration curve.
  • Table 2 free ADH percentage in OAg-ADH samples put in accelerated stabilities at 37°C at different pHs Time OAg- OAg- OAg- OAg- OAg- OAg- OAg- ADH in ADH in ADH in ADH in ADH in 20 mM 20 mM 100 mM 100 mM 20 mM 20 mM AcONa, NaPi, 100 HEPES HEPES NaPi, 100 AcONH 4 , 100 mM mM NaCl (pH 7.2) (pH 7.2) mM NaCl 100 mM NaCl (pH 6.5) and CaCl 2 (pH 8) NaCl (pH 5) (10 mM) (pH 9) 0 weeks 2.8 1.3 4.2 3.3 1.8 4.3 2 weeks 8.4 5.5 11.3 10.9 11.4 28.6 4 weeks
  • Example 5 attempts to improve stability by altering the chemistry used in the conjugation Various alternative conjugation chemistries were explored to see whether they could be used to create a more stable OAg-CRM 197 conjugate, as summarised briefly below. 1.
  • the ADH linker was replaced with a DAH (1,6-Diaminehexane) linker. This modification to the original chemistry allows one to determine if the instability is due to the hydrazide linkage or it is present also with an alifatic amine linkage.
  • a stability study based on the study method described in Example 3 was performed on the OAg-DAH-SIDEA-CRM 197 conjugate.
  • the results (percentage free OAg) are shown in Table 6 below.
  • Table 6 0 days 8 days 14 days 28 days 10 24 26 36 2.
  • the ADH linker was removed, and the Paratyphi OAg was conjugated directly to SIDEA-CRM 197 via a PPetN moiety in the core region of the OAg to understand the stability of linkage through pyrophosphate groups.
  • Example 6 Conjugating OAg to CRM 197 using a random oxidation followed by reductive amination approach
  • Paratyphi OAg (O:2) was conjugated to CRM 197 using a random oxidation with periodate followed by reductive amination approach, which introduces multiple linkages between OAg chains and CRM 197 protein molecules, as described in the following paragraphs.
  • the chemistry is described in Figure 3.
  • Oxidation O:210 mg/mL was oxidized (to O:2 ox) with 10 mM NaIO 4 in buffer AcONa 10mM pH 5. The solution was kept at 25 °C in the dark, for 2 hours. After that, NaIO 4 excess was quenched with 20 mM Na2SO3 in H2O.
  • Conjugation mixture was added with NaBH 4 to reach O:2ox : NaBH 4 1 : 1 w/w ratio, to quench residual oxidized free groups. The mixture was maintained at 37 °C for 2 hours.
  • the conjugate was purified through HIC chromatography.
  • a stability study based on the study method described in Example 3 (but measuring free OAg from samples taken at 0 days, 14 days and 28 days; separating free OAg from conjugate with solid phase extraction method with C4 disposable column and quantifying it using HPAEC-PAD analysis on sugar) was performed on the OAg- CRM 197 conjugate produced using random oxidation followed by reductive amination using the method described in Micoli F et al. PLoS One.
  • O:2 OH groups are activated with CDAP, using O:2 to CDAP w/w ratio of 1:0.3 in 150 mM NaCl solution. pH is adjusted to 9-10 with 10% v/v triethylamine and the solution is incubated at room temperature for 3 ⁇ 0.5 minutes on stirring.
  • Activated cyanoester groups of O:2 are covalently bound with hydrazide/amino groups of CRM197ADH/CRM197 to form O:2-CDAP-ADH-CRM197/O:2-CDAP-CRM197.
  • CRM 197 ADH/CRM 197 is added in the equal w/w ratio (1:1) of O:2 at a concentration of 10 mg/mL (final concentration of O:2 and CRM 197 ADH/CRM 197 is 5 mg/mL).
  • the pH is maintained to 9.5 ⁇ 0.5 with 10% triethylamine and the solution is mixed for 2-3 hours at room temperature.
  • 1M glycine solution is then added to an equal volume of conjugation mixture and the pH is adjusted to 8.0 ⁇ 0.2 with 10% triethylamine; the solution is incubated at 2-8°C for 15 ⁇ 5 hours.
  • the crude conjugate is then buffer exchanged and unbound and unreacted O:2 is removed using HIC Phenyl HP resin.
  • a stability study based on the study method described in Example 3 was performed on the OAg-CRM 197 conjugate produced using CDAP chemistry with or without an ADH linker.
  • the results (percentage free OAg) are shown in Table 10 below.
  • Table 10 Conjugate 0 days 8 days 14 days 28 days OAg-CDAP- ⁇ 5 9 10 14 CRM OAg-CDAP- ⁇ 5 7 9 11 ADH-CRM Significantly less free OAg was seen in the stability study compared to OAg-CRM 197 conjugates made using the chemistries described in Examples 2-5.
  • Example 8 Comparison of immunogenicity for bivalent formulations with O:2-CRM 197 produced through ADH-SIDEA or CDAP chemistry.
  • groups of 10 BALB/c mice were immunised subcutaneously at days 0 and 28 with bivalent formulations with a dose of 2.5 ⁇ g (each component (fVi or S. Paratyphi A O-antigen) in 50 ⁇ l without aluminium hydroxide or a dose of 1.25 ⁇ g (each component) with 75 ⁇ g aluminium hydroxide in 50 ⁇ l.
  • the mice were bled at days 28 and 42 and the level of O:2 and Vi IgG (GMT) measured by ELISA.
  • GTT Vi IgG
  • mice were immunized with or without aluminium hydroxide with: - O:2-ADH-SIDEA-CRM 197 (produced by selective reductive amination as in Example 1) - O:2(CDAP)-CRM197 (produced as in Example 7) in each case formulated in bivalent formulations with fVi-CRM 197 (produced as in Example 2 at the doses indicated above).
  • the results obtained are shown in Figure 5.
  • the conjugate involving CDAP conjugation chemistry elicited similar antibody responses and functionality for O:2 with respect to the conjugate employing the ADH-SIDEA chemistry.
  • the CDAP based conjugates were more stable.
  • Example 9 Human clinical trial showing immunogenicity of bivalent formulations with O:2-CRM 197 produced through CDAP chemistry
  • a Phase I, observer-blind, randomised, controlled, single-centre human clinical trial was performed to characterise the safety and immunogenicity profile of a vaccine containing the O:2(CDAP)-CRM 197 conjugate (produced in Example 7) combined with fVi-CRM 197 (produced as in Example 2).
  • the clinical trial has the study number NCT05613205. 96 healthy adults (18-50 years of age) in Europe were administered one of the formulations in Table 11 or the TYPHIM Vi control described below.
  • Blood samples were taken at day 1 (the same day as vaccine administration) and day 29 (i.e. 28 days after the day of vaccine administration).
  • the antibody levels in the samples were examined using ELISA and SBA assays.
  • ELISA and SBA assays For the estimation of anti-Vi IgG, a robust and qualified ELISA method was used. In this method, 96-well ELISA plates were coated with Vi PS diluted in coating buffer (1X PBS) and kept overnight at 2 – 8 °C. The coated plate was washed with wash-buffer and blocked with blocking solution (5% skimmed milk in PBS). After another round of washing, optimally diluted sera samples (in dilution buffer) were added in the designated wells and incubated at 25°C for 2 hours.
  • Serum bactericidal activity against Salmonella Paratyphi A was measured by L-SBA, based on a method previously reported [Necchi, 2017; Rossi, 2020]. Results are expressed in serum titres, defined as serum dilutions giving 50% inhibition of bacterial growth (IC50). The results are provided in Figure 9 to 11. The SBA and ELISA results were used to calculate the percentage of patients having an anti-Vi IgG GMC greater than or equal to 2 ⁇ g/ml (at day 29) and the percentage of patients having an anti-Vi IgG GMC greater than or equal to 4.3 ⁇ g/ml (at day 29), and these data are set out in Tables 12 and 13 below.
  • Glucosylation level was not altered during SEC, while O-acetylation level was partially impacted, decreasing from 60% in the O:2 [16 kDa + 100 kDa] mixed population, to ⁇ 45% in O:2 [16 kDa] and O:2 [100 kDa] ( Figure 12B).
  • Different O:2-CRM 197 conjugates were synthesized using either O:2 [16 kDa] (Conjugates 1-3, Table 13), O:2[100 kDa] (Conjugates 4-6, Table 13), or O:2[16 kDa + 100 kDa] (Conjugates 7-9, Table 13).
  • Anti-O:2 IgG response was determined by ELISA and sera functional activity was determined by their ability to kill S. Paratyphi A bacteria in the presence of complement.
  • All tested conjugates (Table 13), regardless of their specific structural differences, were immunogenic, with a significant booster effect after the second immunization (Figure 13A).
  • Effect of O:2 size O:2-CRM 197 conjugates with a similar O:2/CRM 197 w/w ratio, but differing for O:2 MW (16 kDa, 100 kDa or 100 kDa + 16 kDa) were compared. No significant differences were found among the conjugates after 1 injection.
  • O:2-CRM 197 glycoconjugate in mice Partial de-O-acetylation of O:2 through ammonia treatment Bacterial polysaccharides often contain O-acetyl esters (OAc) which may constitute an important part of the immunodominant epitopes.
  • OAc O-acetyl esters
  • a panel of O:2-CRM 197 conjugates with different O-acetylation levels was synthesized (Table 14). Partial removal of the OAc groups from O:2 was performed before conjugation to CRM 197 by treating O:2 with a weak base as ammonia.
  • the O:2[16 kDa + 100 kDa] mixed population had a starting 60% of O- acetylation.
  • ammonia concentrations range 5 ⁇ 1000 mM
  • NH4OH ammonia
  • a correlation function was established ( Figure 14).
  • Total O:2 de-O- acetylation was obtained by using 1M ammonia.
  • O:2 [16 kDa] was treated with the suitable ammonia concentration, to obtain a gradually de-O-acetylated polysaccharide.
  • O:2-CRM 197 conjugates with different O-acetylation levels
  • a selection of partially de-O-acetylated O:2 were conjugated, resulting in a panel of O:2-CRM 197 conjugates with 45.2%, 35.3% and 18.5 % OAc (conjugates 2, 3 and 4, Table 14).
  • O:2 [16 kDa] and partially de-O- acetylated O:2 [16 kDa] were conjugated to CRM 197 , resulting in two conjugates with 58.5% and 25.2% OAc (conjugates 6 and 7, Table 14).
  • O:2 activation by CDAP, followed by conjugation to CRM 197 did not impact the initial O:2 OAc levels. All conjugates were fully characterized and showed similar O:2/CRM 197 w/w ratio (Table 14) and similar level of cross-linking, as showed by HPLC SEC fluorescence emission profiles. Table 14.
  • O:2 [16 kDa + 100 kDa]
  • conjugates at five different O-acetylation levels were tested.
  • Results were analyzed by performing a regression analysis of ELISA results (log transformed) vs OAc degree. All conjugates were immunogenic, with a booster response after the second immunization ( Figure 15A).
  • ASPECTS 1.
  • a conjugate comprising a polysaccharide comprising a 3-deoxy-D-manno- octulosonic acid (KDO) moiety conjugated to a carrier protein by a random conjugation method, wherein the polysaccharide comprises more than one activated site.
  • KDO 3-deoxy-D-manno- octulosonic acid
  • a conjugate comprising a polysaccharide comprising a KDO moiety conjugated to a carrier protein using a conjugation method comprising a step of: (i) activating the polysaccharide by 1-cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP) chemistry to provide an activated polysaccharide; or (ii) oxidising the polysaccharide to provide an oxidised polysaccharide.
  • CDAP 1-cyano-4-dimethylaminopyridine tetrafluoroborate
  • a conjugate comprising an O-antigen conjugated to a carrier protein using a conjugation method comprising a step of: (i) activating the O-antigen by CDAP chemistry to provide an activated O-antigen; or (ii) oxidising the O-antigen to provide an oxidised O-antigen. 5.
  • a method for producing a conjugate comprising a polysaccharide comprising a KDO moiety conjugated to a carrier protein comprising a step of introducing multiple activated sites into the polysaccharide.
  • a method for producing a conjugate comprising an O-antigen conjugated to a carrier protein comprising a step of introducing multiple activated sites into the O- antigen. 7.
  • a method for producing a conjugate comprising a polysaccharide comprising a KDO moiety conjugated to a carrier protein comprising a step of: (i) activating the polysaccharide by CDAP chemistry to provide an activated polysaccharide; or (ii) oxidising the polysaccharide to provide an oxidised polysaccharide.
  • a method for producing a conjugate comprising an O-antigen conjugated to a carrier protein comprising a step of: (i) activating the O-antigen by CDAP chemistry to provide an activated O-antigen; or (ii) oxidising the O-antigen to provide an oxidised O-antigen.
  • the carrier protein is selected from the group consisting of CRM 197 , tetanus toxoid (TT) or diphtheria toxoid (DT).
  • the carrier protein is CRM 197 .
  • the conjugate or method of any one of the preceding aspects wherein the linker is an adipic acid dihydrazide (ADH) linker. 15. The conjugate or method of aspect 13 or 14, wherein the linker is between the saccharide or O-antigen and the carrier protein. 16. The conjugate or method of any one of aspects 3, 4 or 7 to 15, wherein the activating or oxidising step introduces multiple activated sites into the polysaccharide or O-antigen. 17. The conjugate or method of any one of the preceding aspects, wherein the polysaccharide or O-antigen comprises 1.5 or more, 2.0 or more, or 2.5 or more activated sites or the activating or oxidising step introduces 1.5 or more, 2.0 or more, or 2.5 or more activated sites. 18.
  • ADH adipic acid dihydrazide
  • the conjugate of any one of aspects 1, 2, 5, 6 or 9 to 17, wherein the random conjugation method or introducing multiple activated sites comprises a step of: (i) activating the polysaccharide or the O-antigen by CDAP chemistry to provide an activated polysaccharide or O-antigen; or (ii) oxidising the polysaccharide or the O-antigen to provide an oxidised polysaccharide or O-antigen. 19.
  • activating the O-antigen and/or the polysaccharide by CDAP chemistry comprises mixing the polysaccharide or the O-antigen with CDAP at a w/w ratio of between 0.05:1 and 5:1, between 0.1:1 and 5:1, between 0.2:1 and 2:1, or around 0.3:1 (CDAP to polysaccharide or O-antigen).
  • activating the O-antigen and/or the polysaccharide by CDAP chemistry comprises mixing the O-antigen and/or the polysaccharide comprising a KDO moiety in a solution of NaCl or KCl at a concentration between 50 mM and 1 M, between 100 mM and 250 mM, between 125 mM and 200 mM, or around 150 mM. 21.
  • the conjugate or method of any one of aspects 3, 4, or 7 to 20, wherein activating the O-antigen and/or the polysaccharide by CDAP chemistry comprises adjusting the pH to between 9 and 10 using triethylamine and optionally incubating the solution at room temperature for 1 to 5 minutes with stirring. 22.
  • reacting the activated polysaccharide or O-antigen with hydrazide/amino groups on the carrier protein or the carrier protein-linker compound comprises mixing the activated polysaccharide or O- antigen with the carrier protein or the carrier protein-linker compound at a w/w ratio of between 0.1:1 and 5:1, between 0.2:1 and 3:1, between 0.5:1 and 2:1, or around 1:1 (polysaccharide or O-antigen to carrier protein or carrier protein-linker).
  • the carrier protein is CRM 197 .
  • conjugation method further comprises: (i) a step of adjusting the pH to between 9 and 10 using triethylamine and incubating the solution at room temperature for 1 to 10 hours with stirring after the step of mixing the activated polysaccharide or O-antigen with the carrier protein or the carrier protein-linker compound; and/or (ii) a step of adding glycine solution and adjusting the pH to pH 7 to 9 using triethylamine and incubating the solution at 2-8oC for between 5 and 50, or between 10 and 20 hours; and/or (iii) a step of removing unreacted polysaccharide or O-antigen.
  • the conjugate or method of aspect 26 wherein the step of removing unreacted polysaccharide or O-antigen comprises a step of chromatography, optionally hydrophobic interaction chromatography or anion exchange chromatography.
  • the step of oxidising the polysaccharide or O-antigen comprises mixing the polysaccharide or O- antigen with an oxidising agent, optionally at a pH between 4 and 6. 29.
  • the conjugate or method of aspect 28, wherein the oxidising agent is periodate, optionally sodium periodate.
  • mixing the polysaccharide or O-antigen with an oxidising agent comprises mixing the polysaccharide or O-antigen with an oxidising agent at a ratio of between 1 mg/ml: 10 mM and 100 mg/mL : 10 mM, between 1 mg/mL :10 mM and 50 mg/mL: 10 mM, between 1 mg/mL: 10 mM and 25 mg/mL: 10 mM, or around 10mg/mL:10 mM (polysaccharide or O-antigen to oxidising agent).
  • step of oxidising the polysaccharide or O-antigen comprises: (i) leaving a solution of the polysaccharide or O-antigen and the oxidising agent in the dark at a temperature between 20oC and 30oC for between 1 and 5 hours; and/or (ii) quenching excess oxidising agent, optionally using Na 2 SO 3 ; and/or (iii) desalting the oxidised polysaccharide or O-antigen, optionally with a PD10 column.
  • the step of reacting the oxidised polysaccharide or O-antigen with the carrier protein comprises mixing the oxidised polysaccharide or O-antigen and the carrier protein with a reducing agent.
  • the reducing agent is sodium cyanoborohydride. 35.
  • the conjugate or method of aspect 33 or 34, wherein the step of reacting the oxidised polysaccharide or O-antigen with the carrier protein comprises mixing the oxidised polysaccharide or O-antigen and the carrier protein at a w/w ratio of between 0.5: 1 and 20: 1, between 1:1 and 10: 1, between 1.5:1 and 5:1, or around 2:1 (polysaccharide or O-antigen to carrier protein). 36.
  • conjugate or method of any one of aspects 33 to 35, wherein the step of reacting the oxidised polysaccharide or O-antigen with the carrier protein comprises mixing the carrier protein and the reducing agent at a w/w ratio of between 0.25:1 and 20: 1, between 0.5:1 and 10: 1, between 0.75:1 and 5:1, or around 1:1 (carrier protein to reducing agent). 37.
  • conjugation method further comprises: (i) incubating a reaction mixture comprising the oxidised polysaccharide or O- antigen and the carrier protein at a temperature between 35oC and 39oC for between 4 and 20 hours; (ii) quenching by adding sodium borohydride, optionally wherein the w/w ratio of oxidised polysaccharide or O-antigen to sodium borohydride is between 10:1 and 0.1: 1, between 5:1 and 0.5: 1, or around 1:1 (w/w polysaccharide or O-antigen to sodium borohydride). 38.
  • a conjugate obtainable by the method of any one of aspects 5 to 37. 39.
  • An immunogenic composition comprising the conjugate of any one of aspects 1 to 4 or 9 to 39.
  • the immunogenic composition of aspect 40 further comprising a pharmaceutically acceptable excipient and/or an adjuvant.
  • the immunogenic composition of aspect 40 or 41, wherein the immunogenic composition further comprises an antigen from Salmonella Typhi (S. Typhi), optionally a Vi polysaccharide.
  • S. Typhi Salmonella Typhi
  • Vi polysaccharide is a fragmented Vi polysaccharide (fVi).
  • the immunogenic composition of aspect 43 wherein the fVi polysaccharide has an average molecular weight of between 10 kDa and 90 kDa, between 25 kDa and 70 kDa, between 40 kDa and 55 kDa, between 41 kDa and 49 kDa, or between 51 kDa and 55 kDa.
  • the immunogenic composition of aspect 46, wherein the carrier protein in the fVi conjugate is CRM 197 or diphtheria toxoid.
  • the immunogenic composition of aspect 47, wherein the carrier protein in the fVi conjugate is CRM 197 .
  • the immunogenic composition of any one of aspects 43 to 48, wherein the fVi polysaccharide is conjugated to the carrier protein by carbodiimide chemistry, optionally via a linker.
  • the immunogenic composition of any one of aspects 43 to 49, wherein the fVi conjugate is obtained by or obtainable by a method comprising the steps of: a.
  • fragmenting Vi polysaccharide to obtain a fragmented Vi (fVi) polysaccharide having an average molecular weight of between 10 kDa and 90 kDa, between 25 kDa and 70 kDa, between 40 kDa and 55 kDa, between 41 kDa and 49 kDa, or between 51 kDa and 55 kDa; b. activating the fVi polysaccharide by reacting the fVi polysaccharide obtained in step a. with a carbodiimide and N-hydroxysuccinimide at a pH of 5 to 6 to form an N-hydroxysuccinimide ester fVi derivative; and c.
  • fVi fragmented Vi
  • the immunogenic composition of aspect 55 or 56 wherein the conjugate is stable in the immunogenic composition for at least 4 weeks, if the amount of polysaccharide or O-antigen released from the conjugate in 4 weeks is substantially less than the amount released in an equivalent immunogenic composition in which the conjugate is an S. Paratyphi O-antigen conjugated to CRM 197 using direct reductive amination via an ADH-SIDEA linker.
  • the immunogenic composition of aspect 57 wherein substantially less is at least 10% less, at least 15% less, at least 20% less, or at least 25% less.
  • a similar level is a level within 25%, within 15%, or within 10%.
  • Paratyphi O-antigen antibodies is measured using an immunogenic assay comprising the following steps: (i) immunise mice at days 0 and 28 with the conjugate subcutaneously at a dose of 25 ⁇ g (O-antigen); (ii) measure the anti-S. Paratyphi O-antigen antibody level by ELISA at day 42.
  • the immunogenic composition of any one of aspects 40 to 64 further comprising: (i) a Salmonella Typhimurium (S. Typhimurium) antigen; and/or (ii) a Salmonella Enteritidis (S. Enteritidis) antigen.
  • the immunogenic composition of aspect 67 or 68, wherein the S. Typhimurium GMMA and/or the S. Enteritidis GMMA comprises detoxified lipid A. 70.
  • the immunogenic composition of aspect 69, wherein the S. Typhimurium GMMA and/or the S. Enteritidis GMMA are derived from S. Typhimurium and/or S.
  • Enteritidis that does not comprise: (i) a gene encoding a functional MsbB protein; and/or (ii) a gene encoding a functional PagP protein.
  • 71. The immunogenic composition of any one of aspects 67 to 70, wherein the S. Typhimurium GMMA and/or the S. Enteritidis GMMA are derived from S. Typhimurium and/or S. Enteritidis that does not comprise gene encoding a functional tolR protein.
  • a vaccine comprising the immunogenic composition of any one of aspects 40 to 71.
  • a method of preventing an infection comprising administering an effective amount of the immunogenic composition or vaccine of any one of aspects 40 to 72 to a subject.
  • 75. Use of the immunogenic composition or vaccine of any one of aspects 40 to 72, for the manufacture of a medicament for use in a method of preventing an infection.
  • 76. The immunogenic composition or vaccine for use of aspect 73, or the use of aspect 75, wherein the method of preventing an infection comprises administering an effective amount of the immunogenic composition or vaccine of any one of aspects 40 to 72 to a subject.
  • the immunogenic composition or vaccine for use, method, or use of any one of aspects 73 to 76, wherein the method of preventing an infection is a method of preventing Salmonella infection.
  • the average molecular weight of the O-antigen is between 10 and 25 kDa, between 10 and 20 kDa, 11 kDa and 19 kDa, 12 kDa and 19 kDa, 13 kDa and 19 kDa, 14 kDa and 18 kDa, 15 and 18 kDa, or between 16 kDa and 18 kDa.
  • O-antigen is 10-100%, 20-100%, 30- 100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, or 90-100%.

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Abstract

La présente invention concerne des conjugués comprenant des polysaccharides comportant des fractions d'acide 3-désoxy-D-manno-octulosonique (KDO), en particulier des conjugués produits à l'aide de procédés de conjugaison aléatoire, des procédés de préparation de tels conjugués, des compositions immunogènes et des vaccins comprenant les conjugués, et des méthodes de traitement ou des utilisations médicales faisant appel aux compositions et vaccins.
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