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WO2025021710A1 - Composition immunogène - Google Patents

Composition immunogène Download PDF

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Publication number
WO2025021710A1
WO2025021710A1 PCT/EP2024/070615 EP2024070615W WO2025021710A1 WO 2025021710 A1 WO2025021710 A1 WO 2025021710A1 EP 2024070615 W EP2024070615 W EP 2024070615W WO 2025021710 A1 WO2025021710 A1 WO 2025021710A1
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WIPO (PCT)
Prior art keywords
paratyphi
immunogenic composition
gmma
bacterium
outer membrane
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PCT/EP2024/070615
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English (en)
Inventor
Rocio CANALS ALVAREZ
Gianmarco GASPERINI
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GlaxoSmithKline Biologicals SA
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GlaxoSmithKline Biologicals SA
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Priority claimed from GBGB2311233.7A external-priority patent/GB202311233D0/en
Priority claimed from GBGB2311231.1A external-priority patent/GB202311231D0/en
Priority claimed from GBGB2318238.9A external-priority patent/GB202318238D0/en
Application filed by GlaxoSmithKline Biologicals SA filed Critical GlaxoSmithKline Biologicals SA
Publication of WO2025021710A1 publication Critical patent/WO2025021710A1/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

  • the present invention relates to immunogenic compositions comprising S. enterica serovar Paratyphi A outer membrane vesicles (OMVs), vaccines comprising the immunogenic compositions and methods and uses of the immunogenic compositions.
  • OMVs outer membrane vesicles
  • the present invention also relates to an S. Paratyphi A bacterium comprising modified lipid A.
  • S. Paratyphi A resides in the human gut and its clinical manifestations are indistinguishable from Typhoid fever.
  • S. Paratyphi A is ranked second as a causative agent of enteric fever, preceded only by Salmonella enterica serovar Typhi (5. 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.
  • both the incidence and relative frequency of Paratyphoid fever have risen in Nepal, Pakistan, and Thailand.
  • OMVs outer membrane vesicles
  • an immunogenic composition comprising S. Paratyphi A membrane vesicles.
  • an S. Paratyphi A bacterium comprising modified lipid A.
  • outer membrane vesicles or GMMA obtainable from the S. Paratyphi A bacterium of the invention.
  • outer membrane vesicles or GMMA obtained from the S. Paratyphi A bacterium of the invention.
  • an immunogenic composition comprising the outer membrane vesicles or GMMA of the invention.
  • a vaccine comprising the immunogenic composition of the invention.
  • an immunogenic composition or vaccine of the invention for use in a method of preventing an infection.
  • an eighth aspect of the invention there is provided a method of preventing an infection comprising administering an effective amount of the immunogenic composition or vaccine of the invention.
  • the invention provides 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.
  • Pan-Salmonella vaccine induces specific serum IgG responses against S. Paratyphi A OAg, and S. Typhi Vi and antibodies are bactericidal in mice.
  • Figures 1(a) and (c) show IgG responses at: one day before immunisation (left-hand column), 27 days after immunisation (middle column) and 42 days after immunisation (right-hand column).
  • Figure 1(b) shows SB A results.
  • the left-hand column is one day before immunisation and the right-hand column is 42 days after immunisation.
  • Figures 2(a), (c), (e) and (g) show IgG responses at: one day before immunisation (left-hand column), 27 days after immunisation (middle column) and 42 days after immunisation (right-hand column).
  • Figures 2(b), (d) and (f) show SBA results.
  • the left-hand column is one day before immunisation and the right-hand column is 42 days after immunisation.
  • each bar is for 42 days after immunisation.
  • Top segment is IgG3
  • next segment is IgG2b
  • next segment is IgG2a
  • bottom segment is IgGl.
  • Figures 4(a) and (c) show IgG responses at: one day before immunisation (left-hand column), 27 days after immunisation (middle column) and 42 days after immunisation (right-hand column).
  • Figure 4(b) shows SBA results, where the left-hand column is one day before immunisation and the right-hand column is 42 days after immunisation.
  • Quadrivalent Pan-Salmonella vaccine elicits bactericidal antibodies against a broad panel of Salmonella strains.
  • Panel includes invasive STm isolates from Africa and Southeast Asia and S. enterica serovars other than STm, SEn, ParA, Typhi.
  • the term “comprising” is intended to mean including but not limited to.
  • the phrase “An immunogenic composition comprising Salmonella Paratyphi A outer membrane vesicles” should be interpreted to mean that the immunogenic composition comprises Salmonella Paratyphi A outer membrane vesicles, but the immunogenic composition may comprise further components.
  • the word “comprising” is replaced with the phrase “consisting of.
  • the term “consisting of” is intended to be limiting.
  • the phrase “An immunogenic composition consisting of Salmonella Paratyphi A outer membrane vesicles” should be understood to mean that the immunogenic composition has Salmonella Paratyphi A outer membrane vesicles and no further components.
  • the word “comprising” is replaced with the phrase “consisting essentially of’ .
  • the term “consisting essentially o means that specific further components can be present, namely those not materially affecting the essential characteristics of the subject matter.
  • 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.
  • S. Paratyphi A bacteria all comprise an outer membrane comprising O-antigen.
  • the S. Paratyphi A OMVs (such as GMMA) in the immunogenic compositions of the invention comprise O-antigen.
  • O-antigen OAg and 0:2 are considered to be interchangeable.
  • the outer membrane of gram-negative bacteria comprise a lipopolysaccharide.
  • This lipopolysaccharide comprises an O-antigen, which is linked via the core domain to a lipid A domain.
  • O-antigen “OAg” and “0:2 ” refer to a polysaccharide made up of the O-antigen alone, or more preferably the O-antigen linked to core domain of the lipopolysaccharide.
  • Salmonella serogroups A, B and D have been described and are thought to share a common backbone: — >2-a-D-Man/?-(l ⁇ 4)-a-L-Rha/?-(l— >3)-a-D-Gal/?-(l— >.
  • the serogroup specificity of Salmonella Paratyphi A is conferred by an a-3,6- dideoxyglucose (a-D-paratose) linked (1 — >3) to the mannose of the backbone.
  • the a-L- rhamnose of the backbone is partially O-acetylated at C-3 (Konadu et al. (1996) Infect. Immun. (7):2709-15).
  • the immunogenic compositions of the invention comprise Salmonella enterica subspecies enterica serovar Paratyphi A (S. Paratyphi A) outer membrane vesicles (such as GMMA).
  • Salmonella enterica subspecies enterica serovar Paratyphi A S. Paratyphi A
  • outer membrane vesicles such as GMMA
  • OMVs include native OMVs.
  • Gram-negative bacteria can spontaneously release outer membrane vesicles (OMVs) during growth due to the turgor pressure of the cell envelope, and these are native OMVs.
  • OMVs are rich in immunogenic cell surface- associated, periplasmic and secreted antigens and have been used as vaccines.
  • OMVs of the invention include Generalised Modules for Membrane Antigens (GMMA), native OMVs (‘NOMVs’ (see Katial et al. 2002, Infect Immun, 70: 702-707), microvesicles (MVs (see WO 02/09643)), detergent-extracted OMVs (DOMVs), mutant- derived OMVs (m-OMV), and blebs, which are outer-membrane protrusions that remain attached to bacteria prior to release as MVs (see Beveridge, 1999, J. Bacteriol. 181: 4725- 4733)).
  • GMMA Generalised Modules for Membrane Antigens
  • NOMVs native OMVs
  • MVs microvesicles
  • DOMVs detergent-extracted OMVs
  • m-OMV mutant- derived OMVs
  • blebs which are outer-membrane protrusions that remain attached to bacteria prior to release as MVs (see Beveridge, 1999, J. Bacteriol
  • GMMA Generalised Modules for Membrane Antigens
  • NOMV native outer membrane vesicles
  • the membrane structure has been modified by the deletion of genes encoding key structural components, such as tolR (leading to hyperblebbing).
  • GMMA large quantities of outer membrane “bud off' (or “hyperbleb'''’) to provide a practical source of membrane material for vaccine production.
  • GA/M4 refers to OMVs which are released spontaneously from bacteria modified to hyperbleb (such as S. Paratyphi A bacteria which are modified such that they do not comprise a gene encoding functional TolR).
  • the OMVs in the immunogenic compositions of the invention are derived from S. Paratyphi A bacteria. “Derived’ optionally means “purified’ i.e. the OMVs in the immunogenic compositions of the invention are purified from S. Paratyphi A bacteria. Suitable purification methods are known in the art, and include a variety of filtration and chromatography methods. A preferred two-step filtration purification process is described in WO 2011/036562, herein incorporated by reference.
  • the S. Paratyphi A OMVs are, comprise or consist of GMMA, i.e. S. Paratyphi A GMMA.
  • the A Paratyphi A GMMA may be derived from A Paratyphi A bacteria that comprise modified lipid A.
  • the A Paratyphi A bacterium of the invention may comprise modified lipid A.
  • a modified lipid A is a lipid A that has a different structure compared to a corresponding wild type lipid A.
  • the structure of lipid A may be determined using MALDI-TOF analysis of lipid A isolated from the GMMA. For the assay, the lipid A is separated after treatment of GMMA with acetic acid and then assayed by MALDI-TOF.
  • GMMA with a protein concentration of about 1 mg/mL (micro BCA calibration curve) or a cell bank suspension with an OD600 of about 3 (4 mL sample) are treated with 1% acetic acid (final concentration) for 2 or 6 hours, respectively, at 100°C to obtain a precipitate containing the lipid A.
  • the precipitate is then collected, washed with water and the lipid A is extracted in chloroform / methanol 4:1.
  • the final solution which contains the lipid A, is mixed 1 : 1 with Super DHB (Fluka, 50862) saturated solution (acetonitrile / water 1:1).
  • Two microliters of the mixture are loaded onto the target plate and after the spot is dried at room temperature, the plate is inserted in the mass spectrometer.
  • the spectra (negative reflectron mode) generally show peaks corresponding to the lipid A molecular species and contain several peaks due to fragmentation of the lipid A (i.e. loss of one or more fatty acid chains), sodium adduct (+22 m/z) and lipid A dephosphorylation (- 80 m/z).
  • the species of lipid A is identified by comparison of the molecular peak mass m/z to what is expected for the sample in analysis.
  • the lipid A is modified to be detoxified (i.e. the modified lipid A is detoxified lipid A).
  • “Detoxified' means that the lipid A is less toxic than wildtype lipid A.
  • the wildtype lipid A used in the comparison is a corresponding wildtype lipid A.
  • “Tbxzczty” or “toxic” in this context refers to the extent to which the innate immune system is activated by lipid A, particularly through the Toll-like receptor 4 pathway. Highly toxic lipid A can lead to uncontrolled inflammation, apoptosis, and in extreme cases septic shock, among other effects.
  • a modified lipid A is less toxic if it is less reactogenic than a corresponding wildtype lipid A. For example, one can determine whether a modified lipid A is less toxic by administering it to an animal such as a rabbit, and determining whether it activated more monocytes compared to a corresponding wildtype lipid A using a monocyte activation test.
  • Corresponding wildtype lipid A refers to lipid A that can be found in the corresponding wildtype bacterium and strain.
  • Paratyphi A GMMA is interpreted to mean a lipid A that is modified (e.g. such that it is less toxic) relative to lipid A found in wildtype S. Paratyphi A.
  • the modified lipid A is penta-acylated lipid A.
  • the S. Paratyphi A GMMA may be derived from S. Paratyphi A bacteria that comprise any suitable modification that leads to production of GMMA comprising lipid A that is less toxic than wildtype lipid A.
  • the S. Paratyphi A bacterium may comprise any suitable modification that leads to production of GMMA comprising lipid A that is less toxic than wildtype lipid A.
  • HtrB, MsbB and PagP are proteins that are involved in production of lipid A in Gramnegative bacteria. Of these, MsbB and PagP are important in Salmonella. Salmonella bacteria that do not express functional versions of MsbB and/or PagP will not produce native lipid A, but rather will produce modified, detoxified lipid A. Thus, the S. Paratyphi A GMMA may be derived from S. Paratyphi A bacteria that do not express functional versions of MsbB and/or PagP. Similarly, the S. Paratyphi A bacterium may not express functional versions of MsbB and/or PagP. Optionally, the S. Paratyphi A GMMA are derived from S.
  • S. Paratyphi A from which GMMA is derived or the S. Paratyphi A bacterium express functional versions of MsbB and/or PagP or comprise a gene encoding a functional MsbB and/or PagP protein may be determined by isolating lipid A from the GMMA and analysing its structure by MALDI-TOF as described above. If the lipid A is detoxified then the S. Paratyphi A from which the GMMA is derived do not or the S. Paratyphi A bacterium does not express functional versions of Msb and/or PagP or comprise a gene encoding a functional MsbB and/or PagP protein.
  • the S. Paratyphi A bacteria from which the GMMA are derived do not or the S. Paratyphi A bacterium does not comprise a gene (such as htrB, msbB, and/or pagP) encoding a functional protein because it comprises a mutation in that gene.
  • the S. Paratyphi A bacteria from which the GMMA are derived do not or the S. Paratyphi A bacterium does not comprise a gene encoding a functional HtrB, MsbB, and/or PagP protein.
  • Paratyphi A bacterium comprise(s) a gene encoding at least a portion of the HtrB, MsbB, and/or PagP protein, but either the gene is mutated such that the HtrB, MsbB, and/or PagP protein encoded is missing one or more important amino acids or a portion of the gene is deleted.
  • the S. Paratyphi A bacteria from which the GMMA are derived or the S. Paratyphi A bacterium may comprise a substitution or deletion mutation in the htrB, msbB, and/or pagP gene.
  • Paratyphi A bacterium may have an addition mutation in the htrB, msbB and/or PagP gene, for example an addition mutation causing a frame shift.
  • the S. Paratyphi A bacteria from which the GMMA are derived or the S. Paratyphi A bacterium comprise(s) a deletion mutation in the htrB, msbB, and/or pagP gene.
  • the htrB, msbB, and/or pagP gene comprises a deletion mutation, and at least 10%, at least 20%, at least 25%, at least 30%, at least 50% or at least 75% of the htrB, msbB, and/or pagP gene is deleted.
  • Paratyphi A bacteria from which the GMMA are derived or the S. Paratyphi A bacterium lacks a htrB, msbB, and/or pagP gene (for example because the complete htrB, msbB, and/or pagP gene has been deleted (a AhtrB, AmsbB, and/or ApagP mutation).
  • the S. Paratyphi A OMVs or GMMA are derived from S. Paratyphi A where at least a part of the msbB and/or pagP gene has been replaced by a different gene.
  • the S. Paratyphi A OMVs or GMMA are derived from S.
  • Paratyphi A where at least a part of the msbB and/or pagP gene has been replaced by a tetracycline (tet) or kanamycin (kan) gene respectively.
  • the S. Paratyphi A OMVs or GMMA are derived from S. Paratyphi A where at least a part of the msbB and/or pagP gene has been replaced by a tetracycline (tet) or kanamycin (kan) gene respectively.
  • the S. Paratyphi A OMVs or GMMA are derived from S. Paratyphi A which is pagP::kan and/or msbB::tet. refers to the gene before the being replaced by the gene after Therefore, “pagP::kan” means that the pagP gene is replaced by kanamycin.
  • the S. Paratyphi A GMMA of the invention are derived from S. Paratyphi A GMMA bacteria that include one or more mutations resulting in deletion of pagP and/or msbB.
  • the S. Paratyphi A bacterium of the invention may include one or more mutations resulting in deletion of pagP and/or msbB.
  • suitable S. Paratyphi A strains may be selected from the group consisting of ApagP and AmsbB (ApagP refers to a Salmonella enterica strain which has the pagP gene deleted and/or replaced with a different gene such as an antibiotic resistance gene).
  • the S. Paratyphi A bacteria from which the GMMA are derived or the S. Paratyphi bacterium may have been modified (for example genetically modified) to hyperbleb i.e. more quantities of outer membrane “bud off' compared to a corresponding Gram-negative bacterium that does not have the genetic mutation.
  • the S. Paratyphi A bacteria from which the GMMA are derived or the S. Paratyphi bacterium may comprise any suitable modification that leads to hyperblebbing.
  • the modification is a mutation, for example the S. Paratyphi A bacteria from which the GMMA are derived or the S. Paratyphi bacterium may not comprise a gene (such as tolR) encoding a functional protein because it comprises a mutation in that gene.
  • the S. Paratyphi A bacteria from which the GMMA are derived or the S. Paratyphi bacterium do(es) not comprise a gene encoding a functional TolR protein.
  • Paratyphi bacterium comprise(s) a gene encoding at least a portion of the TolR protein, but either the gene is mutated such that the TolR protein encoded is missing one or more important amino acids or a portion of the gene is deleted.
  • the S. Paratyphi A bacteria from which the GMMA are derived or the S. Paratyphi bacterium may comprise a substitution or deletion mutation in the tolR gene.
  • the S. Paratyphi A bacteria from which the GMMA are derived or the S. Paratyphi bacterium may have an addition mutation in the tolR gene, for example an addition mutation causing a frame shift.
  • Paratyphi bacterium comprise(s) a deletion mutation in the tolR gene.
  • the tolR gene comprises a deletion mutation, and at least 10%, at least 20%, at least 25%, at least 50% or at least 75% of the tolR gene is deleted.
  • the S. Paratyphi A bacteria from which the GMMA are derived or the S. Paratyphi bacterium lacks a tolR gene (for example because the complete tolR gene has been deleted (a AtolR mutation).
  • the S. Paratyphi A OMVs or GMMA are derived from S. Paratyphi A where at least a part of the tolR gene has been replaced by a different gene.
  • Paratyphi A OMVs or GMMA are derived from S. Paratyphi A where at least a part of the tolR gene has been replaced by a chloramphenicol acetyltransferase (cat) gene.
  • the S. Paratyphi A OMVs or GMMA are derived from S. Paratyphi A where the tolR gene has been replaced by a chloramphenicol acetyltransferase (cat) gene.
  • the S. Paratyphi A OMVs or GMMA are derived from S. Paratyphi A where the tolR gene has been replaced by a chloramphenicol acetyltransferase (cat) gene.
  • Paratyphi A OMVs or GMMA are derived from S. Paratyphi A which is tolR::cat.
  • a genetic modification causes a S. Paratyphi A bacteria from which the GMMA are derived or the S. Paratyphi bacterium to hyperbleb may be tested using the following hyperblebbing assay.
  • the user should prepare two cultures of bacterium. The first culture should comprise the bacterium having the genetic modification to be tested (the test culture), and the second culture should comprise an equivalent bacterium which is identical but for the genetic modification to be tested (the reference culture). The user should grow the test culture and the reference culture under identical conditions and determine the number of outer membrane vesicles released from the bacteria in the test culture and bacteria in the reference culture.
  • the genetic modification causes the bacterium to hyperbleb.
  • the level of outer membrane vesicles released may be determined by O-Antigen quantification, for example by using HPAEC-PAD, as described in Example 5.
  • the immunogenic composition comprises S. Paratyphi A GMMA derived from S. Paratyphi A strain ED 199 (see e.g. Mylona E, Sanchez-Garrido J, Hoang Thu TN, Dongol S, Karkey A, Baker S, Shenoy AR, Frankel G. Very long O-antigen chains of Salmonella Paratyphi A inhibit inflammasome activation and pyroptotic cell death. Cell Microbiol. 2021 May; 23(5):el3306).
  • Paratyphi A bacterium may be an ED 199 S. Paratyphi A bacterium.
  • An immunogenic composition comprises S. Paratyphi A GMMA derived from S. Paratyphi A strain ED 199 or the S. Paratyphi A bacterium is an ED 199 S. Paratyphi A bacterium, if the strain used was based on S. Paratyphi A strain ED 199 even if modifications to strain ED 199 have been made (for example mutation of msbB, pagP or tolR genes).
  • the S. Paratyphi A strain may be tolR::cat pagP::kan msbB::tet.
  • the immunogenic composition of the invention may comprise a dose (O-antigen) of between 1 pg and 50 pg, between 2 pg and 25 pg, between 2 pg and 10 pg, between 15 pg and 25 pg, around 20 pg, or around 4 pg of S. Paratyphi A GMMA.
  • the dose of GMMA may be quantified as an O-antigen dose, i.e. if the immunogenic composition comprises a 1 pg (O-antigen) dose of GMMA then the immunogenic composition comprises sufficient GMMA to provide 1 pg of the O-antigen associated with that GMMA (e.g. the immunogenic composition comprises GMMA containing a total of 1 ig of S. Typhimurium O-antigen).
  • the immunogenic composition comprises GMMA rich in O-antigen, the actual amount of GMMA present to achieve a dose of I pg (O-antigen) may be lower than the amount required if the GMMA is poor in O-antigen.
  • the amount of S. Paratyphi A O-antigen present in an immunogenic composition may be determined by mild hydrolysis of the O-antigen in the immunogenic composition (to provide the monosaccharide Paratose) and detecting the amount of Paratose using HPAEC- PAD. Assuming that no ‘Tree” S. Paratyphi A O-antigen has been added, the amount of O- antigen in an S. Paratyphi A GMMA composition will correspond to the O-antigen dose of the 5. Paratyphi A GMMA.
  • the O-antigen/protein ratio of the S. Paratyphi A OMVs or GMMA present in an immunogenic composition may be at least 0.2, 0.3, 0.4, 0.5 or at least 0.6, typically at least 0.4.
  • the O-antigen/total protein ratio may be at most 0.8, 0.9, 1.0, or 2.0.
  • the O-antigen content can be quantified by HPAEC-PAD, for example as described in Example 5.
  • the protein concentration is quantified by micro-BCA, for example as described in PCT/EP2022/073501.
  • Outer membrane vesicles or GMMA obtainable or obtained from the S. Paratyphi A bacterium
  • outer membrane vesicles or GMMA obtained by or obtainable by the methods of the invention.
  • Such outer membrane vesicles or GMMA may comprise any of the features of the outer membrane vesicles or GMMA described here.
  • such outer membrane vesicles or GMMA may comprise penta-acylated lipid A.
  • the immunogenic compositions of the invention or used in the invention may comprise additional components, such as a pharmaceutically acceptable excipient(s), 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.
  • 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. a lyophilised composition or a spray-freeze dried composition).
  • 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-fdled 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 or used in the invention may be packaged in unit dose form or in multiple dose form.
  • 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.
  • composition will be sterile.
  • Immunogenic compositions of or used in the invention may be isotonic with respect to humans.
  • immunogenic compositions of or used in 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. Salmonella Typhi antigen
  • the immunogenic composition may further comprise an antigen from Salmonella Typhi (an S. Typhi antigen).
  • an S. Typhi antigen is a Vi polysaccharide.
  • Fz or “Fz 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 the Vi monomeric repeating unit is shown below.
  • the fragmented Vi preferably no changes in the structure of the repeating unit is observed compared to native Vi. This can be confirmed byl H 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 asl H NMR or the Hestrin colorimetric method.
  • the IVi polysaccharide In its native size, the IVi polysaccharide has an average molecular weight measured by HPLC size exclusion chromatography (HPLC-SEC) of about 165kDa.
  • 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. it has been made by a method that typically generates fVi having a molecular weight within this range).
  • the molecular weight of the Vi polysaccharide may be determined by HPLC-SEC.
  • 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 pm; cod. 808021) with a TSK gel PWXL guard column (4.0 cm x 6.0 mm; particle size 12 pm; cod. 808033) (Tosoh Bioscience) using dextrans as standards (5, 25, 50, 80, 150 kDa).
  • the mobile phase is 0.1 M NaCI, 0.1 M NaH2 PO4 , 5% CH3 CN, pH 7.2, at the flow rate of 0.5 mL/min (isocratic method for 30 min). Void and bed volume calibration is performed with k-DNA (k-DNA Molecular Weight Marker III 0.12-21.2 kb; Roche) and sodium azide (NaN3; Merck), respectively.
  • Fragmented Vi polysaccharide can further be separated into pools of different average molecular weight ranges. This can be achieved by methods known in the art such as anion exchange chromatography, size exclusion chromatography, and tangential flow filtration.
  • 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
  • PDI Mw / Mn where Mw is the weight average molecular weight and Mn is the number average molecular weight.
  • 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. It may have an avMW distribution characterised in that at least 50% of the pool has an avMW between 35 kDa and 60 kDa. It may have an avMW distribution characterised in that at least 30% of the pool has an avMW between 41 kDa and 55 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 90kDa, 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. Also, hydrolysis with hydrogen peroxide could enable the formation of fragmented Vi having a lower average molecular weight than when using mechanical methods.
  • 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, CRM197, or diphtheria toxoid.
  • the carrier protein is CRM197.
  • 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. Where the fVi polysaccharide has a free aldehyde group, 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. Alternatively, 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).
  • 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.
  • 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.
  • 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.
  • linkers include P-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.
  • step b. reacting the N-hydroxysuccinimide ester fVi derivative obtained in step b. with the carrier protein (optionally derivatised carrier protein) to produce the fVi conjugate.
  • the carrier protein may be derivatised by reacting it with a carbodiimide and a linker.
  • the carbodiimide is l-ethyl-3 -(3 -Dimethylaminopropyl) carbodiimide (EDAC).
  • 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 CRM197 and derivatising the carrier protein comprises one or more of the following steps:
  • CRM 197 is an appropriate buffer, optionally MES buffer;
  • 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 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 20°C and 30°C 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):
  • 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.
  • the immunogenic compositions of or used in the invention may comprise an adjuvant.
  • Any suitable adjuvant may be used.
  • 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. gel, crystalline, amorphous, etc.), and with adsorption being preferred.
  • the mineral containing compositions may also be formulated as a particle of metal salt.
  • the immunogenic compositions of or used in the invention may comprise an aluminium adjuvant, i.e. any compound comprising Al 3+ ions.
  • the aluminium adjuvant may comprise or consist of aluminium phosphate (any compound comprising Al 3+ and PO4 3 ' ions) and/or aluminium hydroxide (any compound comprising Al 3+ and OH' ions).
  • the aluminium adjuvant comprises or consists of aluminium hydroxide.
  • the aluminium hydroxide adjuvant may comprise or be an aluminium oxyhydroxide salt.
  • the aluminium hydroxide adjuvant may comprise or be an aluminium oxyhydroxide salt that is at least partially crystalline.
  • Aluminium oxyhydroxide salt which can be represented by the formula AIO(OH), can be distinguished from other aluminium compounds, such as aluminium hydroxide salt (Al(OH)s), 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.
  • aluminium hydroxide adjuvants will be apparent to one of skill in the art, for example ALHYDROGEL®.
  • the aluminium adjuvant may comprise or consist of between 0.1 mg and 10 mg Al 3+ , between 0.1 mg and 5 mg Al 3+ , between 0.3 mg and 0.4 mg Al 3+ , or around 0.35 mg Al 3+ .
  • the Examples show that the immunogenic composition of the invention demonstrates good immunogenicity. Immunogenicity can be measured according to the assays set out in Example 4.
  • the immunogenic compositions of the invention may induce at least 10 3 EU/ml of S. Paratyphi A O-antigen polysaccharide antibodies in an immunogenicity assay comprising the following steps:
  • a suitable ELISA may involve: coating ELISA plates with S. Paratyphi A O-antigen; applying a blood sample taken from the mice at day 42 to the coated ELISA plates and then washing the plates to remove antibodies not bound to the S. Paratyphi A O- antigen; and detecting the amount of anti- S. Paratyphi A O-antigen antibodies bound to the S.
  • Paratyphi A O-antigen on the ELISA plates using an anti-IgG antibody conjugated to a detection moiety such as alkaline phosphatase.
  • the immunogenic compositions of the invention may demonstrate cross-protection.
  • the immunogenic compositions of the invention may induce antibodies against three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more or all ten of the following strains:
  • the immunogenic compositions of the invention may induce antibodies against:
  • An immunogenic composition “induces ” antibodies against (for example) S. Typhimurium ST34 if it is able to induce these antibodies when used to immunise mice. Whether or not it is able to induce these antibodies when used to immunise mice may be determined by testing a sample of the immunogenic composition using the following cross-protection assay. Whether or not an immunogenic composition induces antibodies against one or more of the strains described above may be determined by carrying out a cross-protection assay.
  • the user may: immunise mice intraperitoneally with 500pL of the immunogenic composition; and measure the level of bactericidal antibodies raised against the relevant strain using a serum bactericidal assay (SBA), wherein the relevant strain is one of strains (a) to (j) above.
  • SBA serum bactericidal assay
  • the SBA assay may be based on the assay described in Example 4, except that the user should measure the bactericidal activity against the relevant strain listed above (rather than, for example Salmonella Paratyphi A NVGH308).
  • the immunogenic composition induces antibodies against one more of the strains described above, if the IC50 (serum dilution giving 50% inhibition of the ATP level) obtained in the SBA assay is above 10 2 .
  • the immunogenic compositions of the invention may induce anti-5.
  • Paratyphi A antibodies in each of classes IgG3, IgG2b, IgG2a, and IgGl, as determined using an antibody class assay comprising the following steps:
  • An immunogenic composition “induces ” anti-5 An immunogenic composition “induces ” anti-5.
  • Paratyphi A antibodies in each of classes IgG3, IgG2b, IgG2a, and IgGl if it is able to induce these antibodies when used to immunise mice. Whether or not it is able to induce these antibodies when used to immunise mice may be determined by testing a sample of the immunogenic composition using an ELISA assay to measure the anti-5.
  • Paratyphi A O-antigen antibody subtype level as set out below. A suitable ELISA to measure the anti-5.
  • Paratyphi A O-antigen antibody subtype level may involve: coating ELISA plates with S.
  • Paratyphi A O-antigen; applying a blood sample taken from the mice at day 42 to the coated ELISA plates and then washing the plates to remove antibodies not bound to the S.
  • Paratyphi A O- antigen; and detecting the amount of anti- S.
  • Paratyphi A O-antigen IgG3 antibodies bound to the S.
  • Paratyphi A O-antigen on the ELISA plates using an anti-IgG3 antibody conjugated to a detection moiety such as alkaline phosphatase; repeating the first three steps but using an anti-IgG2b antibody in place of the anti- IgG3 antibody, then again using an anti-IgG2a antibody in place of the anti-IgG3 antibody and finally using an anti-IgGl antibody in place of the anti-IgG3 antibody.
  • a detection moiety such as alkaline phosphatase
  • 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.
  • the method of preventing an infection is a method of preventing invasive non-typeable Salmonella infection.
  • the method of preventing an infection is a method of preventing infection by S. Typhimurium, S. Entcritidis, S. Typhi and/or S. Paratyphi A.
  • 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 human may be a child i.e. below 18 years old.
  • 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.
  • Salmonella enterica serovar Typhimurium wild-type (WT) strain 2192 was provided by the Salmonella Genetic Stock Center (SGSC) at the University of Calgary, Canada, which belongs to the global Salmonella reference collection A (SARA 12).
  • SGSC Salmonella Genetic Stock Center
  • Salmonella enterica serovar Enteritidis WT strain 618 was provided by Quotient Bioresearch Limited, UK. The strain of animal origin was isolated by the European Antimicrobial Susceptibility Surveillance in Animals (EASSA).
  • EASSA European Antimicrobial Susceptibility Surveillance in Animals
  • Salmonella Typhimurium olR ApagP AmsbB and Salmonella Enteritidis AtolR ApagP AmsbB recombinant mutants for each strain were generated as previously reported (Rossi O, Caboni M, Negrea A, Necchi F, Alfini R, Micoli F, et al. Toll-Like Receptor Activation by Generalized Modules for Membrane Antigens from Lipid A Mutants of Salmonella enterica Serovars Typhimurium and Enteritidis. Clin Vaccine Immunol. 2016;23(4):304-14).
  • GMMA derived from the Salmonella strains above (Salmonella Typhimurium GMMA (STmGMMA) and Salmonella Enteritidis GMMA (SEnGMMA)) were purified and isolated. GMMA were purified using similar methods previously reported for S. sonnei GMMA (Gerke C, Colucci AM, Giannelli C, Sanzone S, Vitali CG, Sollai L, et al. Production of a Shigella sonnei Vaccine Based on Generalized Modules for Membrane Antigens (GMMA), 1790GAHB. PLoS One. 2015;10(8):e0134478. doi:
  • GMMA released into the fermentation broth were purified using two consecutive Tangential Flow Filtration (TFF) steps: a microfiltration in which the culture supernatant containing the GMMA is separated from the bacteria, and an ultrafiltration, in which the GMMA are separated from soluble proteins and nucleic acids.
  • TFF Tangential Flow Filtration
  • Example 2 Production of fVi-CRMi97 conjugate for immunisation against S. Typhi The following protocol was used to produce a bivalent composition comprising an S. Paratyphi A OAg-CRMi97 conjugate made as described above and a conjugate of fragmented Vi polysaccharide from S. Typhi (fVi) conjugated to CRM197.
  • 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.
  • a calculated volume of 10 mM FeSO4 and H2O2 is added to get a final concentration of 0.5 mM FeSO4 and 0.5% v/v H2O2 in the reaction mixture respectively.
  • the reaction mixture is incubated at 15 ⁇ 5°C for 120 +/- 10 min.
  • the reaction is stopped by adding equal volumes of 250 mM EDTA to get a final EDTA concentration of 10 mM and is stirred.
  • Step 2 Buffer Exchange: Buffer exchange is performed by Tangential Flow Filtration (TFF) with lOOmM Sodium phosphate (pH: 7.2 ⁇ 0.2) using 30 kDa cassettes to remove residual H2O2. 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 JVi 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 IVi/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 pS/cm.
  • TFF Tangential Flow Filtraion
  • Step 6 0.2 pm Filtration of fVi polysaccharide'.
  • the fVi polysaccharide is fdtered through a 0.22 pm filter.
  • the purified fVi polysaccharide is stored in PETG bottles.
  • Stepl 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, CRM197 is filtered using 0.5 pm filter.
  • MES Methyl MES
  • 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 CRM197 thawing.
  • Step 3 CRM197 Derivatization'.
  • the required concentration of CRM197 is diluted with 100 mM MES buffer followed by addition of calculated quantity of ADH (Adipic Acid Dihydrazide) and ED AC (l-Ethyl-3 -(3 -Dimethylaminopropyl) carbodiimide) to make a CRM : ADH : ED AC 1 : 3.5 : 0.15 w/w/w ratio.
  • ADH Adipic Acid Dihydrazide
  • ED AC l-Ethyl-3 -(3 -Dimethylaminopropyl) carbodiimide
  • Step 4 Purification of CRM197'. Post reaction, CRM197 is purified by TFF using a 10 KDa cassette with 5 mM MES buffer.
  • Step 5 Filtration ofDia-Filtered CRM197: 0.2-micron filtration is performed for dia- filtered CRM197 solution followed by storage at 2-8°C in glass bottle.
  • Step 1 jVi 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 jVi 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 CRM197 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 ED AC addition to have an ED AC /fVi RU molar ratio of 5 : 1.
  • EDAC solution is added after addition of NHS to ensure complete dissolution. The reaction is incubated at room temperature with slow mixing for 1 h.
  • Step 1 Conjugation of jVi with CRMwADI ⁇ :
  • the conjugation reaction creates a covalent bond between the activated fVi and CRM197-ADH.
  • the activated and derivatized reaction mixture is diluted with lOOmM MES pH: 6.0 and CRM197-ADH is added in a w/w ratio of 1 : 1 (fVi : CRM197) to reach the final fVi concentration of the activated fVi and CRM197- 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-CRMi97 crude conjugate is 0.65 filtered.
  • Step 4 Purification of jVi-CRMi97 Crude Conjugate: Purification of the conjugate from the conditioned reaction mixture is performed through a HIC Phenyl Sepharose High Performance (HP) column. Column integrity is performed for every 5-10 cycles as per standard procedure. The column is equilibrated by using Phenyl Sepharose HP Buffer A Tris 50 mM NaCl 3M pH 8. After addition of conditioning buffer and NaCl, crude conjugate is loaded on to the column. Column washing is done using Phenyl Sepharose HP Buffer-A followed by product elution using Phenyl Sepharose HP Buffer-B Tris 50mM pH: 8. Fractions are collected and stored at 2-8°C till further usage. All the fractions from multiple runs are pooled.
  • HP Phenyl Sepharose High Performance
  • 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 jVi-CRMm Conjugate using 0.2 pm filter: fVi-CRMi97 conjugate is filtered through a 0.2 pm filter for bioburden reduction.
  • Step 7 Sterile Filtration of fVi-CRMi97 Conjugate using 0.2 p cellulose acetate filter: The fVi-CRMi97 conjugate is filtered through a 0.2 pm cellulose acetate filter. The purified fVi-CRMi97 conjugate is sampled and stored at 2-8°C.
  • a S. Paratyphi A strain ED 199 comprising StolR tspagP tsmsbB mutations was prepared using a protocol based on that described for S. Enteritidis and S. Typhimurium in Example 1, except the specific mutations used to delete tolR, pagP and msbB were tolR::cat pagP::kan msbB::tet. GMMA were isolated from that bacterium as described in Example 1.
  • Anti-OAg and anti-Vi antigen-specific IgG levels were measured 2 weeks after the second immunization (day 42) by ELISA as previously reported (Rondini et al, Evaluation of the immunogenicity and biological activity of the Citrobacter freundii Vi-CRM197 conjugate as a vaccine for Salmonella enterica serovar Typhi. Clin Vaccine Immunol. 2011 Mar;18(3):460-8). Briefly, 96-well round-bottom MaxiSorp microtiter plates (Nunc, Roskilde, Denmark) were coated with 100 ml/well antigen overnight at 4°C. OAg purified from S.
  • Paratyphi A (0:2) or S. Enteritidis (0:9) and Vi purified from C. freundii s.l. were used at 15mg/ml and 2mg/ml in carbonate or at Img/ml in phosphate buffer, respectively (Micoli et al, A scalable method for O-antigen purification applied to various Salmonella serovars. Anal Biochem. 2013 Mar 1 ;434(1): 136-45; Micoli et al, Production of a conjugate vaccine for Salmonella enterica serovar Typhi from Citrobacter Vi. Vaccine. 2012 Jan 20;30(5):853 - 61).
  • ELISA units were expressed relative to a mouse antigen-specific antibody standard serum curve composed by 10 standard points and 2 blank wells (run in duplicate on each plate), with the best five-parameter fit determined by a modified Hill plot.
  • One ELISA unit is defined as the reciprocal of the dilution of the standard serum that gives an absorbance value equal to 1 in this assay.
  • reaction mixture containing the target bacterial cells (around 100,000 CFU/ml), BRC (50% for S. Enteritidis, 20% for S. Paratyphi A, and 5% for C.freundii s.l.), and buffer (PBS) was added to SBA plates containing HI serum dilutions and incubated for 3 h at 37°C.
  • BRC 50% for S. Enteritidis, 20% for S. Paratyphi A, and 5% for C.freundii s.l.
  • PBS buffer
  • the plates were centrifuged for lOmin at 4,000 x g, the supernatant was discarded to remove ATP derived from dead bacteria, and live bacterial pellets resuspended in PBS were transferred to a white roundbottom 96-well plate (Greiner) and mixed 1:1 (vol/vol) with BacTiter-Glo reagent (Promega).
  • the reaction mixture was incubated for 5 min at RT in an orbital shaker, and the luminescence signal was measured using a luminometer (Viktor).
  • Example 7 Post-second immunization individual sera isolated from the blood samples taken from the mice immunized as described in the section entitled “Example 7 - Antibody subclasses raised by the quadrivalent Pan-Salmonella vaccine in mice” were tested to determine the isotype of the antibodies produced using a ELISA-based assay working with the same principle as described under the heading “ELISA”. The assay was repeated to determine the EU/mL on each individual sera at the dose tested using as secondary antibody antimouse-IgGl, antimouse-IgG2a, antimouse-IgG2b and antimouse-IgG3 antibodies in standard assay. Results are shown in Example 12 and expressed as calculated as subclass/subclasses total %.
  • Example 5 Formulation of quadrivalent (Pan-Salmonella) vaccine against S. Typhimurium, S. Enteritidis, S. Typhi, and S. Paratyphi for preclinical studies
  • a quadrivalent vaccine called Pan-Salmonella ParA GMMA, was formulated.
  • the vaccine contained GMMA from S. Enteritidis and S. Typhimurium (as described in Example 1), S. Paratyphi A GMMA (as set out in Example 3) and fVi-CRMi97 conjugate (as set out in Example 2).
  • Concentration of phosphate buffer was optimized to obtain the highest adsorption, while quenching to ensure optimal particles size.
  • Final formulation contains sufficient GMMA to provide 40 pg/ml of STm, SEn, and Paratyphi A O-Antigens (sPa), 50 pg/ml of Vi polysaccharide in a phosphate buffered saline matrix containing 0.7mg/mL of Aluminum Hydroxide.
  • Each single OAg is hydrolysed to release its relative di-deoxy monosaccharide (SEn OAg to Tyvelose; STm OAg to Abequose; SPa OAg to Paratose), corresponding to the chromatographic peak, before analysis by HPAEC-PAD.
  • di-deoxy are the only sugar that differs from the others among the ones composing SEn, STm and SPa OAg repeating units (RU).
  • the sample is diluted by volume (450 pL) or by weight on analytical balance, with milliQ water, in order to be within the calibration curve range for each OAg. 120 pL of TFA 1 M is added to vials containing standards or samples and incubated at 75°C for 1.5 hours.
  • pan-Salmonella vaccines induced specific serum IgG responses against S. Paratyphi A O-antigen, and the antibodies are bactericidal in mice.
  • pan-Salmonella vaccines also induced specific serum IgG responses against S. Typhi Vi.
  • ParA GMMA (produced as described in Example 3) adsorbed to Alhydrogel at 1.17 pg (Vi polysaccharide) dose
  • fVi-CRMi97 conjugate (produced as described in Example 2) at 1.25 pg (Vi polysaccharide) dose.
  • the immunisations involved intraperitoneal immunisation of the corresponding dose for each product in 200 pl at days 0 and 28. Blood samples were collected at days 27 and 42, and the antibodies raised were measured using the ELISA assay and SBA assay described above in Example 4. Results are shown in Figure 2.
  • Salmonella ParAGMMA is slightly different (about 7%) from the 0:2 dose used in the Pan-Salmonella_O:2-CRM.
  • Example 7 Antibody subclasses raised by the quadrivalent Pan-Salmonella vaccines in mice
  • the immunisations involved intramuscular immunisation of 500 pl of the formulation at day 0 and day 28. Blood samples were collected at days -1, 27 and 42, and the antibodies raised were studied using the assays described in Example 4 above. The results are set out in Figure 4.
  • the Pan-Salmonella vaccine induced specific serum IgG responses against S. Paratyphi A O-antigen, and the antibodies are bactericidal in rabbits.
  • the Pan-Salmonella vaccine also induced specific serum IgG responses against S. Typhi Vi.
  • Example 9 - Antibodies induced by GMMA-based vaccine demonstrates crossprotection
  • CD1 mice were immunised intraperitoneally on days 0 and 28 using the quadrivalent Pan- Salmonella vaccine described in Example 5 at the following dose:
  • An immunogenic composition comprising S. enterica serovar Paratyphi A outer membrane vesicles.
  • An S. Paratyphi A bacterium comprising modified lipid A.
  • S. Paratyphi A bacterium of embodiment 9, 16 or 17, which is msbB::tet.
  • Outer membrane vesicles or GMMA obtainable from the S. Paratyphi A bacterium of any one of embodiments 3, 5, 6, 9 to 12, 16, 21 to 25, 28, or 29, 34 to 37 or 39.
  • Outer membrane vesicles or GMMA obtained from the S. Paratyphi A bacterium of any one of embodiments 3, 5, 6, 9 to 12, 16, 21 to 25, 28, or 29, 34 to 37 or 39.
  • An immunogenic composition comprising the outer membrane vesicles or GMMA of embodiment 44 or 45.
  • immunogenic composition of any one of embodiments 1, 2, 4 to 8, 11 to 13, 19, 20, 23 to 25, 31 to 33, 36 to 38, or 46, wherein the immunogenic composition induces at least 10 3 EU/ml of S.
  • Paratyphi A O-antigen polysaccharide antibodies in an immunogenicity assay comprising the following steps:
  • immunogenic composition of any one of embodiments 1, 2, 4 to 8, 11 to 13, 19, 20, 23 to 25, 31 to 33, 36 to 38, or 46 to 48, wherein the immunogenic composition further comprises an adjuvant.
  • the pharmaceutically acceptable excipient comprises phosphate buffered saline.
  • a vaccine comprising the immunogenic composition of any one of embodiments 1, 2, 4 to 8, 11 to 13, 19, 20, 23 to 25, 31 to 33, 36 to 38, or 46 to 56
  • a method of preventing an infection comprising administering an effective amount of the immunogenic composition or vaccine of any one of embodiments 1, 2, 4 to 8, 11 to 13, 19, 20, 23 to 25, 31 to 33, 36 to 38, or 46 to 57to a subject.
  • the immunogenic composition or vaccine for use of embodiment 58, or the use of embodiment 60, wherein the method of preventing an infection comprises administering an effective amount of the immunogenic composition or vaccine of any one of embodiments 1, 2, 4 to 8, 11 to 13, 19, 20, 23 to 25, 31 to 33, 36 to 38, or 46 to 57to a subject.
  • the immunogenic composition or vaccine for use, method, or use of any one of embodiments 58 to 61, wherein the method of preventing an infection is a method of preventing Salmonella infection.
  • the immunogenic composition or vaccine for use, method, or use of any one of embodiments 58 to 62, wherein the method of preventing an infection is a method of preventing invasive non-typeable salmonella infection.
  • the immunogenic composition or vaccine for use, method, or use of any one of embodiments 58 to 63, wherein the method of preventing an infection is a method of preventing infection by S. Paratyphi A.
  • the immunogenic composition, immunogenic composition or vaccine for use, method, or use of any one of embodiments 1, 2, 4 to 8, 11 to 13, 19, 20, 23 to 25, 31 to 33, 36 to 38, or 46 to 64, wherein the O-antigen/protein ratio of the S. Paratyphi A outer membrane vesicles or GMMA is at least 0.4.
  • the immunogenic composition, immunogenic composition or vaccine for use, method, or use of any one of embodiments 1, 2, 4 to 8, 11 to 13, 19, 20, 23 to 25, 31 to 33, 36 to 38, or 46 to 65, wherein the immunogenic composition or vaccine further comprises an S. Typhi antigen.
  • immunogenic composition for use, method, or use of embodiment 69, wherein the carrier protein is CRM197 or diphtheria toxoid.

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Abstract

La présente invention concerne des compositions immunogènes comprenant des vésicules membranaires externes (OMV) de S. enterica serovar Paratyphi A, des vaccins comprenant les compositions immunogènes et des méthodes et des utilisations des compositions immunogènes. La présente invention concerne également une bactérie S. Paratyphi A comprenant un lipide A modifié.
PCT/EP2024/070615 2023-07-21 2024-07-19 Composition immunogène Pending WO2025021710A1 (fr)

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