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WO2012049662A1 - Souches hyper bourgeonnantes de salmonella - Google Patents

Souches hyper bourgeonnantes de salmonella Download PDF

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Publication number
WO2012049662A1
WO2012049662A1 PCT/IB2011/054569 IB2011054569W WO2012049662A1 WO 2012049662 A1 WO2012049662 A1 WO 2012049662A1 IB 2011054569 W IB2011054569 W IB 2011054569W WO 2012049662 A1 WO2012049662 A1 WO 2012049662A1
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WIPO (PCT)
Prior art keywords
protein
bacterium
salmonella
blebs
putative
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PCT/IB2011/054569
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English (en)
Inventor
Laura Martin
Allan Saul
Christiane Gerke
Sara Sa Silva
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GSK Vaccines Institute for Global Health SRL
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Novartis Vaccines Institute for Global Health SRL
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/255—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
    • A61K39/02—Bacterial antigens
    • A61K39/025—Enterobacteriales, e.g. Enterobacter
    • A61K39/0275—Salmonella
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • C12P1/04—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using bacteria
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated
    • 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/543—Mucosal route intranasal
    • 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

  • This invention is in the field of immunisation against Salmonella species.
  • Salmonella are Gram-negative facultative anaerobic bacteria. Nomenclature systems have varied over the years but there are currently two recognized species: S. enterica and S. bongori [1]. Within these species there are various subspecies, including enterica, salamae, arizonae, diarizonae, houtenae, and indica. Under the new nomenclature the bacteria previously known as S. paratyphi, S. typhi, and S. typhimitri m are instead serovars within S. enterica, for example S. enterica serovar Typhimurium, abbreviated: S. Typhimurium.
  • the Salmonella cause a variety of diseases in many animal hosts (although S. Typhi and S. Paratyphi A are human-specific serovars), mainly via ingestion of contaminated foods. Human diseases include gastroenteritis, bacteremia, typhoid fever and paratyphoid fever.
  • the invention uses Salmonella blebs as the immunogenic component for vaccination.
  • Salmonella are known to form blebs spontaneously (e.g. the IkyD mutants of S. Typhimurium disclosed in reference 5 and various strains disclosed in reference 6), but in some circumstances a chemical treatment may be required (e.g. treatment with polymyxin B [7]).
  • the inventors have created mutants of Salmonella in which the Tol-Pal system has been disrupted to disturb the bacterial envelope structure. During normal growth these mutants release into their culture medium large quantities of blebs which are rich in immunogenic outer membrane proteins, and these blebs can thus be used as immunogens. They can be particularly useful for raising cross-reactive responses which can protect against multiple serovars of Salmonella.
  • the invention provides a Salmonella bacterium which expresses no more than 4 of TolA, TolB, TolQ, TolR and Pal proteins.
  • at least one protein from the natural five-protein Tol-Pal system is absent, resulting in a bacterium which, during growth in culture medium, releases greater quantities of outer membrane blebs into the medium than the same bacterium expressing all 5 Tol-Pal proteins.
  • TolR is not expressed, but the other four proteins may be expressed.
  • the bacterium should express no more than 3 of TolA, TolB, TolQ, TolR and Pal i.e. there is no expression of TolA and also of at least one other Tol-Pal protein.
  • the invention also provides a Salmonella bacterium which does not express a TolR protein.
  • the invention also provides a AtolR strain of Salmonella (particularly of a S. enterica), such as a AtolRAmsbB strain, a AtolRAwbaP strain, or a AtolR msbBAwbaP strain.
  • the invention also provides a Salmonella bacterium which expresses TolA, TolB, TolQ, TolR and Pal proteins, wherein the TolA, TolQ, TolR and/or Pal protein (a) is located in the bacterium's inner or outer membrane, and (b) includes one or more amino acid sequence mutation(s) such that, compared to the same bacterium without said mutation(s), the bacterium releases greater quantities of outer membrane blebs when growing in culture medium.
  • the invention also provides a Salmonella bacterium (a) in which one or more components of its Tol- Pal system has a modification such that, during growth in culture medium, the bacterium releases greater quantities of outer membrane blebs into the medium than the same bacterium lacking the modification, and (b) which does not express a native Salmonella lipopolysaccharide.
  • the invention also provides a method of preparing a hyperblebbing Salmonella bacterium, comprising a step of modifying gene(s) encoding one or more components of a starting bacterium's Tol-Pal system such that the modification causes the bacterium, when grown in culture medium, to release greater quantities of outer membrane blebs into the medium than the starting bacterium, and wherein the modification involves mutating one or more of the starting bacterium's tolA, tolB, tolQ, tolR and/or pal genes.
  • the mutating step may delete the gene.
  • the method may also involve modification of gene(s) encoding a protein required for synthesis of the bacterium's lipopolysaccharide. Mutation of at least tolR is preferred.
  • the invention also provides a bleb isolated or obtainable from a bacterium of the invention. These blebs are useful as components of Salmonella vaccines.
  • the invention also provides a process for preparing Salmonella blebs, comprising a step of separating the blebs from a culture medium comprising bacteria of the invention which have been grown under conditions which permit the release of blebs into the medium by the bacteria.
  • Blebs prepared by this process can be used as components of Salmonella vaccines.
  • the invention also provides a culture medium comprising bacteria of the invention which have been grown under conditions which permit the release of blebs into the medium by the bacteria. Blebs may be purified from this culture medium.
  • the invention also provides a composition comprising blebs that, during culture of bacteria of the invention, are released into the culture medium. This composition does not comprise any living and/or whole bacteria. This composition and/or its components can be used for Salmonella vaccine preparation.
  • the invention also provides a composition comprising blebs, wherein the blebs are present in the filtrate obtainable after filtration through a 0.22 ⁇ filter of a culture medium in which a bacterium of the invention has been grown.
  • This composition and/or its components can be used for Salmonella vaccine preparation.
  • the invention also provides a Salmonella bleb which includes one or more ⁇ i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or 51) of: (a) a protein consisting of an amino acid sequence selected from SEQ ID NOs: 1 to 52, 60 to 355 and 366 to 648; (b) a protein comprising an amino acid sequence having at least j% identity to one of SEQ ID NOs: 1 to 52, 60 to 355 and 366 to 648, where j is 50 or more ⁇ e.g.
  • n is 7 or more ⁇ e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
  • Preferred fragments comprise an epitope from one of SEQ ID NOs: 1 to 52, 60 to 355 and 366 to 648.
  • Other preferred fragments lack one or more amino acids ⁇ e.g.
  • bleb-free immunogenic composition comprising a bleb protein comprising: (a) one or more ⁇ e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30.
  • amino acid sequences SEQ ID NOs 1 to 52, 60 to 355 and 366 to 648 (b) an amino acid sequence having at least j% identity to one of SEQ ID NOs: 1 to 52, 60 to 355 and 366 to 648, where j is 50 or more ⁇ e.g.
  • n is 7 or more ⁇ e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).
  • Preferred fragments comprise an epitope from one of SEQ ID NOs: 1 to 52, 60 to 355 and 366 to 648, and more preferred fragments are immunogenic fragments.
  • Other preferred fragments lack one or more amino acids ⁇ e.g.
  • the invention provides a Salmonella bleb which includes one or more of: (a) a protein consisting of an amino acid sequence selected from SEQ ID NOs: 1 to 52, 60 to 355 and 366 to 648; (b) a protein comprising an amino acid sequence having at least 85% identity to one of SEQ ID NOs: 1 to 52, 60 to 355 and 366 to 648 and/or comprising a fragment of at least 7 consecutive amino acids of any one of SEQ ID NOs: 1 to 52, 60 to 355 and 366 to 648, wherein the fragment comprises an epitope (and, preferably, the blebs do not include a native Salmonella lipopolysaccharide and lack a native Salmonella O antigen).
  • the invention provides a bleb- free immunogenic composition
  • a protein comprising: (a) amino acid sequences SEQ ID NOs 1 to 52, 60 to 355 and 366 to 648; or (b) an amino acid sequence having at least 85% identity to one of SEQ ID NOs: 1 to 52, 60 to 355 and 366 to 648 and/or comprising a fragment of at least 7 consecutive amino acids of any one of SEQ ID NOs: 1 to 52, 60 to 355 and 366 to 648, said fragment comprising an epitope from one of SEQ ID NOs: 1 to 52, 60 to 355 and 366 to 648.
  • a bleb is defined around SEQ ID NOs: 1 -52 or SEQ ID NOs: 60-355, it is ideally a S. Typhimurium bleb; where a bleb is defined around SEQ ID NOs: 356-648 it is ideally a S. Enteritidis bleb.
  • a preferred subset within SEQ ID NOs: 1 to 52, 60-355 and 366-648 is SEQ ID NOs: 1 to 52.
  • the Salmonella naturally possess a Tol-Pal system which is made up of TolA, TolB, TolQ, TolR and Pal proteins.
  • the natural Tol-Pal system is disrupted, thereby causing the bacterium to release greater quantities of outer membrane blebs into its culture medium during bacterial replication.
  • Various disruptions can be made.
  • At least one of the five Tol-Pal proteins is removed e.g. by deletion or inactivation of the gene encoding the protein.
  • the bacterium may express 0, 1 , 2, 3 or 4 of TolA, TolB, TolQ, TolR and Pal proteins. Removal of one of the five proteins can suffice, in which case the bacterium expresses only 4 of these proteins.
  • the TolR protein is removed e.g. by inactivation of a starting strain's tolR gene.
  • the bacterium may be tolA + tolB + tolQ + TolR Pat.
  • the bacterium expresses all five Tol-Pal proteins, but at least one is mutated to cause hyperblebbing.
  • the TolA, TolQ, TolR and/or Pal protein may be mutated such that the protein retains its membrane localisation but its interactions with other members of the Tol- Pal system are disrupted.
  • the bacterium will thus retain TolA, TolQ and TolR as transmembrane proteins in the inner membrane, and Pal protein as a periplasm-facing lipoprotein in the outer membrane, but at least one of the TolA, TolQ, TolR and/or Pal proteins is mutated.
  • the Salmonella bacterium The Salmonella bacterium
  • the invention can be used with any of S. enterica or S. bongori, but preferably uses S. enterica. Within the S. enterica species, the invention can be used with any subspecies, including subspp. enterica, salamae, arizonae, diarizonae, houtenae, and/or indica.
  • the invention can be used with any serovar, including but not limited to serovars Typhimurium and Enteritidis.
  • the invention can be used with any of the bacteria commonly known as S. Paratyphi, S. Typhi, S. Enteritidis, S. Typhimurium and S. Choleraesuis. It can also be used with serovars such as S. Dublin and S. Minnesota.
  • the invention is used with the serovars that most commonly infect humans, namely S. Typhi, S. Paratyphi , S. Typhimurium and S. Enteritidis.
  • a Salmonella of the invention can advantageously include one or more further changes relative to a wild-type strain. These changes can be used in particular to remove components from the bacterium which would be toxic or undesirable in a human vaccine.
  • a bacterium may not express native Salmonella lipopolysaccharide (LPS), thereby reducing endotoxic activity.
  • LPS Salmonella lipopolysaccharide
  • Various modifications can be made to prevent synthesis of native LPS, and these may disrupt the native lipid A structure, the oligosaccharide core, or the O antigen.
  • Various mutant forms of LPS are known in Salmonella (such as the "rough” and "deep rough” mutants) and these have various genetic causes e.g. from mutations in any of rfaB, rfaC, rfaF, rfaG, rfal, rfaJ, rfbJ, rfl)P, rfaL, pmrB, pmrF, galE, etc. Absence of hexa-acylated lipid A in the LPS is preferred e.g. with a penta-acylated or tetra-acylated lipid A.
  • One useful strain does not express an active MsbB enzyme (an acyltransferase involved in secondary acylation of lipid A; also known as LpxM or WaaN), as msbB mutants produce LPS with reduced toxicity.
  • Another useful strain does not express an active HtrB enzyme (another acyltransferase involved in secondary acylation of lipid A; also known as LpxL or WaaM), as htrB mutants produce LPS with reduced toxicity. Mutation of MsbB is preferred to HtrB because the latter can result in temperature sensitivity in Salmonella [8]. Absence of O antigen in LPS is useful, thereby avoiding a serospecific response.
  • Preferred strains are inactivated for both msbB and wbaP. Some useful strains have penta- or tetra-acylated LPS which includes attached O antigen. More generally, though, preferred strains have penta- or tetra-acylated LPS which lacks attached O antigen.
  • a S. enterica strain with tolR, msbB and wbaP knockouts is useful.
  • a Salmonella of the invention may hyper-express a Salmonella protein.
  • expression of an immunogenic outer membrane protein can be increased by providing a second copy (chromosomal or episomal) or by providing the endogenous gene with a stronger promoter (e.g. a constitutive or inducible promoter) or by inactivating a repressor.
  • a Salmonella of the invention may express one or more heterologous proteins e.g. proteins which are not naturally found in Salmonella. If the heterologous protein is an outer membrane protein then blebs from the strain can be used as a delivery system for presenting non-Salmonella antigens to the immune system.
  • heterologous proteins e.g. proteins which are not naturally found in Salmonella.
  • the heterologous protein is an outer membrane protein then blebs from the strain can be used as a delivery system for presenting non-Salmonella antigens to the immune system.
  • Salmonella bacteria of the invention can be prepared conveniently from wild-type or other starting strains using conventional techniques of mutagenesis. Inactivation of a gene can be achieved in various ways e.g. by deletion or mutation in its promoter, by deletion or mutation of its start codon, by introduction of a premature stop codon, by deletion of the complete coding region, by knockout, etc. Isogenic knockout mutants are preferred. In the resulting Salmonella bacterium mRNA encoding the desired gene is absent and/or its translation is inhibited (e.g. to less than 1% of wild-type levels).
  • a Salmonella bacterium of the invention may contain a marker gene in place of the inactivated gene e.g. an antibiotic resistance marker. This can be achieved using homologous recombination. Preferably, though, unmarked deletions (i. e. deletion without introduction of a marker gene) are used.
  • Salmonella strains possess a virulence plasmid which mediates virulence properties [ 10].
  • a Salmonella of the invention possesses a virulence plasmid; in other embodiments it does not possess a virulence plasmid.
  • Culture conditions for growing Salmonella are well known in the art. For example, they may be grown using an organic nitrogen source (such as amino acid mixtures e.g. containing Ala, Arg, Asn, Asp; casamino acids may be used), glycerol as a carbon source, etc. Inclusion of L-aspartic acid in the medium is particularly useful and may function as both a nitrogen and carbon source.
  • an organic nitrogen source such as amino acid mixtures e.g. containing Ala, Arg, Asn, Asp; casamino acids may be used
  • glycerol as a carbon source
  • Salmonella of the invention may be grown under iron- limiting conditions as this has may up-regulate iron-regulated proteins which are immunogenic and highly-conserved among Salmonella spp.
  • the bacteria may be grown in the presence of a compound such as desferal or 2,2'-dipyridyl or 8-hydroxyquinoline.
  • Salmonella bacteria of the invention are, relative to their corresponding wild-type strains, hyperblebbing i.e. they release into their culture medium larger quantities of blebs than the wild-type strain. These blebs are useful as components of Salmonella vaccines.
  • the blebs typically have a diameter of 35- 120 nm, and sometimes 30-120 nm e.g. 50 nm diameter. Diameters in the range of 30-50 nm are commonly seen, and these are useful blebs of the invention.
  • the blebs are released spontaneously during bacterial growth and can be purified from the culture medium.
  • the purification ideally involves separating the blebs from living and/or intact Salmonella bacteria e.g. by size-based filtration using a filter, such as a 0.22 ⁇ filter, which allows the blebs to pass through but which does not allow intact bacteria to pass through [1 1], or by using low speed centrifugation to pellet cells while leaving blebs in suspension.
  • bleb-containing compositions of the invention will generally be substantially free from whole bacteria, whether living or dead.
  • the size of the blebs means that they can readily be separated from whole bacteria by filtration e.g. as typically used for filter sterilisation.
  • blebs will pass through a standard 0.22 ⁇ filters, these can rapidly become clogged by other material, and so it may be useful to perform sequential steps of filter sterilisation through a series of filters of decreasing pore size before using a 0.22 ⁇ filter. Examples of preceding filters would be those with pore size of 0.8 ⁇ , 0.45 ⁇ , etc.
  • Blebs of the invention contain lipids and proteins. The protein content of the blebs has been analysed, and they include the proteins listed in Tables 1 to 3 and discussed below.
  • Tables 1 to 3 list the GenBank name and GI number for 641 proteins which were detected in Salmonella blebs of the invention. These 641 proteins may be used as immunogenic components in purified form, separate from blebs. 52 proteins were detected in a AtolR knockout of S. Typhimurium strain SL 1344; 296 in a AtolRAwbaP knockout of S. Typhimurium strain SL 1344; and 293 in a AtolRAwbaP knockout of S. Enteritidis strain PI 25109.
  • Polypeptides can be prepared by various means e.g. by chemical synthesis (at least in part), by digesting longer polypeptides using proteases, by translation from RNA, by purification from cell culture (e.g. from recombinant expression or from Salmonella culture), etc. Heterologous expression in an E.coli host is a preferred expression route.
  • Polypeptides of the invention may be attached or immobilised to a solid support.
  • Polypeptides of the invention may comprise a detectable label e.g. a radioactive label, a fluorescent label, or a biotin label. This is particularly useful in immunoassay techniques.
  • Polypeptides can take various forms (e.g. native, fusions, glycosylated, non-glycosylated, lipidated, disulfide bridges, etc.).
  • Polypeptides are preferably Salmonella polypeptides.
  • Polypeptides are preferably prepared in substantially pure or substantially isolated form (i.e. substantially free from other Salmonella or host cell polypeptides) or substantially isolated form.
  • the polypeptides are provided in a non-naturally occurring environment e.g. they are separated from their naturally-occurring environment.
  • the subject polypeptide is present in a composition that is enriched for the polypeptide as compared to a control.
  • purified polypeptide is provided, whereby purified is meant that the polypeptide is present in a composition that is substantially free of other expressed polypeptides, where by substantially free is meant that less than 50%, usually less than 30% and more usually less than 10% of the composition is made up of other expressed polypeptides.
  • polypeptide refers to amino acid polymers of any length.
  • the polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component.
  • polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids, etc.
  • Polypeptides can occur as single chains or associated chains.
  • the invention provides a pharmaceutical composition comprising (a) blebs of the invention and (b) a pharmaceutically acceptable carrier.
  • the invention also provides a process for preparing such a composition, comprising the step of admixing blebs of the invention with a pharmaceutically acceptable carrier.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising (a) the bleb-free immunogenic composition defined above and (b) a pharmaceutically acceptable carrier.
  • the immunogenic composition may include a pharmaceutically acceptable carrier, which can be any substance that does not itself induce the production of antibodies harmful to the patient receiving the composition, and which can be administered without undue toxicity.
  • Pharmaceutically acceptable carriers can include liquids such as water, saline, glycerol and ethanol.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering substances, and the like (e.g. stabilisers, preservatives), can also be present in such vehicles.
  • suitable carriers is available in ref. 12.
  • compositions of the invention may be prepared in various forms.
  • the 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.
  • the composition may be prepared for topical administration e.g. as an ointment, cream or powder.
  • the composition be prepared for oral administration e.g. as a tablet or capsule, or as a syrup (optionally flavoured).
  • the 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 composition may be prepared for nasal, aural or ocular administration e.g. as drops. Administration via skin patch is also possible.
  • a physiological salt such as a sodium salt.
  • Sodium chloride (NaCl) is preferred, which may be present at between 1 and 20 mg/ml e.g. about 10+2 mg/ml NaCl.
  • Other salts that may be present include potassium chloride, potassium dihydrogen phosphate, disodium phosphate dehydrate, magnesium chloride, calcium chloride, etc.
  • the composition is preferably sterile. It is preferably pyrogen-free. It is preferably buffered e.g. at between pH 6 and pH 8, generally around pH 7.
  • Compositions of the invention may be isotonic with respect to humans.
  • a final vaccine product may be a suspension with a cloudy appearance. This appearance means that microbial contamination is not readily visible, and so the vaccine may contain an antimicrobial agent. This is particularly important when the vaccine is packaged in multidose containers.
  • Preferred antimicrobials for inclusion are 2-phenoxyethanol and thimerosal. It is preferred, however, not to use mercurial preservatives ⁇ e.g. thimerosal), and it is preferred that the composition contains less than about 25 ng/ml mercury. More preferably, the composition is mercury- free.
  • a composition may include a temperature protective agent.
  • a liquid temperature protective agent may be added to an aqueous vaccine composition to lower its freezing point e.g. to reduce the freezing point to below 0°C.
  • the temperature protective agent also permits freezing of the composition while protecting any mineral salt adjuvants against agglomeration or sedimentation after freezing and thawing, and may also protect the composition at elevated temperatures e.g. above 40°C.
  • a starting aqueous vaccine and the liquid temperature protective agent may be mixed such that the liquid temperature protective agent forms from 1 -80% by volume of the final mixture. Suitable temperature protective agents should be safe for human administration, readily miscible/soluble in water, and should not damage other components ⁇ e.g.
  • antigen and adjuvant in the composition.
  • examples include glycerin, propylene glycol, and/or polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • Suitable PEGs may have an average molecular weight ranging from 200-20,000 Da.
  • the polyethylene glycol can have an average molecular weight of about 300 Da ('PEG-300').
  • compositions of the invention can include immunogens in addition to a bleb of the invention.
  • a composition can include a combination of blebs from at least two different Salmonella species, subspecies or serovars.
  • Another useful composition can include a combination of Salmonella blebs of the invention with blebs from another bacterial genus e.g. a combination of Salmonella blebs and Shigella blebs (such as those disclosed in reference 14 e.g. from a AtolR or AtolRAmsbB strain of Shigella).
  • the invention provides a composition comprising blebs from a S.
  • Immunogenic compositions comprise an immunologically effective amount of immunogen, as well as any other of other specified components, as needed.
  • 'immunologically effective amount it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. This amount varies depending upon the health and physical condition of the individual to be treated, age, the taxonomic group of individual to be treated (e.g.
  • blebs in compositions of the invention will generally be between 10 and 500 ⁇ g/ml, preferably between 25 and 200 g/ml, and more preferably about 50 ⁇ g/ml or about 100 ⁇ g/ml (expressed in terms of total protein in the blebs).
  • concentration of blebs in compositions of the invention can be in the range of 1 ng/ml to 10 ⁇ g/ml, or 1 ng/ml to 1 ⁇ g/ml, or 0.5 ⁇ g/ml to 50 ⁇ g/ml.
  • a dosage volume of 0.5ml is typical for injection.
  • composition may be administered in conjunction with other immunoregulatory agents.
  • Adjuvants which may be used in compositions of the invention (particularly in bleb-free compositions) include, but are not limited to: A. Mineral-containing compositions
  • Mineral containing compositions suitable for use as adjuvants in the invention include mineral salts, such as aluminium salts and calcium salts.
  • the invention includes mineral salts such as hydroxides
  • the mineral containing compositions may also be formulated as a particle of metal salt.
  • aluminium hydroxide typically aluminium oxyhydroxide salts, which are usually at least partially crystalline.
  • Aluminium oxyhydroxide which can be represented by the formula AIO(OH)
  • AIO(OH) 3 aluminium hydroxide Al(OH) 3
  • I infrared
  • the degree of crystallinity of an aluminium hydroxide adjuvant is reflected by the width of the diffraction band at half height (WHH), with poorly-crystalline particles showing greater line broadening due to smaller crystallite sizes.
  • WHH diffraction band at half height
  • the surface area increases as WHH increases, and adjuvants with higher WHH values have been seen to have greater capacity for antigen adsorption.
  • a fibrous morphology e.g. as seen in transmission electron micrographs
  • the pi of aluminium hydroxide adjuvants is typically about 1 1 i.e. the adjuvant itself has a positive surface charge at physiological pH. Adsorptive capacities of between 1.8-2.6 mg protein per mg Al +++ at pH 7.4 have been reported for aluminium hydroxide adjuvants.
  • the adjuvants known as "aluminium phosphate” are typically aluminium hydroxyphosphates, often also containing a small amount of sulfate (i.e. aluminium hydroxyphosphate sulfate). They may be obtained by precipitation, and the reaction conditions and concentrations during precipitation influence the degree of substitution of phosphate for hydroxyl in the salt. Hydroxyphosphates generally have a P0 4 /A1 molar ratio between 0.3 and 1.2. Hydroxyphosphates can be distinguished from strict AIPO 4 by the presence of hydroxyl groups. For example, an I spectrum band at 3164cm "1 (e.g. at 200°C) indicates the presence of structural hydroxyls [ch. 9 of ref. 18].
  • the P(VA1 3+ molar ratio of an aluminium phosphate adjuvant will generally be between 0.3 and 1.2, preferably between 0.8 and 1 .2, and more preferably 0.95+0.1.
  • the aluminium phosphate will generally be amorphous, particularly for hydroxyphosphate salts.
  • a typical adjuvant is amorphous aluminium hydroxyphosphate with PO 4 /AI molar ratio between 0.84 and 0.92, included at 0.6mg Al 3+ /ml.
  • the aluminium phosphate will generally be particulate (e.g. plate-like morphology as seen in transmission electron micrographs). Typical diameters of the particles are in the range 0.5- 20 ⁇ (e.g. about 5- 10 ⁇ ) after any antigen adsorption.
  • Adsorptive capacities of between 0.7-1.5 mg protein per mg Al +++ at pH 7.4 have been reported for aluminium phosphate adjuvants.
  • Suspensions of aluminium salts used to prepare compositions of the invention may contain a buffer (e.g. a phosphate or a histidine or a Tris buffer), but this is not always necessary.
  • the suspensions are preferably sterile and pyrogen-free.
  • a suspension may include free aqueous phosphate ions e.g. present at a concentration between 1.0 and 20 mM, preferably between 5 and 15 mM, and more preferably about 10 mM.
  • the suspensions may also comprise sodium chloride.
  • an adjuvant component includes a mixture of both an aluminium hydroxide and an aluminium phosphate.
  • there may be more aluminium phosphate than hydroxide e.g. a weight ratio of at least 2 : 1 e.g. >5 : 1 , >6 : 1 , >7 : 1 , >8 : 1 , >9 : 1 , etc.
  • the concentration of Al +++ in a composition for administration to a patient is preferably less than lOmg/ml e.g. ⁇ 5 mg/ml, ⁇ 4 mg/ml, ⁇ 3 mg/ml, ⁇ 2 mg/ml, ⁇ 1 mg/ml, etc.
  • a preferred range is between 0.3 and lmg/ml.
  • a maximum of ⁇ 0.85mg/dose is preferred.
  • Oil emulsion compositions suitable for use as adjuvants in the invention include squalene-water emulsions, such as MF59 [Chapter 10 of ref. 18; see also ref. 15] (5% Squalene, 0.5% Tween 80, and 0.5% Span 85, formulated into submicron particles using a microfluidizer). Complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IF A) may also be used.
  • CFA Complete Freund's adjuvant
  • IF A incomplete Freund's adjuvant
  • oil-in-water emulsions typically include at least one oil and at least one surfactant, with the oil(s) and surfactant(s) being biodegradable (metabolisable) and biocompatible.
  • the oil droplets in the emulsion are generally less than 5 ⁇ in diameter, and advantageously the emulsion comprises oil droplets with a sub-micron diameter, with these small sizes being achieved with a microfluidiser to provide stable emulsions. Droplets with a size less than 220nm are preferred as they can be subjected to filter sterilization.
  • the invention can be used with oils such as those from an animal (such as fish) or vegetable source.
  • Sources for vegetable oils include nuts, seeds and grains. Peanut oil, soybean oil, coconut oil, and olive oil, the most commonly available, exemplify the nut oils.
  • Jojoba oil can be used e.g. obtained from the jojoba bean. Seed oils include safflower oil, cottonseed oil, sunflower seed oil, sesame seed oil and the like. In the grain group, corn oil is the most readily available, but the oil of other cereal grains such as wheat, oats, rye, rice, teff, triticale and the like may also be used.
  • 6-10 carbon fatty acid esters of glycerol and 1 ,2-propanediol may be prepared by hydrolysis, separation and esterification of the appropriate materials starting from the nut and seed oils.
  • Fats and oils from mammalian milk are metabolizable and may therefore be used in the practice of this invention.
  • the procedures for separation, purification, saponification and other means necessary for obtaining pure oils from animal sources are well known in the art.
  • Most fish contain metabolizable oils which may be readily recovered. For example, cod liver oil, shark liver oils, and whale oil such as spermaceti exemplify several of the fish oils which may be used herein.
  • a number of branched chain oils are synthesized biochemically in 5-carbon isoprene units and are generally referred to as terpenoids.
  • Shark liver oil contains a branched, unsaturated terpenoid known as squalene, 2,6, 10, 15, 19,23-hexamethyl-2,6,10, 14, 18,22-tetracosahexaene.
  • Other preferred oils are the tocopherols (see below). Oil in water emulsions comprising sqlauene are particularly preferred. Mixtures of oils can be used.
  • Surfactants can be classified by their 'HLB' (hydrophile/lipophile balance). Preferred surfactants of the invention have a HLB of at least 10, preferably at least 15, and more preferably at least 16.
  • the invention can be used with surfactants including, but not limited to: the polyoxy ethylene sorbitan esters surfactants (commonly referred to as the Tweens), especially polysorbate 20 and polysorbate 80; copolymers of ethylene oxide (EO), propylene oxide (PO), and/or butylene oxide (BO), sold under the DOWFAXTM tradename, such as linear EO/PO block copolymers; octoxynols, which can vary in the number of repeating ethoxy (oxy-l ,2-ethanediyl) groups, with octoxynol-9 (Triton X-100, or t-octylphenoxypolyethoxyethanol) being of particular interest; (octylphenoxy)polyethoxyethanol (IGEPAL CA-630/NP-40); phospholipids such as phosphatidylcholine (lecithin); polyoxyethylene fatty ethers derived from lauryl, cetyl,
  • Preferred surfactants for including in the emulsion are Tween 80 (polyoxyethylene sorbitan monooleate), Span 85 (sorbitan trioleate), lecithin and Triton X-100.
  • detergents such as Tween 80 may contribute to the thermal stability seen in the examples below.
  • surfactants can be used e.g. Tween 80/Span 85 mixtures.
  • a combination of a polyoxyethylene sorbitan ester such as polyoxyethylene sorbitan monooleate (Tween 80) and an octoxynol such as t-octylphenoxypolyethoxyethanol (Triton X-100) is also suitable.
  • Another useful combination comprises laureth 9 plus a polyoxyethylene sorbitan ester and/or an octoxynol.
  • Preferred amounts of surfactants are: polyoxyethylene sorbitan esters (such as Tween 80) 0.01 to 1 %, in particular about 0.1 %; octyl- or nonylphenoxy polyoxyethanols (such as Triton X-100, or other detergents in the Triton series) 0.001 to 0.1 %, in particular 0.005 to 0.02%; polyoxyethylene ethers (such as laureth 9) 0.1 to 20 %, preferably 0.1 to 10 % and in particular 0.1 to 1 % or about 0.5%.
  • High levels of surfactants should be avoided, however, as these may affect the stability and/or integrity of bacterial blebs.
  • oil-in-water emulsion adjuvants useful with the invention include, but are not limited to:
  • a submicron emulsion of squalene, Tween 80, and Span 85 A submicron emulsion of squalene, Tween 80, and Span 85.
  • the composition of the emulsion by volume can be about 5% squalene, about 0.5% polysorbate 80 and about 0.5% Span 85. In weight terms, these ratios become 4.3% squalene, 0.5% polysorbate 80 and 0.48% Span 85.
  • This adjuvant is known as 'MF59' [15- 17], as described in more detail in Chapter 10 of ref. 18 and chapter 12 of ref. 19.
  • the MF59 emulsion advantageously includes citrate ions e.g. lOmM sodium citrate buffer.
  • An emulsion comprising squalene, an a-tocopherol, and polysorbate 80.
  • These emulsions may have from 2 to 10% squalene, from 2 to 10% tocopherol and from 0.3 to 3% Tween 80, and the weight ratio of squalene tocopherol is preferably ⁇ 1 (e.g. 0.90) as this provides a more stable emulsion.
  • Squalene and Tween 80 may be present volume ratio of about 5:2, or at a weight ratio of about 1 1 :5.
  • One such emulsion can be made by dissolving Tween 80 in PBS to give a 2% solution, then mixing 90ml of this solution with a mixture of (5g of DL-a-tocopherol and 5ml squalene), then microfluidising the mixture.
  • the resulting emulsion may have submicron oil droplets e.g. with an average diameter of between 100 and 250nm, preferably about 180nm.
  • An emulsion of squalene, a tocopherol, and a Triton detergent e.g. Triton X-100
  • the emulsion may also include a 3d-MPL (see below).
  • the emulsion may contain a phosphate buffer.
  • An emulsion comprising a polysorbate (e.g. polysorbate 80), a Triton detergent (e.g. Triton X- 100) and a tocopherol (e.g. an a-tocopherol succinate).
  • the emulsion may include these three components at a mass ratio of about 75 : 1 1 : 10 (e.g.
  • the emulsion may also include squalene.
  • the emulsion may also include a 3d-MPL (see below).
  • the aqueous phase may contain a phosphate buffer.
  • An emulsion of squalane, polysorbate 80 and poloxamer 401 ("PluronicTM L 121 ").
  • the emulsion can be formulated in phosphate buffered saline, pH 7.4.
  • This emulsion is a useful delivery vehicle for muramyl dipeptides, and has been used with threonyl-MDP in the "SAF-1 " adjuvant [20] (0.05- 1% Thr-MDP, 5% squalane, 2.5% Pluronic L 121 and 0.2% polysorbate 80). It can also be used without the Thr-MDP, as in the "AF" adjuvant [21] (5% squalane, 1.25% Pluronic L121 and 0.2% polysorbate 80). Microfluidisation is preferred.
  • An emulsion comprising squalene, an aqueous solvent, a polyoxyethylene alkyl ether hydrophilic nonionic surfactant (e.g. polyoxyethylene ( 12) cetostearyl ether) and a hydrophobic nonionic surfactant (e.g. a sorbitan ester or mannide ester, such as sorbitan monoleate or ' Span 80').
  • the emulsion is preferably thermoreversible and/or has at least 90% of the oil droplets (by volume) with a size less than 200 nm [22].
  • the emulsion may also include one or more of: alditol; a cryoprotective agent (e.g. a sugar, such as dodecylmaltoside and/or sucrose); and/or an alkylpolyglycoside. Such emulsions may be lyophilized.
  • An emulsion having from 0.5-50% of an oil, 0.1-10% of a phospholipid, and 0.05-5% of a non- ionic surfactant.
  • preferred phospholipid components are phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidyl inositol, phosphatidylglycerol, phosphatidic acid, sphingomyelin and cardiolipin. Submicron droplet sizes are advantageous.
  • Additives may be included, such as QuilA saponin, cholesterol, a saponin-lipophile conjugate (such as GPI-0100, described in reference 24, produced by addition of aliphatic amine to desacylsaponin via the carboxyl group of glucuronic acid), dimethyidioctadecylammonium bromide and/or N,N-dioctadecyl-N,N-bis (2-hydroxyethyl)propanediamine.
  • a non-metabolisable oil such as light mineral oil
  • surfactant such as lecithin, Tween 80 or Span 80.
  • Additives may be included, such as QuilA saponin, cholesterol, a saponin-lipophile conjugate (such as GPI-0100
  • An emulsion comprising a mineral oil, a non-ionic lipophilic ethoxylated fatty alcohol, and a non-ionic hydrophilic surfactant (e.g. an ethoxylated fatty alcohol and/or polyoxyethylene- polyoxypropylene block copolymer) [25].
  • a non-ionic lipophilic ethoxylated fatty alcohol e.g. an ethoxylated fatty alcohol and/or polyoxyethylene- polyoxypropylene block copolymer
  • An emulsion comprising a mineral oil, a non-ionic hydrophilic ethoxylated fatty alcohol, and a non-ionic lipophilic surfactant (e.g. an ethoxylated fatty alcohol and/or polyoxyethylene- polyoxypropylene block copolymer) [25].
  • a saponin e.g. QuilA or QS21
  • a sterol e.g. a cholesterol
  • Antigens and adjuvants in a composition will typically be in admixture at the time of delivery to a patient.
  • the emulsions may be mixed with antigen during manufacture, or extemporaneously, at the time of delivery.
  • the adjuvant and antigen may be kept separately in a packaged or distributed vaccine, ready for final formulation at the time of use.
  • the antigen will generally be in an aqueous form, such that the vaccine is finally prepared by mixing two liquids.
  • the volume ratio of the two liquids for mixing can vary (e.g. between 5: l and 1 :5, or 10: 1 and 1 : 10) but is generally about 1 : 1.
  • Saponin formulations may also be used as adjuvants in the invention.
  • Saponins are a heterogeneous group of sterol glycosides and triterpenoid glycosides that are found in the bark, leaves, stems, roots and even flowers of a wide range of plant species. Saponin from the bark of the Quillaia saponaria Molina tree have been widely studied as adjuvants. Saponin can also be commercially obtained from Smilax ornata (sarsaprilla), Gypsophilla paniculata (brides veil), and Saponaria offwianalis (soap root).
  • Saponin adjuvant formulations include purified formulations, such as QS21 , as well as lipid formulations, such as ISCOMs. QS21 is marketed as StimulonTM.
  • Saponin compositions have been purified using HPLC and RP-HPLC. Specific purified fractions using these techniques have been identified, including QS7, QS 17, QS 18, QS21 , QH-A, QH-B and QH-C.
  • the saponin is QS21 .
  • a method of production of QS21 is disclosed in ref. 27.
  • Saponin formulations may also comprise a sterol, such as cholesterol [28].
  • ISCOMs immunostimulating complexs
  • phospholipid such as phosphatidylethanolamine or phosphatidylcholine.
  • Any known saponin can be used in ISCOMs.
  • the ISCOM includes one or more of QuilA, QHA & QHC.
  • the ISCOMS may be devoid of additional detergent [31].
  • Adjuvants suitable for use in the invention include bacterial or microbial derivatives such as non-toxic derivatives of enterobacterial lipopolysaccharide (LPS), Lipid A derivatives, immunostimulatory oligonucleotides and ADP-ribosylating toxins and detoxified derivatives thereof.
  • LPS enterobacterial lipopolysaccharide
  • Lipid A derivatives Lipid A derivatives
  • immunostimulatory oligonucleotides and ADP-ribosylating toxins and detoxified derivatives thereof.
  • Non-toxic derivatives of LPS include monophosphoryl lipid A (MPL) and 3-O-deacylated MPL (3dMPL).
  • 3dMPL is a mixture of 3 de-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains.
  • a preferred "small particle" form of 3 De-O-acylated monophosphoryl lipid A is disclosed in ref. 34. Such "small particles" of 3dMPL are small enough to be sterile filtered through a 0.22 ⁇ membrane [34].
  • Other non-toxic LPS derivatives include monophosphoryl lipid A mimics, such as aminoalkyl glucosaminide phosphate derivatives e.g. RC-529 [35,36].
  • Lipid A derivatives include derivatives of lipid A from Escherichia coli such as OM-174.
  • OM- 174 is described for example in refs. 37 & 38.
  • Immunostimulatory oligonucleotides suitable for use as adjuvants in the invention include nucleotide sequences containing a CpG motif (a dinucleotide sequence containing an unmethylated cytosine linked by a phosphate bond to a guanosine). Double-stranded RNAs and oligonucleotides containing palindromic or poly(dG) sequences have also been shown to be immunostimulatory.
  • the CpG's can include nucleotide modifications/analogs such as phosphorothioate modifications and can be double-stranded or single-stranded.
  • References 39, 40 and 41 disclose possible analog substitutions e.g. replacement of guanosine with 2'-deoxy-7-deazaguanosine.
  • the adjuvant effect of CpG oligonucleotides is further discussed in refs. 42-47.
  • the CpG sequence may be directed to TLR9, such as the motif GTCGTT or TTCGTT [48].
  • the CpG sequence may be specific for inducing a Thl immune response, such as a CpG- A ODN, or it may be more specific for inducing a B cell response, such a CpG-B ODN.
  • CpG-A and CpG-B ODNs are discussed in refs. 49-51.
  • the CpG is a CpG-A ODN.
  • the CpG oligonucleotide is constructed so that the 5' end is accessible for receptor recognition.
  • two CpG oligonucleotide sequences may be attached at their 3' ends to form "immunomers". See, for example, refs. 52-54.
  • a particularly useful adjuvant based around immunostimulatory oligonucleotides is known as IC-3 1TM [55-57].
  • an adjuvant used with the invention may comprise a mixture of (i) an oligonucleotide (e.g. between 15-40 nucleotides) including at least one (and preferably multiple) Cpl motifs (i.e.
  • a cytosine linked to an inosine to form a dinucleotide and (ii) a polycationic polymer, such as an oligopeptide (e.g. between 5-20 amino acids) including at least one (and preferably multiple) Lys-Arg-Lys tripeptide sequence(s).
  • the oligonucleotide may be a deoxynucleotide comprising 26-mer sequence 5'-(IC)i 3 -3' (SEQ ID NO : 58).
  • the polycationic polymer may be a peptide comprising 1 1-mer amino acid sequence KLKLLLLLKLK (SEQ ID NO: 59). This combination of SEQ ID NOs: 58 and 59 provides the IC-31TM adjuvant.
  • Bacterial ADP-ribosylating toxins and detoxified derivatives thereof may be used as adjuvants in the invention.
  • the protein is derived from E. coli (E.coli heat labile enterotoxin "LT"), cholera ("CT"), or pertussis ("PT").
  • LT E.coli heat labile enterotoxin
  • CT cholera
  • PT pertussis
  • the use of detoxified ADP-ribosylating toxins as mucosal adjuvants is described in ref. 58 and as parenteral adjuvants in ref. 59.
  • the toxin or toxoid is preferably in the form of a holotoxin, comprising both A and B subunits.
  • the A subunit contains a detoxifying mutation; preferably the B subunit is not mutated.
  • the adjuvant is a detoxified LT mutant such as LT-K63, LT-R72, and LT-G 192.
  • LT-K63, LT-R72, and LT-G 192 The use of ADP-ribosylating toxins and detoxified derivatives thereof, particularly LT-K63 and LT-R72, as adjuvants can be found in refs. 60-67.
  • a useful CT mutant is or CT-E29H [68].
  • Numerical reference for amino acid substitutions is preferably based on the alignments of the A and B subunits of ADP-ribosylating toxins set forth in ref. 69, specifically incorporated herein by reference in its entirety. E. Human immunomodulators
  • Human immunomodulators suitable for use as adjuvants in the invention include cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-1 2 [70], etc.) [71], interferons (e.g. interferon- ⁇ ), macrophage colony stimulating factor, and tumor necrosis factor.
  • cytokines such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-1 2 [70], etc.) [71], interferons (e.g. interferon- ⁇ ), macrophage colony stimulating factor, and tumor necrosis factor.
  • interferons e.g. interferon- ⁇
  • macrophage colony stimulating factor e.g. interferon- ⁇
  • tumor necrosis factor e.g. interferon- ⁇
  • a preferred immunomodulator is IL-12.
  • B ioadhesives and mucoadhesives may also be used as adjuvants in the invention.
  • Suitable bioadhesives include esterified hyaluronic acid microspheres [72] or mucoadhesives such as cross-linked derivatives of poly(acrylic acid), polyvinyl alcohol, polyvinyl pyrollidone, polysaccharides and carboxymethylcellulose. Chitosan and derivatives thereof may also be used as adjuvants in the invention [73].
  • Microparticles may also be used as adjuvants in the invention.
  • Microparticles i.e. a particle of ⁇ 100nm to ⁇ 150 ⁇ in diameter, more preferably ⁇ 200nm to ⁇ 30 ⁇ in diameter, and most preferably ⁇ 500nm to - ⁇ ⁇ in diameter
  • materials that are biodegradable and non-toxic e.g. a poly(a-hydroxy acid) , a polyhydroxybutyric acid, a polyorthoester, a polyanhydride, a polycaprolactone, etc.
  • a negatively-charged surface e.g. with SDS
  • a positively-charged surface e.g. with a cationic detergent, such as CTAB
  • liposome formulations suitable for use as adjuvants are described in refs. 74-76.
  • imidazoquinolone compounds suitable for use adjuvants in the invention include Imiquamod and its homologues (e.g. "Resiquimod 3M"), described further in refs. 77 and 78.
  • the invention may also comprise combinations of aspects of one or more of the adjuvants identified above.
  • the following adjuvant compositions may be used in the invention: (1) a saponin and an oil-in-water emulsion [79]; (2) a saponin (e.g. QS21) + a non-toxic LPS derivative (e.g.
  • 3dMPL 3dMPL
  • a saponin e.g. QS21
  • a non-toxic LPS derivative e.g. 3dMPL
  • a saponin e.g. QS21
  • 3dMPL + IL- 12 optionally + a sterol
  • SAF containing 10% squalane, 0.4% Tween 80TM, 5% pluronic-block polymer L 121 , and thr-MDP, either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion.
  • RibiTM adjuvant system (RAS), (Ribi Immunochem) containing 2% squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL + CWS (DetoxTM); and (8) one or more mineral salts (such as an aluminum salt) + a non-toxic derivative of LPS (such as 3dMPL).
  • MPL monophosphorylipid A
  • TDM trehalose dimycolate
  • CWS cell wall skeleton
  • LPS such as 3dMPL
  • An aluminium hydroxide adjuvant is useful, and antigens are generally adsorbed to this salt.
  • Oil-in- water emulsions comprising squalene, with submicron oil droplets, are also preferred, particularly in the elderly.
  • Useful adjuvant combinations include combinations of Thl and Th2 adjuvants such as CpG & an aluminium salt, or resiquimod & an aluminium salt.
  • a combination of an aluminium salt and 3dMPL may be used.
  • the invention also provides a method for raising an immune response in a mammal, comprising administering an immunogenic composition of the invention to the mammal.
  • the immune response will typically include an antibody response.
  • the immune response is preferably a protective immune response.
  • the invention also provides compositions of the invention for use in such methods. Protection can be measured in vivo by various techniques e.g. by immunising test animals, administering a lethal dose of Salmonella, and assessing survival. Suitable models are available in the art e.g. the chicken challenge model of reference 83, the murine models of references 84 & 85, or the murine model used herein.
  • the invention also provides a method for raising an antibody response in a mammal, comprising administering an immunogenic composition of the invention to the mammal.
  • the antibody response is preferably a protective antibody response.
  • the invention also provides compositions of the invention for use in such methods.
  • the invention also provides a method for protecting a mammal against a Salmonella infection and/or disease ⁇ e.g. against gastroenteritis, typhoid fever and/or paratyphoid fever; or against bacteremia), comprising administering to the mammal an immunogenic composition of the invention.
  • the invention provides compositions of the invention for use as medicaments ⁇ e.g. as immunogenic compositions or as vaccines). It also provides the use of vesicles of the invention in the manufacture of a medicament for preventing a Salmonella infection in a mammal e.g. for preventing gastroenteritis, typhoid fever and/or paratyphoid fever (or bacteremia). It also provides the use of a bleb protein (as defined above) in the manufacture of a bleb-free medicament for preventing a Salmonella infection in a mammal e.g. for preventing gastroenteritis, typhoid fever and/or paratyphoid fever (or bacteremia).
  • the mammal is preferably a human.
  • the human may be an adult or, preferably, a child. Where the vaccine is for prophylactic use, the human is preferably a child (e.g. a toddler or infant); where the vaccine is for therapeutic use, the human is preferably an adult. A vaccine intended for children may also be administered to adults e.g. to assess safety, dosage, immunogenicity, etc.
  • the uses and methods are particularly useful for preventing/treating diseases including, but not limited to, gastroenteritis, typhoid fever, bacteremia and/or paratyphoid fever.
  • Efficacy of therapeutic treatment can be tested by monitoring Salmonella infection after administration of the composition of the invention.
  • Efficacy of prophylactic treatment can be tested by monitoring immune responses against immunogenic proteins in the blebs or other antigens after administration of the composition.
  • Immunogenicity of compositions of the invention can be determined by administering them to test subjects (e.g. children 12-16 months age) and then determining standard serological parameters. These immune responses will generally be determined around 4 weeks after administration of the composition, and compared to values determined before administration of the composition. Where more than one dose of the composition is administered, more than one post-administration determination may be made.
  • compositions of the invention will generally be administered directly to a patient.
  • Direct delivery may be accomplished by parenteral injection (e.g. subcutaneously, intraperitoneally, intravenously, intramuscularly, or to the interstitial space of a tissue), or by rectal, oral, vaginal, topical, transdermal, intranasal, ocular, aural, pulmonary or other mucosal administration. Sublingual or buccal administration is also possible.
  • Intramuscular administration to the thigh or the upper arm is preferred. Injection may be via a needle (e.g. a hypodermic needle), but needle-free injection may alternatively be used.
  • a typical intramuscular dose is about 0.5 ml.
  • the invention may be used to elicit systemic and/or mucosal immunity.
  • Dosage treatment can be a single dose schedule or a multiple dose schedule. Multiple doses may be used in a primary immunisation schedule and/or in a booster immunisation schedule. A primary dose schedule may be followed by a booster dose schedule. Suitable timing between priming doses (e.g. between 4- 16 weeks), and between priming and boosting, can be routinely determined.
  • composition comprising X may consist exclusively of X or may include something additional e.g. X + Y.
  • references to a percentage sequence identity between two amino acid sequences means that, when aligned, that percentage of amino acids are the same in comparing the two sequences.
  • This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in section 7.7.18 of reference 86.
  • a preferred alignment is determined by the Smith- Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 2, BLOSUM matrix of 62.
  • the Smith- Waterman homology search algorithm is well known and is disclosed in reference 87.
  • GI numbering is used above.
  • a GI number, or “Genlnfo Identifier” is a series of digits assigned consecutively to each sequence record processed by NCBI when sequences are added to its databases. The GI number bears no resemblance to the accession number of the sequence record.
  • a sequence is updated (e.g. for correction, or to add more annotation or information) then it receives a new GI number. Thus the sequence associated with a given GI number is never changed.
  • this epitope may be a B-cell epitope and/or a T-cell epitope.
  • Such epitopes can be identified empirically (e.g. using PEPSCAN [88,89] or similar methods), or they can be predicted (e.g. using the Jameson- Wolf antigenic index [90], matrix-based approaches [91], MAPITOPE [92], TEPITOPE [93,94], neural networks [95], OptiMer & EpiMer [96, 97] , ADEPT [98], Tsites [99], hydrophilicity [100], antigenic index [ 101 ] or the methods disclosed in references 102-103, etc.).
  • Epitopes are the parts of an antigen that are recognised by and bind to the antigen binding sites of antibodies or T-cell receptors, and they may also be referred to as "antigenic determinants”.
  • Figure 1 shows SDS-PAGE results.
  • the four lanes are MW marker, medium alone, wild-type LT2 and AtolR LT2.
  • Figure 2 shows mean absorbance in ELISA assays of Salmonella bleb-specific IgG, in all cases using 1 : 10000 dilutions of sera.
  • Figure 2A the 3 groups of bars show results after different doses of LT2 AtolR blebs (0.1 , 1 , 10 ⁇ g from left to right). Each group has a bar for 3 different time points (days 0, 14, 32 from left to right).
  • each group includes results for three different time points: day 0 (circles; pre-immune sera), day 20 (triangles; post first immunization), day 45 (squares; post second immunization). Horizontal bars show geometric means for the three time points.
  • Figure 3 shows a competitive ELISA in the presence of exogenous LPS, where sera raised against blebs from LT2 AtolR were mixed with different amounts of LPS ⁇ g/mL on x-axis) from S. Typhimurium and the remaining IgG response to blebs from S. Typhimurium LT2 AtolR was analyzed by ELISA. Plates were coated with LT2 AtolR blebs.
  • Figure 4 shows (A) 2D SDS-PAGE of SL1344 AtolR blebs, with pH from 3-11 on x-axis and MW from 10-250 kDa on y-axis; and (B) a 2D immunoblot of SL 1344 AtolR blebs probed with serum raised against LT2 AtolR blebs.
  • Figure 5 shows SDS-PAGE analysis of samples taken (i) before the first TFF, (ii) after the first TFF, and (iii) after the second TFF.
  • Each panel has three lanes showing, from left to right, total protein, bleb protein and soluble protein.
  • Figure 6 shows a 12% Bis-Tris SDS-PAGE of 10 ⁇ g of Salmonella blebs (a: SL1344Afo/i?, b: SL ⁇ 344AtolRAwbaP, c: SL UUAtolRAwbaPAmsbB) grown in LB under iron-rich (left, a-c) or iron-limiting (right, a-c) conditions. Some iron regulated proteins are highlighted with an arrow.
  • Figure 7 shows SL 1344Ato/i? blebs visualized by transmission electron microscopy.
  • the bar at the bottom-right is 1 OOnm long.
  • Figure 8 shows the analysis by flow cytometry of reactivity of sera raised against blebs from S. Typhimurium SL 1344 AtolR (panels A, C, E) or from SL 1344 AtolRAwbaP (panels B, D, F) with live bacteria of S. Typhimurium (wild-type in panels A & B; AwbaP mutants in panels C & D) and S. Enteritidis AwbaP mutants in panels E & F).
  • Figure 9 shows I D western blots with 2.5, 5 or 10 ⁇ g of blebs generated from S. Typhimurium SL1344 AtolRAwbaP (left in each blot) and S. Enteritidis P 125 109 AtolRAwbaP (right in each blot) and probed with sera raised against S. Typhimurium SL 1344 AtolRAwbaP (left blot) and SL1344 tolRAwbaPAmsbB (right blot).
  • Figure 1 1 shows IgG titers to blebs from (A) SL1344 AtolR and (B) SL 1344 AtolRAwbaP in sera of mice immunized intranasally with blebs from SL 1344 AtolR ( ⁇ g; left); SL 1344 AtolR (5 ⁇ ig; middle), or SL 1344 AtolRAwbaP (1 ⁇ g; right).
  • restriction enzyme cloning regions 500 bp upstream and downstream of the target gene (e.g. the tolR gene) were amplified by PCR with primers containing RE sites at the 5' and 3' end. These fragments were then digested with the respective RE and ligated to a previously RE digested pBlueScript II S (+) (pBS) to generate a plasmid containing the 500 bp upstream and downstream regions of tolR with a restriction site in the middle (e.g. pBS-tolR5'3').
  • RE restriction enzyme
  • This plasmid was subsequently digested with an RE and ligated to a kanamycin resistance cassette that had been amplified from pUC4K with primers corresponding to the same RE site at both the 5' and 3 ' end, to generate a plasmid containing the 500 bp upstream region, the kmR cassette, and the 500 bp downstream region (e.g. pBS-tolR5'-kmR-tolR3').
  • This plasmid was linearised with a RE and a linear DNA fragment was amplified by PCR to yield e.g. tolR5'kmRtolR3'.
  • This fragment was electroporated into the LT2 strain expressing the phage ⁇ red homologous recombination system encoded by plasmid pAJD434, and LT2 mutants where the target gene had been replaced with the kmR cassette (e.g. LT2AtolR::kari) were selected on LB plates containing kanamycin. Gene replacement was confirmed by PCR amplification of the genomic DNA of the mutant colonies using primers annealing upstream and downstream of the regions used for homologous recombination.
  • kmR cassette e.g. LT2AtolR::kari
  • Inactivation of chromosomal genes by PCR was performed as in reference 104.
  • a chloramphenicol-resistance gene was amplified by PCR from pKD3 using primers with 45 bp extensions that were homologous to the regions adjacent to the target gene.
  • msbB a tetracycline-resistance gene was amplified by PCR from E.coli DH5 genomic DNA containing a Tnl 0 using primers with 45bp extensions that were homologous to the regions adjacent to the target gene.
  • PCR products were then transformed into electrocompetent strains expressing the phage ⁇ red recombinase encoded on pSIM18 and successful recombination was checked by PCR on the transformant colonies grown on selective medium (chloramphenicol or hygromycin). Gene deletion was confirmed by PCR screening. Rough mutants (AwbaP) were further confirmed by silver stained LPS SDS-PAGE and resistance to P22 bacteriophage.
  • ⁇ of an overnight culture of the donor strain with ⁇ ⁇ of P22 lysate was incubated at 37°C for 20 mins, 3mL of liquid top LB agar (0.75% agar) were added to the mix, and poured over a pre-warmed LB plate for incubation for 4 hours at 37°C. Once the confluence peak was reached, top of the agar was scrapped, mixed with 3 mL of LB broth, mixed thoroughly in the presence of lOOul of chloroform, incubated for 20 min at room temperature, and the lysate containing the phage was stored at 4°C until further use.
  • FIG. 1 shows a ID gel of TCA-precipitated culture supernatant, after 0.22 ⁇ filtration, after growing wild-type and AtolR LT2 bacteria. A large number of protein bands were visible in the supernatant of AtolR mutant, but most of these bands were not visible in the wild type strain. Thus the mutant strain secretes many more proteins into the culture medium during growth.
  • Mutant strains were grown in LB or HTMC to OD 0.8 or 6.0, respectively. Bacteria were pelleted by centrifugation at 4000 x g and culture media was filtered through a 0.22 ⁇ filter. Filtrates containing blebs were concentrated using a stirred ultrafiltration cell and ultracentrifuged (186,000 x g, 3 hours, 4°C). The bleb-containing pellet was resuspended in PBS . Total protein concentration was determined by Bradford assay. Transmission electron microscopy demonstrated that this purification procedure results in a homogeneous population of blebs -30-50 nm diameter (Figure 7).
  • the knockout strains were grown under the following conditions: pH 7.1 , 37°C, dissolved oxygen maintained at 30% saturation by controlling agitation and setting maximum aeration.
  • the pH was controlled by addition of 30% ammonium hydroxide.
  • Foam was controlled by addition of 0.25g/L of PPG in the fermentation medium.
  • the culture inoculum was 1 % of the fermenter volume. The fermentation process was stopped after 14 hours, when the culture achieved a cell concentration of 29 OD 6 oonm- Culture supernatant containing vesicles was separated from the Salmonella biomass by tangential flow filtration (TFF) through a 0.22 ⁇ pore size filter cassette with a 0.1m 2 filtration area.
  • TMF tangential flow filtration
  • Blebs were also obtained from bacteria grown in the presence of 200 ⁇ of iron chelator 2,2'- dipyridyl to simulate in vivo iron-limiting conditions. Under these conditions Salmonella can upregulate the expression of certain outer membrane proteins which are highly conserved among serovars. Analysis of the resulting blebs did not show any major changes in protein composition. As shown in Figure 6 several proteins of predominantly 70-80 kDa size were induced under iron- limiting conditions, which is in accordance with the known major iron-regulated proteins, including cirA (74kDa), fepA (83 kDa), fhuA (81 kDa), fhuE (81 kDa), and tonB (79 kDa). Bleb immunogenicity
  • mice Three groups of mice were subcutaneous ly immunized twice with a two-week interval (days 0 & 14) using different amounts of llAtolR blebs (0.1 ⁇ g, 1.0 ⁇ g, 10.0 ⁇ g). To assess the immunogenicity by ELISA sera were collected at three time points (day 0, 14, 32). Flat bottom Maxisorp plates were coated with blebs overnight, washed with PBST, and incubated with sera (1 : 1 ,000 in PBS) for 2 hours. Plates were further washed with PBST and incubated with alkaline phosphatase anti-mouse IgG (1 : 10,000) for 1 hour. 4-nitrophenyl phosphate disodium salt hexahydrate substrate was added for 1 hour, and absorbance was measured (490-405nm).
  • mice displayed a dose-dependent antibody response to the llAtolR blebs.
  • the second immunization was important for a suitable antibody response, as seen by the large increase in IgG response between days 14 and 32 at all three doses.
  • the strong ELISA signal for 1 : 1000 dilutions of the sera indicates a strong immunogenicity of the blebs.
  • Competitive ELISA assays in the presence of exogenous LPS from S. Typhimurium (Figure 3) were performed to assess the contribution of IgG directed to the LPS to the overall IgG response to blebs from LT2AtolR. More than 10 ⁇ g/mL of LPS in the assay were needed to reduce the signal in the ELISA, indicating that a significant portion of the IgG is directed to the LPS.
  • SL1344 AtolR and SL 1344 AtolRAwbaP were also tested for their ability to elicit a strong antibody responses by intranasal immunization. Mice were immunized twice with 1 ⁇ g or 5 ⁇ g of SL1344 AtolR blebs, or 1 ⁇ g or 5 ⁇ g of AtolRAwbaP blebs (although the 5 ⁇ g dose with AtolRAwbaP blebs was not well tolerated and so this experiment was stopped). IgG titers were determined by ELISA with serial dilutions of sera. As shown in Figure 1 1, both types of blebs elicited very strong antibody responses.
  • blebs from SL 1344 AtolRAwbaP elicited very high titers against blebs without O antigen suggesting a strong response to the proteins or the LPS core.
  • the bleb approach has a strong potential to produce effective and low-cost vaccines and can be extended to different Salmonella strains towards a broad spectrum vaccine.
  • Blebs were characterised by I D or 2D gel electrophoresis.
  • I D characterisation 20 ⁇ g of blebs were precipitated for 1 hour in TCA 0.4% DOC, washed three times in 100% ethanol, and resuspended in reswelling buffer (7M urea, 2M thiourea, 2% CHAPS, 2% ASB- 14, 0. 1 % DTT, 20mM Tris base, and bromophenol blue) before loading onto a 10% Bis-Tris gel. The gel was run at 120 V in MOPS buffer and stained with Coomassie overnight.
  • Protein spots were excised from the gel and proteins and identified using mass finger printing. 52 proteins identified in the AtolR knockout of strain SL1344 are listed in Table 1. Analysis of blebs from the S. Typhimurium SL 1344 AtolRAwbaP strain revealed the 296 proteins listed in Table 2. Similarly, Table 3 lists 293 proteins which were identified in blebs from the AtolRAwbaP mutant of S. Enteritidis.
  • a 2D separation of the SL1344 to/i? blebs was also analysed by immunoblot using sera obtained from mice immunised with LT2AtolR blebs, thereby identifying immunogenic proteins in the blebs.
  • Gels were transferred to a nitrocellulose membrane using the iBlot dry transfer system, incubated with mouse anti-bleb sera (1 : 1000 in 3% milk in PBS), washed three times for 10 min in 3% milk, 0.1% Tween, PBS, incubated with secondary antibody anti-mouse HRP (1 :5000), washed three times for 10 min in 3% milk, 0.1% Tween, PBS, and developed by chemiluminescence using ImageQuant.
  • the 2D blot in Figure 4B shows three major groups of bands in the middle of the blot, confirming cross-reactivity of antibody between the LT2 and SL 1344 blebs. By comparison with earlier 2D-PAGE results the bands appear to correspond to outer membrane protein A. Cross-reactivity with different Salmonella serovars of sera raised against blebs
  • the blots show that the strong antibody reactivity observed previously by ELISA and flow cytometry is elicited by a wide number of proteins in blebs. Moreover, the blots showed extensive cross reactivity with S. Enteritidis proteins, as seen by the similarities in the protein pattern between the two types of blebs. Slight differences can be observed in the 50-75 kDa region. Protection against S. Typhimurium by blebs in a murine challenge model
  • mice were immunized subcutaneously with 1 ⁇ g of blebs generated under iron-limiting conditions from S. Typhimurium SL 1344 AtolR, SL 1344 AtolRAwbaP, and S. Enteritidis PI 25109 AtolRAwbaP on days 0, 7, and 21.
  • a negative control group was not immunised.
  • mice were immunized orally with 10 9 bacteria of the live attenuated vaccine strain S. Typhimurium SL3261 (S. Typhimurium SL 1344 AaroA) on day 0.
  • mice were challenged on day 63 with 1.1 x l O 6 bacteria of the wild-type strain S. Typhimurium M525 (with intermediate virulence). The mice were sacrificed 3 days after challenge and colonization of spleen, liver, and blood by M525 was determined. As shown in Figure 10A, immunization with all types of bleb significantly reduced colonization of the spleens. Importantly, immunization with blebs from heterologous S. Enteritidis also resulted in a reduction of colonization in the challenged mice, indicating that blebs without O antigen could potentially protect against multiple serovars of Salmonella.
  • SEQ ID NO: 21 is the same as SEQ ID NO: 54;
  • SEQ ID NO: 35 is the same as SEQ ID NO: 57.
  • hsIJ heat shock protein
  • protease III precursor (pitrilysin)
  • DNA-binding protein (histone-like protein Hlp-ll)
  • Vaccine Adjuvants Preparation Methods and Research Protocols (Volume 42 of Methods in Molecular Medicine series). ISBN: 1-59259-083-7. Ed. O'Hagan.

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Abstract

L'invention concerne des souches hyper bourgeonnantes de Salmonella qui sont générées par l'interruption d'un ou plusieurs composants dans le système Tol-Pal, tel que TolR. Les bourgeonnements provenant de ces souches sont des immunogènes utiles pour la vaccination. Les protéines individuelles trouvées dans ces bourgeonnements peuvent également être utilisées en tant qu'immunogènes.
PCT/IB2011/054569 2010-10-15 2011-10-14 Souches hyper bourgeonnantes de salmonella Ceased WO2012049662A1 (fr)

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WO2015142875A1 (fr) * 2014-03-17 2015-09-24 The Trustees Of The University Of Pennsylvania Compositions et procédés utilisant de la salmonelle modifiée
WO2016202872A1 (fr) * 2015-06-16 2016-12-22 Glaxosmithkline Biologicals Sa Compositions immunogènes
WO2019155415A1 (fr) * 2018-02-09 2019-08-15 Consejo Nacional De Investigaciones Cientificas Y Tecnicas (Conicet) Polypeptides immunomodulateurs et immunostimulants pour administration de médicament
EP3581201A1 (fr) * 2018-06-15 2019-12-18 GlaxoSmithKline Biologicals S.A. Escherichia coli o157:h7 polypeptides et leurs utilisations
EP3808372A1 (fr) * 2019-10-17 2021-04-21 GlaxoSmithKline Biologicals S.A. Nouvelles compositions de vaccin
US20210401960A1 (en) * 2018-11-10 2021-12-30 Bharat Biotech International Limited Multivalent glycoconjugates immunogenic compositions
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