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WO2025172892A1 - Protéines modifiées et méthodes - Google Patents

Protéines modifiées et méthodes

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Publication number
WO2025172892A1
WO2025172892A1 PCT/IB2025/051557 IB2025051557W WO2025172892A1 WO 2025172892 A1 WO2025172892 A1 WO 2025172892A1 IB 2025051557 W IB2025051557 W IB 2025051557W WO 2025172892 A1 WO2025172892 A1 WO 2025172892A1
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WO
WIPO (PCT)
Prior art keywords
nanoparticle
seq
modified
species
subunit
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/IB2025/051557
Other languages
English (en)
Inventor
Roberta COZZI
Roberto ROSINI
Roberto ADAMO
Maria Rosaria Romano
Domenico Maione
Luigia CAPPELLI
Paolo CINELLI
Newton Muchugu WAHOME
Sanjay Kumar Phogat
Michael Thomas KOWARIK
Gerd Martin LIPOWSKY
Sandra MARKOVIC-MÜLLER
Maria Paula Carranza Sandmeier
Stefan Herwig
Fabian Oliver MÜLLER
Levi KUHN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
GlaxoSmithKline Biologicals SA
Original Assignee
GlaxoSmithKline Biologicals SA
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Filing date
Publication date
Application filed by GlaxoSmithKline Biologicals SA filed Critical GlaxoSmithKline Biologicals SA
Publication of WO2025172892A1 publication Critical patent/WO2025172892A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • 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
    • 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
    • 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
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/005—Glycopeptides, glycoproteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1081—Glycosyltransferases (2.4) transferring other glycosyl groups (2.4.99)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/99—Glycosyltransferases (2.4) transferring other glycosyl groups (2.4.99)
    • C12Y204/99019—Undecaprenyl-diphosphooligosaccharide—protein glycotransferase (2.4.99.19)

Definitions

  • the present invention relates to the field of modified proteins, immunogenic compositions and vaccines comprising the modified proteins, their manufacture and method of making and the use of such compositions in medicine. More particularly, it relates to a bioconjugated modified nanoparticle (NP) subunit (or a bioconjugated modified carrier protein linked to an optionally modified nanoparticle (NP) subunit) and methods for preparing an assembled bioconjugated modified nanoparticle in a host cell (or an assembled bioconjugated modified carrier protein linked to an optionally modified nanoparticle (NP)). It also relates to methods for preparing an assembled bioconjugated modified nanoparticle (or an assembled bioconjugated modified carrier protein linked to an optionally modified nanoparticle (NP)) in a host cell (e.g.
  • glycans Different protein glycosylation mechanisms are distinguishable by the mode in which the glycans are transferred to proteins.
  • One mechanism involves the transfer of carbohydrates directly from nucleotide-activated sugars to acceptor proteins (e.g. protein O-glycosylation in the Golgi apparatus of eukaryotic cells, and flagellin O-glycosylation in certain types of bacteria).
  • a second mechanism involves the preassembly of a polysaccharide onto a lipid-carrier (e.g. by glycosyltransferases), which is then transferred to a protein acceptor by an oligosaccharyltransferase (OTase) (Faridmoayer et al., J. Bacteriology, pp.
  • N-linked glycosylation For N-linked glycosylation (N-glycosylation), glycans are generally attached to an asparagine residue on the protein acceptor.
  • the N-glycosylation of C. jejuni proteins may be reconstituted by recombinantly expressing the pgl locus and acceptor glycoprotein in E. coli at the same time (W acker et al . (2002) Science 298, 1790-1793).
  • N-glycosylated protein comprising one or more of the following optimized amino acid sequence(s): D/E-X-N-Z-S/T (SEQ ID NO: 49), wherein X and Z may be any natural amino acid except Pro.
  • D/E-X-N-Z-S/T SEQ ID NO: 49
  • X and Z may be any natural amino acid except Pro.
  • Conjugate vaccines have been a successful approach for vaccination against a variety of bacterial infections.
  • Conjugation of T-independent antigens, for example saccharides, to carrier proteins has long been established as a way of enabling T-cell to help to become part of the immune response for a normally T-independent antigen. In this way, an immune response can be enhanced by allowing the development of immune memory and boostability of the response.
  • in vivo methods to produce a “bioconjugate vaccine” have been in development. These in vivo methods leverage the N-glycosylation and O- glycosylation systems discussed above (see, W02009/104074 and W02017/035181).
  • an adaptable bioconjugate nanoparticle platform capable of producing a bioconjugated nanoparticle subunit that self-assembles (or a bioconjugated carrier protein fused directly or indirectly to a nanoparticle subunit that self-assembles) .
  • Such a platform is suitable for preparation of immunogenic compositions and/or vaccines for different nanoparticles and different saccharides (e.g. polysaccharide antigens, oligosaccharide antigens, etc.).
  • such a platform would be capable of producing a bioconjugated nanoparticle (or a bioconjugated carrier protein fused directly or indirectly to a nanoparticle) in a single step.
  • a nanoparticle (NP) based platform for producing a bioconjugated nanoparticle (or a bioconjugated carrier protein fused directly or indirectly to a nanoparticle).
  • Modified nanoparticle subunits are translocated to or expressed in the periplasm of a host cell (e.g. a bacterial host cell such as E. coli) along with a glycosyltransferase (e.g. glycosyltransferase pglB).
  • a host cell e.g. a bacterial host cell such as E. coli
  • glycosyltransferase e.g. glycosyltransferase pglB
  • the glycosyltransferase conjugates antigens (e.g.
  • the consensus sequence comprises or consists of D/E-X-N-Z-S/T (SEQ ID NO: 49), wherein X and Z may be independently any natural amino acid except proline (e.g. the consensus sequence may be D-Q-N-X-T, wherein X may be A or R (e.g. SEQ ID NO:53)).
  • the assembled glycoconjugated-NP or glycoconjugated-carrier protein-NP are generated in a single step (e.g.
  • Individual NP subunit proteins are capable of self-assembly to form nanoparticles, e .g . with a size ranging from about 5 to 100 nm, from about 5 nm to about 75 nm, from about 6 nm to about 75 nm, from about 6 nm to about 50 nm, from about 6 nm to about 25 nm, from about 6 nm to about 24 nm, etc.
  • Self-assembly of NPs refers to the oligomerization or aggregation ofNP subunits or carrier proteins fused to NP subunits into an ordered arrangement driven by non-covalent interactions.
  • the modified NP subunits, the antigen (e.g. bacterial antigen), and the glycosyltransferase (e.g. pglB) are expressed in or translocated to the periplasm of a bacterial host cell (e.g., E. col ).
  • the glycosyltransferase conjugates the antigen (e.g. the bacterial polysaccharide or oligosaccharide) to the modified NP subunit (or to the modified carrier protein fused directly or indirectly to a nanoparticle subunit), which self-assembles into a bioconjugated nanoparticle.
  • multiple copies of antigenic epitopes are displayed on the exterior surface of the assembled NP.
  • the assembled NPs display bacterial antigens (e.g. from gram-positive bacteria or gram-negative bacteria).
  • the bioconjugated NPs may be used for any suitable purpose, such as for inducing an immune response in a subject, and/or preventing or treating a disorder or disease.
  • the present embodiments provide glycoconjugate nanoparticle vaccines against a variety of bacterial pathogens that present cell surface carbohydrates, where an effective immune response may be achieved after one or more administrations of the immunogenic composition or vaccine.
  • NPs may include any suitable protein or virus-like particle capable of self-assembly, including but not limited to dodecin, ferritin (e.g. ferritin Hp, ferritin Pa, etc.), E2p, EPA-ferritin, etc.
  • FIG. 6A shows the last purification step of a KpO3b-ferritin Hp bioconjugate.
  • the ferritin variant used in this experiment contains engineered glycosites at the N-terminus and in loop 4 (SEQ ID NO: 18).
  • the ferritin bioconjugate was subjected to SECon Superose 6 10/300 column . According to SDS-PAGE analysis, fractions 12-16 contained the glycosylated KpO3b-ferritin Hp bioconjugate and were mixed to make a final pool.
  • FIG. 7D shows assembled KpO3b-ferritin nanoparticles according to a negative stain EM.
  • the measured particle size is around 12 nm.
  • Pa ferritin is expressed at a high level in E.coli periplasm, with the DsbA signal sequence yielding the highest periplasmic expression (SEQ ID NO: 23), followed by the Flgl (gives a good ratio of glycosylated with single Spl2F repeat unit versus unglycosylated ferritin), MalE and RBP signal sequences.
  • Glycosite introduced in loop 4 (L4, SEQ ID NO: 25) is more efficiently glycosylated than the glycosite in loop 2 (L2, SEQ ID NO: 24).
  • the glycosite in L4 in Pa ferritin is not tolerated as well as L4 in Hp ferritin, since the expression level of this variant is decreased.
  • FIG. 9A shows analysis of periplasmic expression and glycosylation for dodecin from Mycobacterium tuberculosis by SDS-PAGE (upper panel) and Western blot against Spl2F glycan (lower panel).
  • glycosite insertion at the N-terminus and in the 3 loops is evaluated with DsbA signal sequence.
  • the N-terminus and Mut3 (loop 3) are shown as being suitable for glycosylation.
  • Glycosites may also be combined as shown in the examples of the construct (e.g. DsbAss - dodecin-Mut3+C) (SEQ ID NO: 8).
  • FIG. 14C shows SDS-PAGE analysis of the fractions eluted from SEC for purifying Spl2F-EPA - Ferritin in FIG. 14B. Fractions A24-A34 were enriched in the Spl2F-EPA-Ferritin nanoparticle.
  • FIG. 15A shows samples used in a preclinical study for testing Spl2F immunogenicity in mice using different carrier proteins for bioconjugation.
  • Nanoparticle based carriers include an Spl2F- ferritin bioconjugate (group 4, SEQ ID NO: 18), a Spl2F -dodecin bioconjugate (group 5, SEC ID NO: 7), and an Spl2F-EPA-ferritin bioconjugate (group 6, SEQ ID NO: 52).
  • PBS group 1, negative control
  • Spl2F-EPA bioconjugate group 2 previously shown not to be immunogenic for Spl2F
  • Spl2F-CRM197 group 3, previously shown to give high Spl2F immunogenicity.
  • each conjugate including sugar to protein ratio and particle size are shown in the table as well. While an equal polysaccharide of 0.22 pg was used for all groups, the protein dose was different. Aluminium -phosphate was used as adjuvant in all groups.
  • FIG. 15B shows IgG titers after immunization with the samples listed in FIG. 15 A.
  • IgG serum titers before the first immunization (pre) and after the second (post-II) and third immunization (post-III) have been measured.
  • the highest IgG titers and responder rate was obtained using Spl2F -ferritin (SEQ ID NO: 18), even higher than when using chemical conjugate of Spl2F to CRM 197.
  • Significant Spl2F immunogenicity was obtained when using EPA with 5 engineered glycosites fused to Ferritin (SEQ ID NO: 52) as a carrier.
  • EPA-Ferritin fusion molecule is therefore superior to EPA as a stand-alone protein, suggesting the importance of particle size in generating potent immune response.
  • the responder rate was low, but better than when using EPA as carrier.
  • FIG. 16A shows samples used in a preclinical study for testing KpO3b immunogenicity in mice using different carrier proteins for bioconjugation.
  • Nanoparticle based carriers include a KpO3b- ferritin bioconjugate (group 1, SEQ ID NO: 18), a KpO3b-dodecin bioconjugate (group 2, SEQ ID NO: 7), and a KpO3b-E2p bioconjugate (group 3, SEQ ID NO: 29).
  • KpO3b-EPA bioconjugate group 4
  • PBS buffer group 5
  • the quality attributes of each conjugate including sugar to protein ratio and particle size are shown in the table as well. While an equal polysaccharide of 0.22 pg was used for all groups, the protein dose was different. AS03 was used as adjuvant in all groups.
  • FIG. 16B shows IgG titers after immunization with KpO3b-ferritn (group 1, SEQ ID NO: 18) compared to the control group (group 5) as measured before the first immunization (pre) and after the third immunization (post-III).
  • FIG. 17A shows characterization of a purified Sp33F-ferritin bioconjugate by SDS-PAGE, anti- Sp33F Western blot, DLS and negative stain EM.
  • the ferritin variant used in this example corresponds to SEQ ID NO: 18.
  • the measured average particle diameter by DLS is 21 nm which indicates that the Sp33F glycan is present on the nanoparticle.
  • the EM analysis indicates assembled Sp33F-ferritin nanoparticles with the measured particle size of around 12 nm.
  • FIG. 17B shows characterization of a purified Sp33F-dodecin bioconjugate by SDS-PAGE, anti- Sp33F Western blot, DLS and negative stain EM.
  • the dodecin variant used in this example corresponds to SEQ ID NO: 7.
  • the measured average particle diameter by DLS is 15 nm which indicates that the Sp33F glycan is present on the nanoparticle.
  • the EM analysis indicates assembled Sp33F-dodecin nanoparticles with the measured particle size of around 6 nm.
  • a transferase e.g. PglB
  • a carrier protein optionally a carrier protein
  • a glycosylation unit to produce saccharides is expressed in and/or translocated to the periplasm of the host cell, to produce a self-assembling bioconjugated nanoparticle (e.g. in a single step), is provided herein.
  • proline refers to an amino acid selected from the group consisting of alanine (ala, A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gin, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).
  • naturally occurring amino acid residues refers to amino acids that are naturally incorporated into polypeptides.
  • the 20 amino acids encoded by the universal genetic code alanine (ala, A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gin, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).
  • glycosidic linkages As used herein, the term “glycosyltransferases (GTFs, Gtfs)” refers to enzymes that establish glycosidic linkages. Glycosyltransferases are enzymes that catalyze the formation of the glycosidic linkage to form a glycoside, for example, by catalyzing the transfer of saccharide moieties from an activated nucleotide sugar (also known as the “glycosyl donor”) to a nucleophilic glycosyl acceptor molecule, the nucleophile of which can be oxygen-, carbon-, nitrogen-, or sulfurbased.
  • GTFs, Gtfs activated nucleotide sugar
  • nucleophilic glycosyl acceptor molecule the nucleophile of which can be oxygen-, carbon-, nitrogen-, or sulfurbased.
  • O-Antigens also known as O-specific polysaccharides or O-side chains
  • LPS surface lipopolysaccharide
  • examples include O-antigens from Pseudomonas aeruginosa and Klebsiella pneumoniae.
  • capsule polysaccharide refers to a polysaccharide found on the bacterial cell wall. Examples include but are not limited to capsular polysaccharide from Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis and Staphylcoccus aureus.
  • wzy refers to a polysaccharide polymerase gene encoding an enzyme which catalyzes polysaccharide polymerization.
  • the encoded enzyme transfers oligosaccharide units to the non-reducing end forming a glycosidic bond.
  • the term “waaL ” refers to an O antigen ligase gene encoding a membrane bound enzyme.
  • the encoded enzyme transfers undecaprenyl-diphosphate (UPP)-bound O antigen to the lipid A core oligosaccharide, forming lipopolysaccharide.
  • UFP undecaprenyl-diphosphate
  • “Stabilized” refers to introducing one or more mutations into an amino acid sequence, for example, of a nanoparticle monomer subunit, in order to improve the stability of the nanoparticle (e.g. with regards to self assembly).
  • the term “conservative amino acid substitution” involves substitution of a native amino acid residue with a non-native residue such that there is little or no effect on the size, polarity, charge, hydrophobicity, or hydrophilicity of the amino acid residue at that position, and without resulting in decreased immunogenicity.
  • these may be substitutions within the following groups: valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
  • Conservative amino acid modifications to the sequence of a polypeptide (and the corresponding modifications to the encoding nucleotides) may produce polypeptides having functional and chemical characteristics similar to those of a parental polypeptide.
  • deletion refers to the removal of one or more amino acid residues from the protein sequence. Typically, no more than about from 1 to 10 (e.g. 1 to 7 residues, 1 to 6 residues or 1 to 4 residues) are deleted at any one site within the protein molecule.
  • the Needleman Wunsch algorithm (Needleman and Wunsch 1970, J. Mol. Biol. 48: 443-453) for global alignment, or the Smith Waterman algorithm (Smith and Waterman 1981 , J. Mol. Biol. 147: 195- 197) for local alignment may be used, e.g. using the default parameters (Smith Waterman uses BLOSUM 62 scoring matrix with a Gap opening penalty of 10 and a Gap extension penalty of 1).
  • a preferred algorithm is described by Dufresne et al. in Nature Biotechnology in 2002 (vol. 20, pp. 1269-71) and is used in the software GenePAST (Genome Quest Life Sciences, Inc. Boston, MA).
  • the GenePAST “percent identity” algorithm finds the best fit between the query sequence and the subject sequence and expresses the alignment as an exact percentage. GenePAST makes no alignment scoring adjustments based on considerations of biological relevance between query and subject sequences. Identity between two sequences is calculated across the entire length of both sequences and is expressed as a percentage of the reference sequence.
  • recombinant means artificial or synthetic.
  • a “recombinant protein” refers to a protein that has been made using recombinant nucleotide sequences (nucleotide sequences introduced into a host cell).
  • the nucleotide sequence that encodes a “recombinant protein” is heterologous to the host cell.
  • isolated or purified mean a protein, conjugate (e.g. bioconjugate), polynucleotide, or vector in a form not found in nature. This includes, for example, a protein, conjugate (e.g. bioconjugate), polynucleotide, or vector having been separated from host cell or organism (including crude extracts) or otherwise removed from its natural environment.
  • an isolated or purified protein is a protein essentially free from all other polypeptides with which the protein is innately associated (or innately in contact with).
  • the term “subject” refers to an animal, in particular a mammal such as a primate (e.g. human).
  • the term “effective amount,” in the context of administering a therapy (e.g. an immunogenic composition or vaccine of present embodiments) to a subject refers to the amount of a therapy which has a prophylactic and/or therapeutic effect(s).
  • an “effective amount” refers to the amount of a therapy which is sufficient to achieve one, two, three, four, or more of the following effects: (i) reduce or ameliorate the severity of a bacterial infection or symptom associated therewith; (ii) reduce the duration of a bacterial infection or symptom associated therewith; (iii) prevent the progression of a bacterial infection or symptom associated therewith; (iv) cause regression of a bacterial infection or symptom associated therewith; (v) prevent the development or onset of a bacterial infection, or symptom associated therewith; (vi) prevent the recurrence of a bacterial infection or symptom associated therewith; (vii) reduce organ failure associated with a bacterial infection; (viii) reduce hospitalization of a subject having a bacterial infection; (ix) reduce hospitalization length of a subject having a bacterial infection; (x) increase the survival of a subject with a bacterial infection; (xi) eliminate a bacterial infection in a subject; (xii) inhibit
  • a “modified nanoparticle subunit” refers to a nanoparticle subunit that has been modified for stability (e.g., one or more mutations) and/or to include one or more glycosites, wherein the glycosites may be bioconjugated to one or more antigenic molecules (e.g. polysaccharides or oligosaccharides) as provided herein.
  • the bioconjugated nanoparticle subunit or bioconjugated carrier protein fused to a nanoparticle subunit) self-assembles with other subunits to form an assembled nanoparticle (e.g. composed of multiple subunits).
  • a modified nanoparticle subunit that is bioconjugated to an antigen may be part of a pharmaceutical composition designed to elicit an immune response against the one or more antigen molecules bioconjugated to the carrier protein or nanoparticle subunit.
  • nanoparticles may be used to display polysaccharide antigens to induce a host response such as an effective B cell response and/or a T cell response.
  • the modified nanoparticle subunit may optionally include one or more stabilizing mutations, and optionally may include one or more insertions, substitution or deletions to introduce one or more glycosites.
  • Nanoparticles may undergo self-assembly into highly symmetric stable and organized structures and may be modified to display antigen(s) in order to mimic the surface of a virus or bacteria.
  • carrier protein refers to an immunogenic protein (e.g. such as CRM, DT, TT, EPA, etc.) which, when conjugated to an antigen (e.g. a saccharide antigen, such as a bacterial polysaccharide antigen or oligosaccharide antigen) and administered to an animal, will enhance an immune response in the animal, particularly the production of antibodies that bind specifically to the conjugated polysaccharide or oligosaccharide.
  • an antigen e.g. a saccharide antigen, such as a bacterial polysaccharide antigen or oligosaccharide antigen
  • the carrier protein may be covalently linked directly to the N-terminal amino acid of the nanoparticle subunit (polypeptide monomer) or via a short (20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid long) peptide linker sequence placed between the carrier protein and the nanoparticle subunit.
  • Linkers between a nanoparticle and a conjugated carrier protein include, for example, Glycine/Serine/Alanine linkers (8 to 14 amino acid residues containing repeats of Glycine, Serine, or Alanine (see W02009/109428 (PCI7EP2009/050996)).
  • bioconjugate refers to a conjugate between a protein (e.g. a carrier protein fused to a nanoparticle subunit, or a nanoparticle subunit) and an antigen (e.g. a saccharide antigen, such as a bacterial polysaccharide antigen) prepared in a host cell background, wherein host cell machinery links the antigen to the protein (e.g. N-linked glycosylation).
  • a protein e.g. a carrier protein fused to a nanoparticle subunit, or a nanoparticle subunit
  • an antigen e.g. a saccharide antigen, such as a bacterial polysaccharide antigen
  • a nanoparticle is provided which has undergone bioconjugation to a polysaccharide or oligosaccharide.
  • a carrier protein fused to a nanoparticle wherein the carrier protein has undergone bioconjugation to a polysaccharide or oligosaccharide.
  • modified protein means a protein that is altered (in one or more way) as compared to wild type (e.g. a “modified protein” excludes a wild type protein).
  • nucleic acid molecule comprising a polynucleotide that encodes such modified nanoparticles (comprising glycosylation sites) or fusion carrier protein nanoparticle molecules (e.g. comprising a modified carrier protein, with one or more bioconjugation sites fused to a nanoparticle subunit.
  • Certain embodiments provide a pharmaceutical composition comprising one or more nanoparticle monomers. Certain other embodiments provide a composition comprising a nanoparticle subunit linked to one or more antigens (e.g., saccharides) or a nanoparticle subunit linked to a carrier protein that is conjugated to an antigen). The nanoparticle subunits may assemble into nanoparticles.
  • present embodiments provide a modified nanoparticle subunit protein, wherein the amino acid sequence further comprises a signal sequence which is capable of directing the nanoparticle carrier protein molecule to the periplasm of a host cell (e.g. bacterium), said signal sequence being any suitable sequence.
  • a signal sequence which is capable of directing the nanoparticle carrier protein molecule to the periplasm of a host cell (e.g. bacterium), said signal sequence being any suitable sequence.
  • Techniques for fusing signal peptide to a nanoparticle subunit are known in the art (e.g. a signal peptide of the protein DsbA from E. coli can be genetically fused to the N-terminus of the mature nanoparticle subunit protein sequence) (see, Schulz, H., Hennecke, H., and Thony-Meyer, L., Science, 281, 1197-1200, 1998).
  • amino acid numbers referred to herein correspond to the amino acids in SEQ ID NO: X and as described above, a person skilled in the art can determine equivalent amino acid positions in an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: X by alignment.
  • the addition or deletion of amino acids from the variant and/or fragment of SEQ ID NO: X could lead to a difference in the actual amino acid position of the consensus sequence in the mutated sequence, however, by lining the mutated sequence up with the reference sequence, the amino acid in an equivalent position to the corresponding amino acid in the reference sequence can be identified and hence the appropriate position for addition or substitution of the consensus sequence can be established.
  • mutation positions listed with respect to a sequence may refer to a first sequence (e.g. having mutations) aligned with a reference sequence (e.g. wt sequence).
  • a dodecin subunit protein of present embodiments can be produced by methods provided herein.
  • the amino acid sequence of wildtype dodecin is shown in SEQ ID NO: 1 (Uniprot: Q8VK10). Modifications to dodecin were made to improve stability, with said modifications at one, two or three of positions G25, V50 and A53 (e.g., G25N, V50T, A53T), wherein the positions are with respect to the wt sequence.
  • a nucleotide sequence for the modified amino acid sequence of dodecin is provided at SEQ ID NO: 3.
  • the dodecin subunit protein is modified to introduce glycosylation sites at designated positions in the amino acid sequence. Modifications to introduce a glycosylation site may be at one or more of the N-terminus, the C terminus, or internally within the dodecin sequence (e.g. amino acid residues 49-55, 50-54, 50-53, 50-52, 51) to produce a modified dodecin subunit protein, which is expressed in the periplasm wherein it undergoes glycosylation.
  • the modified glycosylated dodecin subunit protein further undergoes selfassembly in the periplasm to form an assembled glycosylated nanoparticle.
  • the modified dodecin subunit protein may comprise stabilizing mutations at one or more of glycine to asparagine (G25N), valine to threonine (V50T), and alanine to threonine (A53T) with reference to the amino acid sequence of SEQ ID NO: 1 (or an equivalent position in an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1).
  • G25N glycine to asparagine
  • V50T valine to threonine
  • A53T alanine to threonine
  • the modified dodecin subunit protein of the invention may be the amino acid sequence of SEQ ID NO: 1 (or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1) and comprising substitution of alanine to threonine (A53T).
  • the modified dodecin subunit protein of present embodiments includes amino acids 2-70 of SEQ ID NO: 2 or a self-assembling fragment thereof (or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acids 2-70 of SEQ ID NO: 1 or a self-assembling fragment thereof) comprising one or more of substitution of glycine to asparagine (G25N), valine to threonine (V50T), and alanine to threonine (A53T).
  • G25N glycine to asparagine
  • V50T valine to threonine
  • A53T alanine to threonine
  • Ferritin may be modified by introducing one or more glycosites into amino acid residue(s) exposed at the nanoparticle surface. Sites for modification are selected so as to not interfere with nanoparticle formation but are present on the nanoparticle surface and suitable for antigen display. For example, suitable positions for introduction of glycosites include the N-terminus, loop 1 (LI), the long loop between helix 2 and 3 (L2), the short loop between helix 3 and 4 (L3), and the loop between helix 4 and (5) L4.
  • a ferritin nanoparticle may assemble from 24 subunits to form a spherical shape with a 12 nm diameter. For 2 and 3 glycosites per ferritin subunit, this leads to 48 and 72 positions for glycan attachment per ferritin nanoparticle. Present embodiments encompass any ferritin molecule from any species; representative examples are below.
  • H. pylori bacterial ferritin (see Protein Data Bank (PDB) Accession Number Q9ZLI1) has been investigated for use as a pharmaceutically acceptable carrier.
  • H. pylori bacterial ferritin consists of 24 identical polypeptide subunits that self-assemble into a spherical nanoparticle. Li et al. reported preparation of a nucleotide sequence encoding a fusion of bacterial (H. pylori) ferritin subunit polypeptide and a rotavirus antigen, with expression in a prokaryotic (E. coli) system.
  • a signal sequence may replace a start methionine of SEQ ID NO: 11 (or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 11).
  • the modified ferritin subunit protein may be the amino acid sequence of SEQ ID NO: 20 or a selfassembling fragment thereof (or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 20 or a self-assembling fragment thereof) comprising substitution of methionine to isoleucine (Ml 441).
  • the modified ferritin subunit protein may be the amino acid sequence of SEQ ID NO: 20 or a self-assembling fragment thereof (or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 20 or a self-assembling fragment thereof) comprising substitution of isoleucine to methionine (I154M).
  • the modified ferritin subunit protein may comprise one, two, three, four or more of mutations M3 II, K120L, A124R, M144I and/or I154M or a self-assembling fragment thereof.
  • the modified ferritin subunit comprises a signal sequence followed by a glycosylation sequence, followed by a histidine tag followed by ferritin (SEQ ID NO:23), or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 23.
  • the modified ferritin subunit comprises a signal sequence followed by a histidine tag followed by ferritin, with insertion of a glycosylation site at T80 in Loop 2 of ferritin (SEQ ID NO:24), or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 24.
  • the modified ferritin subunit comprises a signal sequence followed by a histidine tag followed by ferritin, with insertion of a glycosylation site at G145 in Loop 4 of ferritin (SEQ ID NO:25), or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 25.
  • the modified ferritin subunit protein may include a modification in Loop 2 to residue 80 (counting from the methionine of native ferritin sequence UNIPROT ID: Q9HWF9).
  • T80 is substituted with a glycotag (SEQ ID NO: 31) or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 24.
  • G145 is substituted with a glycotag (SEQ ID NO: 31) or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 25.
  • ferritin may be modified to include one or more of a glycosite in L2, a glycosite in L4, and a glycosite at the N- terminus as recited herein.
  • the ferritin subunit protein may comprise a signal sequence at the N-terminus.
  • Any suitable signal sequence may be appended to the N terminus of ferritin subunit protein (e.g. DsbA (SEQ ID NO: 10), TolB (SEQ ID NO: 41), PelB (SEQ ID NO: 45), Figi (SEQ ID NO: 42), LtIIB_(SEQ ID NO: 46)).
  • a signal sequence e.g. SEQ ID NO: 10
  • SEQ ID NO: 41 TolB
  • PelB SEQ ID NO: 45
  • Figi SEQ ID NO: 42
  • a signal sequence e.g. SEQ ID NO: 10
  • SEQ ID NO: 10 may be appended at the N-terminus (e.g.
  • an assembled glycosylated nanoparticle in a single step.
  • the method comprises the steps of providing a host cell; expressing a modified protein (e.g. a modified ferritin subunit comprising an antigen) and glycosyltransferase pglB to produce the glycoprotein nanoparticle.
  • a modified protein e.g. a modified ferritin subunit comprising an antigen
  • glycosyltransferase pglB glycoprotein nanoparticle.
  • Polysaccharides are produced by the host cell based on the techniques provided herein along with any other proteins needed to express and/or translocate the polysaccharides to the periplasm.
  • E2p is a protein found in G. stearothermophilus .
  • An E2p nanoparticle may assemble to form a 60-mer hollow dodecahedron with about a 24 nm diameter.
  • Recombinant E2p nanoparticles produced using recombinant gene expression in a bacterial expression system may be purified from bacterial homogenate by size exclusion chromatography (Kozlovska et al. 1993) or by a combination of fractionated ammonium sulphate precipitation and size exclusion chromatography (Vasiljeva et al (1998); Ciliens et al. (2000)).
  • E2p nanoparticle subunits may be linked to saccharides and selfassemble to form nanoparticles.
  • the wild type amino acid sequence (SEQ ID NO: 26) and nucleotide sequence (SEQ ID NO: 28) for E2p are provided herein.
  • the modified E2p subunit protein includes amino acids 185-426 of SEQ ID NO: 26 or a self-assembling fragment thereof (or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acids 185-426 of SEQ ID NO: 26 or a selfassembling fragment thereof) or a fragment thereof.
  • the amino acid sequence encoding E2p has been modified for stability.
  • the modified E2p subunit protein may be the amino acid sequence of amino acids 185-426 of SEQ ID NO: 26 or a self-assembling fragment thereof (or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acids 185-426 of SEQ ID NO: 26 or a self-assembling fragment thereof) comprising substitution of alanine to threonine (A187T).
  • the modified E2p subunit protein may be the amino acid sequence of amino acids 185-426 of SEQ ID NO: 26 or a self-assembling fragment thereof (or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acids 185-426 of SEQ ID NO: 26 or a self-assembling fragment thereof) comprising substitution of threonine to asparagine (T281N).
  • the modified E2p subunit protein may be the amino acid sequence of amino acids 185-426 of SEQ ID NO: 26 or a self-assembling fragment thereof (or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acids 185-426 of SEQ ID NO: 26 or a self-assembling fragment thereof) comprising substitution of alanine to valine (A352V).
  • the modified E2p subunit protein may be the amino acid sequence of amino acids 185-426 of SEQ ID NO: 26 or a self-assembling fragment thereof (or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acids 185-426 of SEQ ID NO: 26 or a self-assembling fragment thereof) comprising substitution of leucine to isoleucine (L425I).
  • the modified E2p subunit protein may be the amino acid sequence of amino acids 185-426 of SEQ ID NO: 26 or a self-assembling fragment thereof (or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acids 185-426 of SEQ ID NO: 26 or a self-assembling fragment thereof) comprising deletion of amino acids at positions 427 and 428 (A427-428).
  • the modified E2p subunit protein may comprise one or more of mutations A187T, F196Y, T281N, P314S, A352V, L425I, A427-428 of amino acids 185-426 of SEQ ID NO: 26. In aspects, the modified E2p subunit protein may comprise all the mutations A187T, F196Y, T281N, P314S, A352V, L425I, A427-428 of amino acids 185-426 of SEQ ID NO: 26.
  • E2p may be modified to include a glycosylation site at the N-terminus.
  • the consensus sequence comprises D/E-X-N-Z-S/T (SEQ ID NO: 49), wherein X and Z may be independently any natural amino acid except proline (e.g. the consensus sequence may be D-Q-N-X-T, wherein X may be A or R (SEQ ID NO:53)).
  • the glycosylation site is SEQ ID NO:36.
  • the modified E2p subunit protein may comprise a signal sequence, optionally followed by a histidine tag, followed by a glycosylation site and optional linker, followed by E2p.
  • the modified sequence optionally may include a poly His tail (e.g. a string of 5 to 7 histidine residues, or 6 histidine resides) appended to the N-terminus for purification of the amino acid sequence of SEQ ID NO: 29 (or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 29).
  • a poly His tail e.g. a string of 5 to 7 histidine residues, or 6 histidine resides
  • sequence alignment tools are not limited to Clustal Omega (www(.)ebi(.)ac(.)ac(.)uk) MUSCLE (www(.)ebi(.)ac(.)uk), or T-coffee (www(.)tcoffee(.)org).
  • sequence alignment tool used is Clustal Omega (www(.)ebi(.)ac(.)ac(
  • nanoparticle subunits such as ferritin may be fused to a carrier protein such as EPA, according to techniques known in the art.
  • the fusion of the carrier protein with ferritin may undergo expression and glycosylation in the cell and may self-assemble in the periplasm.
  • the modified EPA carrier protein of present embodiments may be a recombinant modified EPA carrier protein fused to a ferritin nanoparticle subunit (see, e.g. SEQ ID NO: 52).
  • the modified EPA protein of present embodiments may be an isolated recombinant modified EPA protein fused to a ferritin nanoparticle subunit.
  • the modified nanoparticle subunit protein may further comprise a “peptide tag” or “tag”, i.e. a sequence of amino acids that allows for the isolation and/or identification of the modified subunit protein.
  • a tag i.e. a sequence of amino acids that allows for the isolation and/or identification of the modified subunit protein.
  • adding a tag to a modified nanoparticle subunit protein can be useful in the purification of that protein and, hence, the purification of conjugate (e.g. bioconjugate) vaccines comprising the tagged modified nanoparticle subunit protein.
  • Exemplary tags that can be used herein include, without limitation, histidine (HIS) tags (e.g. hexa histidine- tag, or 6Xhis-Tag), FLAG-TAG, and HA tags.
  • the tag is a hexa-histidine tag.
  • the modified nanoparticle subunit protein may further comprise a peptide tag.
  • the peptide tag is located at the C- terminus of the amino acid sequence.
  • the tag comprises six histidine residues at the N- terminus or at the C-terminus of the amino acid sequence.
  • the modified nanoparticle subunit protein comprises (or consists of) an amino acid sequence which is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to any one of the nanoparticle subunit sequences provided herein and a peptide tag (e.g. six histidine residues at the N- or C-terminus of the amino acid sequence).
  • a peptide tag e.g. six histidine residues at the N- or C-terminus of the amino acid sequence.
  • An embodiment comprises a modified nanoparticle (or a modified carrier protein fused to an optionally modified nanoparticle) displaying one or more poly- or oligo- saccharide antigens on the exterior surface of the modified nanoparticle (or modified carrier protein).
  • the poly- or oligo- saccharide antigens may be conjugated at glycosylation sites of the nanoparticle, wherein the nanoparticle is not conjugated to another polypeptide.
  • the poly- or oligo- saccharide antigens are conjugated at glycosylation sites of a modified carrier protein fused to the nanoparticle, such as a modified CRM (e.g. CRM-197), a modified EPA, a modified Diptheria Toxoid (DT), a modified OMPC, or a modified Tetanis Toxoid (TT), etc.
  • the antigen bioconjugated to the modified nanoparticle or modified carrier protein via a glycosylation site may be a saccharide antigen, for example, a bacterial poly- or oligo- saccharide, a yeast polysaccharide or a mammalian polysaccharide.
  • Polysaccharides may comprise two or more monosaccharides, typically greater than ten monosaccharides. Oligo- saccharides may comprise a few monosaccharides, for example, less than ten monosaccharides.
  • the antigen is a bacterial polysaccharide antigen such as O-antigen from a gram-negative bacterium, or a capsular polysaccharide from a gram -positive bacterium.
  • the antigen displayed on the nanoparticle is any O- antigen or capsular polysaccharide or immunogenic fragment thereof, or combinations of such of any suitable serotype.
  • the antigen (e.g. bioconjugate) displayed on the modified nanoparticle surface is a polysaccharide antigen from Escherichia species, Shigella species, Klebsiella species, Salmonella species, Yersinia species, Helicobacter species, Proteus species, Pseudomonas species, Corynebacterium species, Streptomyces species, Streptococcus species, Enterococcus species, Staphylococcus species, Bacillus species, Clostridium species, Listeria species, or Campylobacter species.
  • Escherichia species Shigella species, Klebsiella species, Salmonella species, Yersinia species, Helicobacter species, Proteus species, Pseudomonas species, Corynebacterium species, Streptomyces species, Streptococcus species, Enterococcus species, Staphylococcus species, Bacillus species, Clostridium species, Listeria species, or Campy
  • the antigen in a conjugate (e.g. bioconjugate) of present embodiments is a bacterial polysaccharide selected from Shigella flexneri, Klebsiella pneumoniae and Streptococcus pneumoniae.
  • the antigen is an O-antigen e.g. from a gram-negative bacterium (e.g. Salmonella species, Shigella species, Pseudomonas species or Klebsiella species such as Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Pseudomonas aeruginosa, or Klebsiella pneumoniae (see, Dmitriev, B.A., et al. Somatic Antigens of Shigella Eur J. Biochem, 1979. 98: p. 8; Liu et al Structure and genetics of Shigella O antigens FEMS Microbiology Review, 2008. 32: p. 27)).
  • a gram-negative bacterium e.g. Salmonella species, Shigella species, Pseudomonas species or Klebsiella species such as Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Pseudomon
  • the antigen is an O-antigen from Pseudomonas aeruginosa.
  • the antigen may be an O-antigen from Pseudomonas aeruginosa serotypes 1-20 (Raymond et al., J Bacteriol. 2002 184( 13) :3614-22).
  • the antigen is an O- antigen from Klebsiella pneumoniae.
  • the antigen is a capsular polysaccharide from a gram-negative or gram-positive bacteria such as Neisseria meningitidis serogroup A (MenA), N. meningitidis serogroup C (MenC), N. meningitidis serogroup Y (MenY), N. meningitidis serogroup W (MenW), H. influenzae type b (Hib), Group B Streptococcus (GBS), Streptococcus pneumoniae, or Staphylococcus aureus.
  • the antigen is a capsular polysaccharide from Streptococcus species or Staphylococcus species (e.g.
  • the antigen is a capsular polysaccharide from Staphylococcus aureus.
  • the antigen may be a capsular polysaccharide from Staphylococcus aureus type 5 and 8.
  • the antigen is a capsular polysaccharide from Streptococcus pneumoniae (e.g. any of the 90+ serotypes (see, Shoji et al., Infection and Drug Resistance (2016) vol. 11, pp 1387-1400).
  • Antigen-displaying modified nanoparticles preferably display multiple copies of antigenic molecules in an ordered array, such an ordered array presented on a nanoparticle surface, to allow multiple binding events to occur simultaneously between the nanoparticle and host cell, which may favor the induction of a potent host immune response (see e.g. Lopcz-Sagascta ⁇ ++//.. (2016)). Presentation of antigens on nanoparticles has been exploited to improve the immunogenicity of subunit protein antigens (see, Jardine et al, (2013); Correira et al, (2014)).
  • heterologous nucleotide sequences are introduced into the host cells using a plasmid, e.g. the heterologous nucleotide sequences are expressed in the host cells by a plasmid (e.g. an expression vector).
  • a plasmid e.g. an expression vector
  • a host cell comprising: i) one or more nucleotide sequences comprising polysaccharide synthesis genes, optionally for producing a bacterial polysaccharide antigen (e.g. an O-antigen from a gramnegative bacterium optionally from Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Pseudomonas aeruginosa, Klebsiella pneumoniae, or a capsular polysaccharide from a gram- positive bacterium optionally from Streptococcus pneumoniae or Staphylcoccus aureus or a capsular polysaccharide from Neisseria meningitidis) or a yeast polysaccharide antigen or a mammalian polysaccharide antigen, optionally integrated into the host cell genome; ii) a nucleotide sequence encoding a heterologous oligosaccharyl transferase (e.g.
  • Host cells that can be used to produce the bioconjugate nanoparticles or bioconjugate carrier proteins fused to nanoparticles of present embodiments include archea, prokaryotic host cells, and eukaryotic host cells.
  • the host cell is a non-human host cell.
  • Exemplary prokaryotic host cells for use in production of the bioconjugates of present embodiments include Escherichia species, Shigella species, Klebsiella species, Xhantomonas species, Salmonella species, Yersinia species, Lactococcus species, Lactobacillus species, Pseudomonas species, Corynebacterium species, Streptomyces species, Streptococcus species, Staphylococcus species, Bacillus species, and Clostridium species.
  • the host cell is E. coli.
  • Disclosures of methods for making such host cells suitable for use with the methods provided herein are WO 06/119987, WO 09/104074, WO 11/62615, WO 11/138361, WO 14/57109, WO14/72405 and WO 16/20499.
  • Host cells may be modified to delete or modify genes in the host cell genetic background (genome) that compete or interfere with the synthesis of the polysaccharide of interest (e.g. compete or interfere with one or more heterologous polysaccharide synthesis genes that are recombinantly introduced into the host cell).
  • These genes can be deleted or modified in the host cell background (genome) in a manner that makes them inactive/dysfunctional (i.e. the host cell nucleotide sequences that are deleted/modified do not encode a functional protein or do not encode a protein whatsoever) .
  • nucleotide sequences are deleted from the genome of the host cells of present embodiments, they are replaced by a desirable sequence, e.g.
  • genes that can be deleted in host cells include genes of host cells involved in glycolipid biosynthesis, such as waaL (see, e.g. Feldman et al. 2005, PNAS USA 102:3016-3021), the O antigen cluster (rft> or wb), enterobacterial common antigen cluster (wee), the lipid A core biosynthesis cluster (waa), galactose cluster (gal), arabinose cluster (ara), colonic acid cluster (wc), capsular polysaccharide cluster, undecaprenol-pyrophosphate biosynthesis genes (e.g.
  • uppS Undecaprenyl pyrophosphate synthase
  • uppP Undecaprenyl diphosphatase
  • Und-P recycling genes metabolic enzymes involved in nucleotide activated sugar biosynthesis, enterobacterial common antigen cluster, and prophage O antigen modification clusters like the gtrABS cluster.
  • one or more of the waciL gene, gtrA gene, gtrB gene, gtrS gene, or a gene or genes from the wee cluster or a gene or genes from the rfb gene cluster are deleted or functionally inactivated from the genome of a prokaryotic host cell of present embodiments.
  • the host cell of present embodiments is E. coli, wherein the native enterobacterial common antigen cluster (ECA, wee) with the exception of wecA. the colanic acid cluster (wca), and the 016-antigen cluster have been deleted.
  • ECA enterobacterial common antigen cluster
  • wca colanic acid cluster
  • 016-antigen cluster 016-antigen cluster
  • the native lipopolysaccharide O-antigen ligase waaL may be deleted from the host cell of present embodiments.
  • the native gtrA gene, gtrB gene and gtrS gene may be deleted from the host cell of present embodiments.
  • the host cells of the present invention are engineered to comprise heterologous nucleotide sequences.
  • the host cells of the present invention are engineered to comprise a nucleotide sequence that encodes a modified nanoparticle subunit protein (or a modified carrier protein fused to a nanoparticle subunit protein), optionally within a plasmid.
  • the host cells of present embodiments also comprise one or more nucleotide sequences comprising polysaccharide synthesis genes.
  • host cells of present embodiments can produce a bioconjugate comprising an antigen, for example a saccharide antigen (e.g.
  • a bacterial, yeast or mammalian polysaccharide antigen which is attached to a modified nanoparticle subunit protein (or a modified carrier protein fused to a nanoparticle subunit protein).
  • One or more heterologous nucleotide sequences may encode for the polysaccharide synthesis proteins to produce the bacterial polysaccharide antigen, yeast polysaccharide antigen or mammalian polysaccharide antigen.
  • the present invention also provides a host cell comprising: i) one or more heterologous nucleotide sequences comprising polysaccharide synthesis genes for producing a bacterial polysaccharide antigen (e.g.
  • an O-antigen from a gramnegative bacterium optionally from Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Pseudomonas aeruginosa, Klebsiella pneumoniae, or a capsular polysaccharide from a grampositive bacterium optionally from Streptococcus pneumoniae or Staphylcoccus aureus, or a capsular polysaccharide from N.
  • a yeast polysaccharide antigen or a mammalian polysaccharide antigen optionally integrated into the host cell genome; ii) a nucleotide sequence encoding a heterologous oligosaccharyl transferase (e.g. pglB), optionally within a plasmid; iii) a nucleotide sequence that encodes a modified nanoparticle subunit protein (or a modified carrier protein fused to a nanoparticle subunit protein), optionally within a plasmid.
  • a heterologous oligosaccharyl transferase e.g. pglB
  • the host cells of present embodiments may comprise one or more nucleotide sequences sufficient for producing a saccharide antigen (e.g. abacterial polysaccharide antigen), in particular for producing a saccharide antigen (e.g. a bacterial polysaccharide antigen) that is heterologous to the host cell.
  • a saccharide antigen e.g. abacterial polysaccharide antigen
  • the host cell may comprise one or more nucleotide sequences comprising polysaccharide synthesis genes sufficient for producing a bacterial polysaccharide antigen of a bacteria which is not an E. coli polysaccharide antigen.
  • the bacterial polysaccharide antigen may be an O-antigen or a capsular polysaccharide antigen.
  • present embodiments also provide a host cell comprising: i) one or more nucleotide sequences comprising polysaccharide synthesis genes, for producing a bacterial polysaccharide antigen (e.g. an O-antigen from a gram-negative bacterium, optionally from Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Pseudomonas aeruginosa, Klebsiella pneumoniae, or a capsular polysaccharide from a gram-positive bacterium optionally from Streptococcus pneumoniae or Staphylcoccus aureus), optionally integrated into the host cell genome; ii) a nucleotide sequence encoding a heterologous oligosaccharyl transferase (e.g.
  • a heterologous oligosaccharyl transferase e.g.
  • Polysaccharide synthesis genes encode proteins involved in synthesis of a polysaccharide (polysaccharide synthesis proteins).
  • the host cells may comprise one or more nucleotide sequences comprising polysaccharide synthesis genes for producing an O-antigen from a gram-negative bacterium selected from Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Pseudomonas aeruginosa and Klebsiella pneumoniae, or a capsular polysaccharide from a grampositive bacterium selected from Streptococcus pneumoniae and Staphylcoccus aureus.
  • the host cells may comprise one or more nucleotide sequences comprising polysaccharide synthesis genes for producing an O-antigen from a gram-negative bacterium selected from Shigella flexneri and Klebsiella pneumoniae, or a capsular polysaccharide from a gram-positive bacterium selected from Streptococcus pneumoniae and Staphylcoccus aureus.
  • the host cell comprises one or more nucleotide sequences comprising polysaccharide synthesis genes for producing an O-antigen from Shigella species or Klebsiella species, (e.g. Shigella dysenteriae, Shigella flexneri, Shigella sonnei or Klebsiella pneumoniae) .
  • the host cell comprises one or more nucleotide sequences comprising polysaccharide synthesis genes for producing an O-antigen from Shigella dysenteriae, Shigella flexneri or Shigella sonnei.
  • the host cell may comprise one or more nucleotide sequences comprising polysaccharide synthesis genes for producing an O-antigen from .S', dysenteriae type 1, .S', sonnei, and .S', flexneri type 6, and .S', flexneri 2a and 3a 0 (Dmitriev, B.A., et al Somatic Antigens of Shigella Eur J. Biochem, 1979. 98: p.
  • the host cell comprises one or more nucleotide sequences comprising polysaccharide synthesis genes for producing an O-antigen from Pseudomonas aeruginosa, e.g. Pseudomonas aeruginosa serotypes 1-20.
  • the host cell comprises one or more nucleotide sequences comprising polysaccharide synthesis genes for producing an O-antigen from Klebsiella pneumoniae.
  • the host cells of present embodiments may comprise one or more nucleotide sequences comprising polysaccharide synthesis genes for producing a capsular polysaccharide.
  • the host cell comprises one or more nucleotide sequences comprising polysaccharide synthesis genes for producing a capsular polysaccharide from N. meningitidis serogroup A (MenA), N. meningitidis serogroup C (MenC), N. meningitidis serogroup Y (MenY), N. meningitidis serogroup W (MenW), H.
  • the host cell comprises one or more nucleotide sequences comprising polysaccharide synthesis genes for producing a capsular polysaccharide from Streptococcus species, or Staphylococcus species (e.g. Streptococcus pneumoniae or Staphylcoccus aureus) .
  • the host cell comprises one or more nucleotide sequences comprising polysaccharide synthesis genes for producing a capsular polysaccharide from Staphylococcus aureus, e.g. from Staphylococcus aureus type 5 and 8.
  • the host cell comprises one or more nucleotide sequences comprising polysaccharide synthesis genes for producing a capsular polysaccharide from Streptococcus pneumoniae.
  • Host cells comprising heterologous nucleotide sequences for producing a bacterial polysaccharide antigen
  • the host cells of the present invention may naturally express one or more nucleotide sequences comprising polysaccharide synthesis genes for production of a saccharide antigen (e.g. a bacterial polysaccharide antigen), or the host cells may be engineered to express one or more such nucleotide sequences.
  • host cells of the present invention may utilize endogenous or heterologous glycosyltransferases for sequential assembly of oligosaccharides in the cytosol (cytosolic glycosyltransferases).
  • Heterologous nucleotide sequences e.g. nucleotide sequences that encode carrier proteins and/or nucleotide sequences that encode other proteins, e.g.
  • heterologous nucleotide sequences are introduced into the host cells of present embodiments using a plasmid, e.g. the heterologous nucleotide sequences are expressed in the host cells by a plasmid (e.g. an expression vector).
  • heterologous nucleotide sequences are introduced into the host cells of present embodiments using the method described in WO14/037585.
  • the host cell comprises one or more nucleotide sequences comprising polysaccharide synthesis genes which are heterologous to the host cell.
  • one or more of said nucleotide sequences comprising polysaccharide synthesis genes which are heterologous to the host cell are integrated into the genome of the host cell.
  • the heterologous nucleotide sequences may encode, without limitation, glycosyltransferases, oligosaccharyl transferases, epimerases, flippases, and/or polymerases.
  • the host cells of present embodiments comprise one or more heterologous nucleotide sequences encoding glycosyltransferase(s), which can be derived from, e.g. Escherichia species, Shigella species, Klebsiella species, Salmonella species, Pseudomonas species, Streptococcus species, or Staphylococcus species.
  • glycosyltransferase(s) can be derived from, e.g. Escherichia species, Shigella species, Klebsiella species, Salmonella species, Pseudomonas species, Streptococcus species, or Staphylococcus species.
  • the host cells of present embodiments may comprise one or more heterologous nucleotide sequences comprising polysaccharide synthesis genes for producing an O-antigen.
  • the host cell comprises one or more nucleotide sequences from Salmonella species, Shigella species, Pseudomonas species or Klebsiella species that encode polysaccharide synthesis proteins for producing an O-antigen.
  • the host cell comprises one or more nucleotide sequences from Shigella species, Pseudomonas species or Klebsiella species (e.g.
  • the host cell comprises one or more nucleotide sequences from Shigella species or Klebsiella species (e.g. Shigella dysenteriae, Shigella flexneri, Shigella sonnei, or Klebsiella pneumoniae) that encode polysaccharide synthesis proteins for producing an O-antigen.
  • Shigella species or Klebsiella species e.g. Shigella dysenteriae, Shigella flexneri, Shigella sonnei, or Klebsiella pneumoniae
  • the host cell comprises one or more nucleotide sequences from Shigella dysenteriae, Shigella flexneri or Shigella sonnei that encode polysaccharide synthesis proteins for producing an O-antigen.
  • the host cell may comprise one or more nucleotide sequences from .S'. dysenteriae type 1, .S', sonnei, and .S', flexneri type 6, and .S', flexneri 2a and 3a that encode polysaccharide synthesis proteins for producing an O-antigen.
  • the host cell comprises one or more nucleotide sequences from Pseudomonas aeruginosa, e.g.
  • the host cell comprises one or more nucleotide sequences from Klebsiella pneumoniae that encode polysaccharide synthesis proteins for producing an O-antigen.
  • the nucleotide sequences that encode an O-antigen may be a rft> cluster.
  • rft> cluster refers to a gene cluster that encodes enzymatic machinery capable of synthesis of an O antigen.
  • the host cells may comprise a rfb gene cluster from Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Pseudomonas aeruginosa, Klebsiella pneumoniae.
  • the host cells of present embodiments may comprise one or more heterologous nucleotide sequences comprising polysaccharide synthesis genes for producing a capsular saccharide.
  • the host cell comprises one or more nucleotide sequences from N. meningitidis serogroup A (MenA), N. meningitidis serogroup C (MenC), N. meningitidis serogroup Y (MenY), N. meningitidis serogroup W (MenW), H.
  • the host cell comprises one or more nucleotide sequences from Streptococcus species, or Staphylococcus species (e.g. Streptococcus pneumoniae or Staphylcoccus aureus) that encode polysaccharide synthesis proteins for producing a capsular polysaccharide.
  • the host cell comprises one or more nucleotide sequences from Staphylococcus aureus, e.g.
  • the host cell comprises one or more nucleotide sequences comprising polysaccharide synthesis genes for producing a capsular polysaccharide from Streptococcus pneumoniae.
  • the nucleotide sequences may be a capsular polysaccharide gene cluster.
  • the host cells may comprise a capsular polysaccharide gene cluster from a Streptococcus strain (e.g. S. pneumoniae, S. pyrogenes, S. agalacticae), a Staphylococcus strain (e.g. S. aureus).
  • the capsular polysaccharide gene cluster for Streptococcus pneumoniae maps between dexB and aliA in the pneumococcal chromosome (see, Llull et al., 1999, J. Exp. Med. 190, 241-251). There are typically four relatively conserved genes: (wzg), (wzh), (wzd), (wze) at the 5' end of the capsular polysaccharide gene cluster (Jiang et al., 2001, Infect. Immun. 69, 1244-1255). Also included in the capsular polysaccharide gene cluster of .S', pneumoniae are wzx (polysaccharide flippase gene) and wzy (polysaccharide polymerase gene).
  • the CP gene clusters of at least 90 .S', pneumoniae serotypes have been sequenced by Sanger Institute (http://www.sanger.ac.uk/Projects/S_pneumoniae/CPS/), and wzx and wzy of at least 89 serotypes have been annotated and analyzed (Kong et al., 2005, J. Med. Microbiol. 54, 351-356).
  • the capsular biosynthetic genes of .S', pneumoniae are further described in Bentley et al. (PloS Genet. 2006 Mar; 2(3): e31 and the sequences are provided in GenBank.
  • the host cells of present embodiments may further comprise a nucleotide sequence encoding a polymerase (e.g. wzy), a flippase (e.g. wzx) and optionally a nucleotide sequence encoding and/or a chain length regulator (e.g. wzz).
  • a polymerase e.g. wzy
  • a flippase e.g. wzx
  • a nucleotide sequence encoding and/or a chain length regulator e.g. wzz
  • the host cells may also comprise heterologous nucleotide sequences that are located outside of a rft> cluster or a capsular polysaccharide cluster.
  • heterologous nucleotide sequences that are located outside of a rft> cluster or a capsular polysaccharide cluster.
  • nucleotide sequences encoding glycosyltransferases and acetyltransferases that are found outside of rfb clusters or capsular polysaccharide clusters and that modify recombinant polysaccharides can be introduced into the host cells.
  • N-linked protein glycosylation (the addition of carbohydrate molecules to an asparagine residue in the polypeptide chain of the target protein) is the most common type of post-translational modification occurring in the endoplasmic reticulum of eukaryotic organisms.
  • the process is accomplished by the enzymatic oligosaccharyltransferase complex (OST) responsible for the transfer of a preassembled oligosaccharide from a lipid carrier (dolichol phosphate) to an asparagine residue of a nascent protein within the conserved sequence Asn-X-Ser/Thr (where X is any amino acid except proline) in the endoplasmic reticulum.
  • OST enzymatic oligosaccharyltransferase complex
  • the host cells of the present invention comprise a nucleotide sequence encoding a heterologous oligosaccharyl transferase, optionally within a plasmid.
  • the oligosaccharyl transferase is an oligosaccharyl transferase from Campylobacter.
  • the oligosaccharyl transferase is a pglB, optionally from Campylobacter jejuni (i.e. pglB; see, e.g. Wacker et al. 2002, Science 298: 1790-1793; see also, e.g. NCBI Gene ID: 3231775, UniProt Accession No. 086154) SEQ ID NO: 54:
  • host cells of the present invention may comprise a nucleotide sequence encoding pglB, optionally pglB from Campylobacter jejuni, optionally a nucleotide sequence encoding pglB from Campylobacter jejuni having a sequence at least 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 54, optionally within a plasmid.
  • Host cells of the present invention may also comprise a nucleotide sequence that encodes a polymerase (e.g. wzy).
  • the polymerase e.g. wzy
  • the polymerase is introduced into a host cell of present embodiments (i.e. the polymerase is heterologous to the host cell).
  • the polymerase is a bacterial polymerase.
  • the polymerase is a capsular polysaccharide polymerase (e.g. wzy) or an O antigen polymerase (e.g. wzy).
  • the polymerase is an O-antigen polysaccharide polymerase (e.g. wzy), e.g.
  • the polymerase is a capsular polysaccharide polymerase (e.g. wzy), e.g. from N. meningitidis serogroup A (MenA), N. meningitidis serogroup C (MenC), N. meningitidis serogroup Y (MenY), N. meningitidis serogroup W (MenW), H.
  • MenA meningitidis serogroup A
  • MenC N. meningitidis serogroup C
  • MenY N. meningitidis serogroup W
  • H H.
  • the polymerase is a capsular polysaccharide polymerase (e.g. wzy) of Streptococcus pneumoniae.
  • Said wzy polymerase may be incorporated (e.g. inserted into the genome or expressed by a plasmid) in said host cell as part of a rfb cluster or capsular polysaccharide cluster.
  • a host cell of present embodiments may further comprise a nucleotide sequence encoding a heterologous wzy polymerase.
  • a host cell of present embodiments may also comprise a nucleotide sequence encoding a flippase (e.g. wzx), e.g. a heterologous flippase.
  • Flippases translocate wild type repeating units and/or their corresponding engineered (hybrid) repeat units from the cytoplasm into the periplasm of host cells (e.g. E. coli).
  • the flippase is a bacterial flippase, e.g. a flippase of the polysaccharide biosynthetic pathway of interest.
  • the host cell of present embodiments comprises a nucleotide sequence encoding a flippase (e.g.
  • the flippase is a capsular polysaccharide flippase (e.g. wzx) of Streptococcus pneumoniae.
  • Other flippases that can be introduced into the host cells of present embodiments are for example from Campylobacter jejuni (e.g. pglK).
  • nucleotide sequences encoding one or more accessory enzymes are introduced into the host cells of present embodiments.
  • a host cell of present embodiments may further comprise one or more of these accessory enzymes.
  • Such nucleotide sequences encoding one or more accessory enzymes can be either plasmid-borne or integrated into the genome of the host cells of present embodiments.
  • Exemplary accessory enzymes include, without limitation, epimerases (see e.g. WO2011/062615), branching, modifying (e.g. to add cholins, glycerolphosphates, pyruvates), amidating, chain length regulating, acetylating, formylating, polymerizing enzymes.
  • a host cell of present embodiments may also comprise a nucleotide sequence encoding a chain length regulator (e.g. wzz), e.g. a heterologous chain length regulator.
  • a chain length regulator e.g. wzz
  • the chain length regulator is a capsular polysaccharide chain length regulator (e.g. wzz) of Streptococcus pneumoniae.
  • present embodiments provide a bioconjugate comprising a modified nanoparticle subunit (or a modified carrier protein fused to a nanoparticle subunit) linked to an antigen (e.g. a bacterial polysaccharide antigen or a yeast polysaccharide antigen or a mammalian polysaccharide antigen).
  • an antigen e.g. a bacterial polysaccharide antigen or a yeast polysaccharide antigen or a mammalian polysaccharide antigen.
  • said antigen is an O-antigen or a capsular polysaccharide.
  • the antigen is an O-antigen from a gram-negative bacterium.
  • a bioconjugate comprising a modified nanoparticle subunit (or a modified carrier protein fused to a nanoparticle subunit) conjugated to an antigen
  • the antigen is a saccharide, optionally a bacterial polysaccharide (e.g. from Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae or Staphylcoccus aureus).
  • the present invention provides a bioconjugate comprising a modified nanoparticle subunit (or a modified carrier protein fused to a nanoparticle subunit) linked to an antigen wherein the antigen is a bacterial polysaccharide (e.g. from Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Klebsiella pneumoniae, or Streptococcus pneumoniae) .
  • a bacterial polysaccharide e.g. from Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Klebsiella pneumoniae, or Streptococcus pneumoniae
  • the present invention provides a bioconjugate comprising a modified nanoparticle subunit (or a modified carrier protein fused to a nanoparticle subunit) linked to an antigen wherein the antigen is a bacterial polysaccharide from Shigella flexneri, Klebsiella pneumoniae or Streptococcus pneumoniae.
  • the antigen is linked to an amino acid on the modified nanoparticle subunit (or a modified carrier protein fused to a nanoparticle subunit) selected from asparagine, aspartic acid, glutamic acid, lysine, cysteine, tyrosine, histidine, arginine or tryptophan (e.g. asparagine).
  • Bioconjugates, as described herein have advantageous properties over chemical conjugates of antigen-carrier protein, in that they require less chemicals in manufacture and are more consistent in terms of the final product generated.
  • a further aspect of present embodiments is a process for producing a bioconjugate nanoparticle subunit that comprises or consists of a modified nanoparticle subunit linked to a saccharide, said process comprising (i) culturing the host cell of present embodiments under conditions suitable for the production of glycoproteins, glycotransferases, and nanoparticle subunits, and (ii) isolating the bioconjugate nanoparticle produced by said host cell, optionally isolating the bioconjugate nanoparticle from a periplasmic extract from the host cell.
  • a further aspect of present embodiments is a process for producing a bioconjugate carrier nanoparticle subunit that comprises (or consists of) a modified carrier protein (linked to a saccharide) fused to a nanoparticle subunit, said process comprising (i) culturing the host cell of present embodiments under conditions suitable for the production of glycoproteins, glycotransferases, and nanoparticle carrier proteins fused to subunits, and (ii) isolating the bioconjugate carrier nanoparticle product produced by said host cell, optionally isolating the bioconjugate carrier nanoparticle from a periplasmic extract from the host cell.
  • bioconjugate nanoparticles can be made using the shakeflask process, e.g. in a LB shake flask.
  • a fed-batch process for the production of recombinant glycosylated proteins in bacteria can be used to produce bioconjugate nanoparticles.
  • the aim is to increase glycosylation efficiency and recombinant protein yield per cell and while maintaining simplicity and reproducibility in the process.
  • Bioconjugate nanoparticles can be manufactured on a commercial scale by developing an optimized manufacturing method using typical E. coli production processes.
  • feed strategies such as batch, chemostat and fed-batch can be used.
  • bioconjugate nanoparticles of present embodiments can be purified for example, by chromatography (e.g. ion exchange, anionic exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins (see, e.g. Saraswat et al. 2013, Biomed. Res. Int. ID#312709 (p. 1-18); see also the methods described in WO 2009/104074). Further, the bioconjugates may be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.
  • nanoparticles or carrier proteins fused to nanoparticle subunits
  • displayed molecules may be incorporated onto or attached to the nanoparticles (or carrier proteins fused to nanoparticle subunits) by any suitable means.
  • amino acid side-chain groups used for conjugation include an amino group on lysine, thiol on cysteine, carboxylic acid on aspartic acids and glutamic acids, and hydroxyl moiety on tyrosine with different chemistries known in the art. Homo- or hetero-bifunctional crosslinkers are available for conjugation.
  • the side-chain amino groups of lysine residues are nucleophiles, so lysine residues exposed at the nanoparticle surface have large solvent accessibility and can be used as sites for conjugation to display molecules.
  • One or more selected amino acid residues within a nanoparticle subunit polypeptide sequence may be modified using methods known in the art to provide a site suitable for chemical bioconjugation at the nanoparticle exterior surface (or carrier protein attached to the nanoparticle exterior surface), where such modification does not disrupt nanoparticle assembly.
  • a modified nanoparticle wherein one or more display molecules (e.g. antigens) are chemically conjugated to residues present at the exterior surface of the nanoparticle.
  • the display molecule(s) may be an oligosaccharide, a polysaccharide, a glycan, or a glycoconjugate, or combinations thereof.
  • Covalent conjugation of saccharides to modified carrier proteins or modified nanoparticles enhances the immunogenicity of saccharides as it converts them from T-independent antigens to T-dependent antigens, thus allowing priming for immunological memory.
  • Conjugation is useful for pediatric and adult vaccines ((see, Ramsay et al. (2001); (Lindberg (1999); Buttery & Moxon (2000); Ahmad & Chapnick (1999); Goldblatt (1998); European Patent 0477508; US Patent No. 5,306,492; WO98/42721; Dick et al. in Conjugate Vaccines (1989); Hermanson, (1996)).
  • An alternative conjugation process involves the use of -NH2 groups in the saccharide (either from de-N-acetylation, or after introduction of amines) in conjunction with bifunctional linkers, as described (W02006/082530).
  • a further alternative process is described in WO96/40795 and Michon et al. (2006). In this process, the free aldehydes groups of terminal 2,5-anhydro-D- mannose residues from depolymerization of type II or type III capsular saccharides by mild cleavage through de-N-acetylation/nitrosation are used for conjugation by reductive amination.
  • conjugate comprising (or consisting of) a modified nanoparticle or modified carrier protein fused to a nanoparticle (e.g. a modified EPA fused to a ferritin subunit) linked to an antigen (e.g. a saccharide antigen, optionally a bacterial polysaccharide antigen).
  • the antigen may be a bacterial polysaccharide antigen, or a yeast polysaccharide antigen, or a mammalian polysaccharide antigen.
  • a conjugate comprising (or consisting of) a modified nanoparticle or modified carrier protein fused to a nanoparticle covalently linked to an antigen (e.g. a saccharide antigen, optionally a bacterial polysaccharide antigen), wherein the antigen is linked (either directly or through a linker) to the modified carrier protein or modified nanoparticle.
  • an antigen e.g. a saccharide antigen, optionally a bacterial polysaccharide antigen
  • the antigen is linked (either directly or through a linker) to the modified carrier protein or modified nanoparticle.
  • the antigen is directly linked to the modified nanoparticle or modified carrier protein fused to a nanoparticle.
  • the antigen is directly linked to an amino acid residue of the modified nanoparticle or modified carrier protein fused to a nanoparticle.
  • SARS Severe Acute Respiratory Syndrome
  • HAV Human Immunodeficiency Virus
  • EBV Epstein-Barr virus
  • RSV Respiratory Syncytial Virus
  • HCMV Human Cytomegalovirus
  • the modified nanoparticle or modified carrier protein fused to a nanoparticle may be covalently linked to the antigen through a chemical linkage obtainable using a chemical conjugation method (i.e. the conjugate is produced by chemical conjugation).
  • the chemical conjugation method may be selected from the group consisting of carbodiimide chemistry, reductive animation, cyanylation chemistry (for example CDAP chemistry), maleimide chemistry, hydrazide chemistry, ester chemistry, and N-hydroysuccinimide chemistry.
  • Conjugates can be prepared by direct reductive amination methods as described in, US200710184072 (Hausdorff) US 4365170 (Jennings) and US 4673574 (Anderson).
  • conjugation method may alternatively rely on activation of the saccharide with l-cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP) to form a cyanate ester.
  • CDAP l-cyano-4-dimethylamino pyridinium tetrafluoroborate
  • Such conjugates are described in PCT published application WO 93/15760 Uniformed Services University and WO 95/08348 and WO 96/29094. See also Chu C. etal. Infect. Immunity, 1983 245 256.
  • the functional groups on one or more amino acids of the polypeptide monomer(s) can be used for site-specific conjugation to a display molecule(s) (e.g. to an antigen or an immunostimulant).
  • Amino acid side-chain groups used for conjugation include amino group on lysine, thiol on cysteine, carboxylic acid on aspartic acids and glutamic acids, and hydroxyl moiety on tyrosine.
  • Heterobifunctional crosslinkers are available for protein conjugation.
  • Primary amines on a first polypeptide can be conjugated to carboxylic acids on a second polypeptide using 1 -ethyl - 3-(-3-dimethylaminopropyl) carbodiimide (EDC) crosslinkers, typically in combination with N- hydroxysuccinimide (NHS).
  • EDC carbodiimide
  • NHS N- hydroxysuccinimide
  • the side-chain amino groups of lysine residues are nucleophiles, so lysine residues exposed at the nanoparticle exterior surface have large solvent accessibility and can be used as sites for conjugation to display molecules.
  • Chemical methods also include sitespecific chemical conjugation through engineered cysteines or selenocysteines (see Siegmund et al. 2016 Scientific Reports 6(39291)). In general, the following types of chemical groups on a modified carrier protein or nanoparticle subunit can be used for coupling / conjugation:
  • Carboxyl for instance via aspartic acid or glutamic acid.
  • this group is linked to amino groups on saccharides directly or to an amino group on a linker with carbodiimide chemistry e.g. with EDAC.
  • Sulphydryl for instance via cysteine.
  • this group is linked to a bromo or chloro acetylated saccharide or linker with maleimide chemistry.
  • this group is activated/modified with bis diazobenzidine.
  • Imidazolyl group (for instance via histidine). In one embodiment this group is activated/modified with bis diazobenzidine.
  • Aldehyde groups can be generated after different treatments such as: periodate, acid hydrolysis, hydrogen peroxide, etc.
  • one or more nucleotide constructs are prepared that recombinantly express a polypeptide sequence (e.g. a contiguous amino acid sequence) comprising the monomer amino acid sequence (e.g. a modified nanoparticle subunit) and the display molecule (e.g. display antigen or display immunostimulant saccharide).
  • a polypeptide sequence e.g. a contiguous amino acid sequence
  • the monomer amino acid sequence e.g. a modified nanoparticle subunit
  • the display molecule e.g. display antigen or display immunostimulant saccharide
  • the modified carrier protein fusion construct self-assembles into a nanoparticle.
  • the modified monomeric subunit or the modified carrier protein fusion construct self-assemble into a nanoparticle.
  • tag-capture system The use of peptide “tag” and binding partner (or “capture” or “dock”) pairs for the spontaneous formation of isopeptide bonds between heterologous molecules (“tag-capture system”) is known (see, e.g. Zakeri et al. 2012 PNAS 109(12): E690-E697 of “SpyTag-SpyCatcher” system; WO2011/098772 (PCT/GB2011/000188), M. HOWARTH and Hatlem etal. 2019 Int. J. Mol. Sci. 20(9): 2129, 19 pages; Ma et al. 2018 Nat. Comm. 9: 1489 (DOI: 10.1038/s41467-018-03931-4); Zhang et al.
  • one embodiment of the present invention is a nanoparticle covalently linked to an carrier (e.g. on its exterior surface) that has undergone bioconjugation.
  • the nanoparticles and the carrier may be conjugated using a tag-capture system (e.g. wherein the tag-capture system is the SpyTag-SpyCatcher binding system (SPYBiotech, Oxford, England); see Hatlem et al. 2019 Int. J. Mol. Sci. 20(9): 2129, 19 pages; Zhang et al. 2020 bioRxiv (doi: 10.1101/2020.06.11.147496)).
  • a tag-capture system e.g. wherein the tag-capture system is the SpyTag-SpyCatcher binding system (SPYBiotech, Oxford, England); see Hatlem et al. 2019 Int. J. Mol. Sci. 20(9): 2129, 19 pages; Zhang et al. 2020 bioRxiv (doi: 10.1101/2020.06.11.147496)).
  • one aspect is a nanoparticle conjugated to a tag molecule of a tag-capture system (optionally wherein the nanoparticle monomer, such as the polypeptide subunit monomer, is conjugated to a tag molecule of a tag -capture system).
  • a nanoparticle is attached to a tag molecule, and the tag molecule attaches to a bioconjugate carrier attached to a carrier molecule.
  • a nanoparticle conjugated to a capture molecule of a tag -capture system optionally wherein the monomer, such as the polypeptide monomer, is conjugated to a capture molecule of a tag -capture system).
  • a nanoparticle is attached to a capture molecule, and the capture molecule attaches to a bioconjugate carrier attached to a tag molecule.
  • a bioconjugate carrier attached to a tag molecule.
  • a heterologous molecule e.g. an antigen/carrier or an immunostimulant
  • the heterologous molecule contains the other sequence of the binding pair
  • an isopeptide bond forms and the heterologous molecule becomes covalently attached to the nanoparticle.
  • Conjugates e.g. chemical conjugates, bioconjugates, etc.
  • the actual conditions used to purify a particular conjugate will depend, in past, on the synthesis strategy (e.g. synthetic production vs. recombinant production) and on factors such as net charge, hydrophobicity, and/or hydrophilicity of the bioconjugate.
  • compositions and sugar chain lengths of the bioconjugates of present embodiments can be used to analyze the compositions and sugar chain lengths of the bioconjugates of present embodiments and to determine glycosylation site usage.
  • Hydrazinolysis can be used to analyze glycans.
  • polysaccharides are released from their protein carriers by incubation with hydrazine according to the manufacturer’s instructions (Ludger Liberate Hydrazinolysis Glycan Release Kit, Oxfordshire, UK).
  • the nucleophile hydrazine attacks the glycosidic bond between the polysaccharide and the nanoparticle subunit protein or the carrier protein and allows release of the attached glycans.
  • N-acetyl groups are lost during this treatment and have to be reconstituted by re-N-acetylation.
  • site usage is reflected by an earlier elution time from a SE HPLC column.
  • site usage may be quantified by quantitative densitometry of purified bioconjugates stained with Coomassie Brilliant Blue following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
  • purified refers to the separation or isolation of a defined product (e.g. a recombinantly expressed nanoparticle (or a recombinantly expressed carrier protein fused to a nanoparticle) that is bioconjugated from a composition containing other components (e.g. a host cell or host cell medium).
  • a composition containing other components e.g. a host cell or host cell medium.
  • a composition that has been fractionated to remove undesired components, and which composition retains its biological activity is considered purified.
  • a purified bioconjugate nanoparticle (or purified carrier protein fused to a nanoparticle) retains its biological activity.
  • Purified means removed from its natural environment and substantially free of impurities from that natural environment (such as other chromosomal and extra-chromosomal DNA and RNA, organelles, and proteins (including other proteins, lipids, or polysaccharides which are also secreted into culture medium or result from lysis of host cells)).
  • a molecule such as an antigen or immunostimulant
  • a purified molecule such as a purified antigen or purified immunostimulant
  • a molecule such as an antigen, agent, immunostimulant, additive, vector, or other compound
  • composition to be suitable (i.e., safe) for pharmaceutical or vaccine use (e.g. administration) with a human or non-human mammal (i.e., for the molecule to be pharmaceutically acceptable)
  • it is at least purified (i.e., not crude).
  • chromatography such as High Performance Liquid Chromatography (HPLC), ionexchange chromatography, and size -exclusion chromatography, hydrophobic interaction, ion exchange, affinity, chelating, and size exclusion; electrophoresis such as gel electrophoresis; centrifugation such as density gradient centrifugation; dialysis; filtration; precipitation; antibody capture; solvent extraction, affinity purification, and combinations thereof.
  • Polypeptides NPs may be expressed with a tag operable for affinity purification, such as a 6xHistidine tag as is known in the art.
  • a His-tagged polypeptide may be purified using, for example, Ni-NTA column chromatography or using anti-6xHis antibody fused to a solid support.
  • a “substantially pure” preparation of polypeptides (or nanoparticles, or carrier proteins fused to nanoparticles) or nucleic acid molecules is one in which the desired component represents at least 50% of the total polypeptide (or nucleic acid) content of the preparation.
  • a substantially pure preparation will contain at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more of the total polypeptide (or nucleic acid) content of the preparation.
  • Methods for quantifying the degree of purification of expressed polypeptides include, for example, determining the specific activity of an active fraction, or assessing the number of polypeptides within a fraction by SDS/PAGE analysis.
  • purification or “purifying” herein refers to the process of removing components from a composition or host cell or culture, the presence of which is not desired. Molecules which have not been subjected to any purification steps (i.e., the molecule as it is found in nature or a “crude” molecule) are not suitable for pharmaceutical use (i.e., not suitable for administration to a subject).
  • the nanoparticle subunit polypeptide or carrier protein fused to nanoparticle subunit polypeptide of present embodiments may be produced by any suitable means, including by recombinant expression or by chemical synthesis, and purified (if necessary) using any suitable method known in the art.
  • the nanoparticle product may be analyzed using methods known in the art, e.g. by crystallography, Dynamic Light Scattering (DLS), Nano-Differential Scanning Fluorimetry (Nano-DSF), and Electron Microscopy, to confirm production of suitable nanoparticles.
  • the expressed polypeptide may include a purification tag and/or a protease site.
  • Various expression systems are known in the art, including those using human (e.g. HeLa) host cells, mammalian (e.g. Chinese Hamster Ovary (CHO)) host cells, prokaryotic host cells (e.g. E. coli), or insect host cells.
  • the host cell is typically transformed with the recombinant nucleic acid sequence encoding the desired polypeptide product, cultured under conditions suitable for expression of the product, and the product purified from the cell or culture medium.
  • Cell culture conditions are particular to the cell type and expression vector, as is known in the art.
  • Host cells can be cultured in conventional nutrient media modified as appropriate and as will be apparent to those skilled in the art (e.g. for activating promoters). Culture conditions, such as temperature, pH and the like, may be determined using knowledge in the art, see e.g. Freshney (1994) and the references cited therein.
  • bacterial host cell systems a number of expression vectors are available including, but not limited to, multifunctional E. coll cloning and expression vectors such as BLUESCRIPT (Stratagene) or pET vectors (Novagen, Madison WI).
  • BLUESCRIPT Stratagene
  • pET vectors Novagen, Madison WI
  • mammalian host cell systems a number of expression systems, including both plasmids and viralbased systems, are available commercially.
  • Eukaryotic or microbial host cells expressing nanoparticle subunit polypeptides or carrier proteins fused to nanoparticle subunit polypeptides can be disrupted by any convenient method (including freeze-thaw cycling, sonication, mechanical disruption), and polypeptides and/or selfassembled nanoparticles or carrier proteins fused to nanoparticle subunit polypeptides can be recovered and purified from recombinant cell culture by any suitable method known in the art (including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography (e.g. using any of the tagging systems noted herein), hydroxyapatite chromatography, and lectin chromatography)).
  • HPLC high performance liquid chromatography
  • expression of a recombinantly encoded nanoparticle subunit polypeptide involves preparation of an expression vector comprising a recombinant polynucleotide under the control of one or more promoters, such that the promoter stimulates transcription of the polynucleotide and promotes expression of the encoded polypeptide.
  • “Recombinant Expression” as used herein refers to such a method.
  • Recombinant expression vectors comprise a recombinant nucleic acid sequence operatively linked to control sequences capable of effecting expression of the gene product.
  • Control sequences are nucleic acid sequences capable of effecting the expression of the nucleic acid molecules and need not be contiguous with the nucleic acid sequences, so long as they function to direct the expression thereof.
  • Recombinant host cells comprise such recombinant expression vectors.
  • a method for preparing an assembled glycoprotein nanoparticle comprising the steps of providing a host cell; expressing a nanoparticle subunit or modified carrier protein fused to a nanoparticle subunit in the host cell, wherein the optionally modified nanoparticle subunit or carrier protein comprises one or more glycosylation sites (e.g.
  • the method comprises expressing a modified nanoparticle subunit such as a modified ferritin, a modified dodecin, or a modified E2p, etc.
  • diluents and/or pharmaceutically acceptable excipients are known in the art and are described, e.g. in Remington’s Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975).
  • pharmaceutically acceptable indicates that the diluent or excipient is suitable for administration to a subject (e.g. a human or non-human mammalian subject).
  • a subject e.g. a human or non-human mammalian subject.
  • the nature of the diluent and/or excipient will depend on the particular mode of administration being employed.
  • parenteral formulations usually include injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • a liquid diluent is not employed.
  • nontoxic solid components can be used, including for example, pharmaceutical grades of trehalose, mannitol, lactose, starch or magnesium stearate.
  • Suitable solid components are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles.
  • suitable excipients can be selected by those of skill in the art to produce a formulation suitable for delivery to a subject by a selected route of administration.
  • the immunogenic or pharmaceutical compositions comprising nanoparticles do not further comprise an adjuvant. In another embodiment, the immunogenic or pharmaceutical compositions comprising nanoparticles do further comprise an adjuvant.
  • bioconjugates of present embodiments are particularly suited for inclusion in immunogenic compositions and vaccines.
  • the present embodiments provide an immunogenic composition comprising a nanoparticle, and optionally a pharmaceutically acceptable excipient and/or carrier.
  • an immunogenic composition e.g. a vaccine composition
  • an adjuvant e.g. an adjuvant
  • adjuvant refers to a compound that when administered in conjunction with or as part of an immunogenic composition of the vaccine of present embodiments augments, enhances and/or boosts the immune response to nanoparticles, but when the compound is administered alone does not generate an immune response to the nanoparticles.
  • adjuvants can enhance an immune response by several mechanisms including, e.g. lymphocyte recruitment, stimulation of B and/or T cells, and stimulation of macrophages.
  • adjuvants are oil in water emulsions (such as squalene or peanut oil), optionally in combination with immune stimulants, such as monophosphoryl lipid A (see Stoute et al. N. Engl. J. Med. 336, 86-91 (1997)).
  • Immunogenic compositions of the present invention may additionally include one or more adjuvants.
  • An “adjuvant” is an agent that enhances the production of an immune response in a non-specific manner.
  • Common adjuvants include suspensions of minerals (e.g. alum, aluminum hydroxide, aluminum phosphate); saponins such as QS21; emulsions, including water-in-oil, and oil-in-water (and variants thereof, including double emulsions and reversible emulsions), liposaccharides, lipopolysaccharides, immunostimulatory nucleic acid molecules (such as CpG oligonucleotides), liposomes, Toll Receptor agonists, Toll-like Receptor agonists (particularly, TLR2, TER4, TER7/8 and TER9 agonists), and various combinations of such components.
  • the assembled nanoparticle is not considered an adjuvant.
  • the immunogenic compositions of present embodiments can be included in a container, pack, or dispenser together with instructions for administration.
  • the immunogenic compositions or vaccines of thereof can be stored before use, e.g. the compositions can be stored frozen (e.g. at about -20°C or at about -70°C); stored in refrigerated conditions (e.g. at about 4°C); or stored at room temperature.
  • the immunogenic compositions or vaccines of present embodiments may be stored in solution or lyophilized.
  • the solution is lyophilized in the presence of a sugar such as sucrose, trehalose or lactose.
  • the vaccines of present embodiments are lyophilized and extemporaneously reconstituted prior to use.
  • the immunogenic composition or vaccine of present embodiments is administered by the intramuscular delivery route.
  • Intramuscular administration may be to the thigh or the upper arm. Injection is typically via a needle (e.g. a hypodermic needle), but needle-free injection may alternatively be used.
  • a typical intramuscular dose is 0.5 mb.
  • the immunogenic composition or vaccine of present embodiments is administered by the intradermal administration.
  • Human skin comprises an outer "homy" cuticle, called the stratum comeum, which overlays the epidermis. Underneath this epidermis is a layer called the dermis, which in turn overlays the subcutaneous tissue.
  • the conventional technique of intradermal injection, the "mantoux procedure,” comprises steps of cleaning the skin, and then stretching with one hand, and with the bevel of a narrow-gauge needle (26 to 31 gauge) facing upwards the needle is inserted at an angle of between 10 to 15°. Once the bevel of the needle is inserted, the barrel of the needle is lowered and further advanced whilst providing a slight pressure to elevate it under the skin. The liquid is then injected very slowly thereby forming a bleb or bump on the skin surface, followed by slow withdrawal of the needle.
  • the immunogenic composition or vaccine of present embodiments is administered by the intranasal administration.
  • the immunogenic composition or vaccine is administered locally to the nasopharyngeal area, e.g. without being inhaled into the lungs.
  • an intranasal delivery device which delivers the immunogenic composition or vaccine formulation to the nasopharyngeal area, without or substantially without it entering the lungs.
  • Suitable devices for intranasal administration of the vaccines according to present embodiments are spray devices. Suitable commercially available nasal spray devices include ACCUSPRAYTM (Becton Dickinson).
  • the amount of bioconjugate in each immunogenic composition or vaccine dose is selected as an amount which induces an immunoprotective response without significant, adverse side effects in typical vaccines. Such amount will vary depending upon which specific immunogen is employed and how it is presented.
  • the content of bioconjugate will typically be in the range 1-100 pg, suitably 5-50 pg.
  • the present invention also provides an immunogenic composition of present embodiments, or the vaccine of present embodiments, for use in medicine.
  • a method is provided of inducing an immune response in a subject (e.g. human), the method comprising administering a therapeutically or prophylactically effective amount of bioconjugate nanoparticle (optionally fused to a carrier protein), an immunogenic composition thereof, or a vaccine thereof, to a subject (e.g. human) in need thereof.
  • a bioconjugate nanoparticle thereof, an immunogenic composition thereof or a vaccine thereof for use in inducing an immune response in a subject (e.g. human).
  • a bioconjugate nanoparticle (optionally fused to a carrier protein), the immunogenic composition thereof, or the vaccine thereof for use in the manufacture of a medicament for inducing an immune response in a subject (e.g. human).
  • a further aspect is a method of inducing an immune response for the purpose of treating and/or preventing a bacterial infection of a subject, comprising administering to the subject an immunologically effective amount of the bacterial antigenic molecule displayed on the surface of nanoparticles or carrier molecules fused to nanoparticles that display the antigenic molecule to which an immune response is desired, where said antigens can induce a protective or therapeutic immune response.
  • Such bacterial antigenic molecules displayed on the surface of nanoparticles or carrier molecules fused to nanoparticles may be within an immunogenic or pharmaceutical composition as described herein.
  • a single dose is administered to the subject.
  • the dose may be adjuvant-free, or it may further comprise an adjuvant.
  • the conjugate (e.g. bioconjugate) nanoparticle of present embodiments is an immunogenic composition or a vaccine that can be used to induce an immune response against a bacterium, e.g. Shigella species, Pseudomonas aeruginosa, Klebsiella pneumoniae, N. meningitidis, H. influenzae type b (Hib), Group B Streptococcus (GBS), Streptococcus pneumoniae, or Staphylococcus aureus.
  • a bacterium e.g. Shigella species, Pseudomonas aeruginosa, Klebsiella pneumoniae, N. meningitidis, H. influenzae type b (Hib), Group B Streptococcus (GBS), Streptococcus pneumoniae, or Staphylococcus aureus.
  • glycosyltransferase is glycosyltransferase pglB (optionally derived from Campylobacter jejuni).
  • the capsular polysaccharide or oligosaccharide is selected from Streptococcus or Klebsiella species.
  • the nanoparticle subunit is selected from the group consisting of: an E2p monomer subunit, a ferritin monomer subunit, and a dodecin monomer subunit.
  • a bacterial polysaccharide e.g. from Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae or Staphylcoccus aureus
  • composition of any one of paragraphs 23 to 49, wherein the dodecin subunit is encoded by the nucleic acid comprising SEQ ID NO: 3.
  • amino acids 1-167 of SEQ ID NO: 11 or a self-assembling fragment thereof or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acids 1-167 of SEQ ID NO: 11 or a self-assembling fragment thereof;
  • the one (or more) glycosylation sequence(s) have been added next to (e.g. within about 1-15 amino acids) or substituted for, at the N terminus, amino acid residues 1-10 of SEQ ID NO: 11 (e.g. amino acid residues 1-10, 1-5, 1-4, 1-3, 1-2, 1 of SEQ ID NO: 11),
  • the one (or more) glycosylation sequence(s) have been added next to or substituted for one or more amino acids from amino acid residues 74-84 of SEQ ID NO: 11 (e.g. amino acid residues 76-82 or 78-80 of SEQ ID NO: 11, or residue K79 of SEQ ID NO: 11); (iii) the one (or more) glycosylation sequence(s) have been added next to or substituted for one or more amino acids from amino acid residues 141-154 of SEQ ID NO: 11 (e.g. amino acid residues 143-152 or 145-150 of SEQ ID NO: 11, amino acid residues 146-149 of SEQ ID NO: 11),
  • a composition comprising a modified ferritin nanoparticle, the ferritin NP comprising one or more ferritin subunits, each ferritin nanoparticle subunit comprising:
  • modified amino acid sequence comprises one or more of the following mutations selected from: M3 II, K120L, A124R, M144I, and I154M wherein the numbering of the positions is relative to SEQ ID NO:20.
  • composition of paragraph 64 or paragraph 65, wherein the modified amino acid sequence comprises each of mutations M3 II, K120L, A124R, M144I, and I154M, wherein the numbering of the positions is relative to SEQ ID NO:20.
  • composition of paragraph 64, wherein the modified ferritin subunit comprises the amino acid sequence of SEQ ID NO: 24.
  • a composition comprising a modified exotoxin protein A (EP A) ferritin nanoparticle, wherein each subunit comprises a modified EPA fused directly or indirectly to ferritin.
  • EP A exotoxin protein A
  • composition of paragraph 83 or 84, wherein the modified EPA-ferritin subunit comprises amino acids 36-663 of SEQ ID NO: 52, or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acids 36-663 of SEQ ID NO: 52.
  • the host cell of paragraph 91 comprising: i) one or more nucleotide sequences comprising polysaccharide synthesis genes, optionally for producing a bacterial polysaccharide antigen (e.g. an O-antigen from a gram-negative bacterium optionally from Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Pseudomonas aeruginosa, Klebsiella pneumoniae or a capsular polysaccharide from a gram-positive bacterium optionally from Streptococcus pneumoniae or Staphylcoccus aureus) or a yeast polysaccharide antigen or a mammalian polysaccharide antigen, optionally integrated into the host cell genome; ii) a nucleotide sequence encoding a heterologous oligosaccharyl transferase, such as glycosyltransferase pglB, optionally within a plasm
  • a host cell according to paragraph 91 or 92 further comprising a nucleotide sequence encoding a polymerase (e.g. wzy), a flippase (e.g. wzx) and optionally a nucleotide sequence encoding a chain length regulator (e.g. wzz).
  • a polymerase e.g. wzy
  • a flippase e.g. wzx
  • a nucleotide sequence encoding a chain length regulator e.g. wzz
  • oligosaccharyl transferase is a PglB, optionally derived from Campylobacter jejuni.
  • An immunogenic composition comprising an assembled glycoprotein nanoparticle according to any preceding paragraph.
  • the immunogenic composition of paragraph 95 further comprising an adjuvant.
  • said adjuvant is selected from the group consisting of alum, aluminium hydroxide, aluminium phosphate, a saponin, a water-in-oil emulsion, an oil-in-water emulsion, a liposaccharide, a lipopolysaccharide, an immunostimulatory nucleic acid molecules, a liposome, and a Toll Receptor or Toll- Like Receptor agonist.
  • the immunogenic composition of any one of paragraphs 95 to 97 which does not further comprise an adjuvant.
  • a vaccine comprising the immunogenic composition of any one of paragraphs 95 to 99, and optionally an adjuvant.
  • Use of an assembled glycoprotein nanoparticle according to any one of paragraphs 1 to 22, an immunogenic composition according to any one of paragraphs 95 to 99, or a vaccine according to paragraph 100 for the manufacture of a medicament, for inducing an immune response in a subject.
  • the method comprising administering a therapeutically or prophylactically effective amount of the immunogenic composition of any one of paragraphs 95 to 99 or the vaccine of paragraph 100, to a subject (e.g. human) in need thereof.
  • a subject e.g. human
  • the immunogenic composition of any one of paragraphs 95 to 99 or the vaccine of paragraph 100 for use in treating and/or preventing a yeast or bacterial infection in a subject (e.g. human). .
  • the immunogenic composition of paragraph 111 wherein X is Q (glutamine) and Z is A (alanine). .
  • the immunogenic composition of paragraph 111 or 112 wherein the amino acid sequence further comprises a peptide tag, optionally said peptide tag comprises six histidine residues and optionally said peptide tag is located at the C-terminus of the amino acid sequence.
  • the immunogenic composition of any one of paragraphs 111 to 113 further comprising a conjugate (e.g. a bioconjugate) comprising a optionally modified nanoparticle subunit linked to an antigen (e.g. a saccharide antigen, optionally a bacterial polysaccharide antigen). .
  • a bacterial polysaccharide e.g. from Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae or Staphylcoccus aureus
  • concentrations or levels of a substance such as an antigen
  • concentrations or levels of a substance are intended to be approximate.
  • concentration is indicated to be at least (for example) 200 pg, it is intended that the concentration be understood to be at least approximately (or “about” or “ ⁇ ”) 200 pg.
  • Constructs for insertion of glycosites were designed, made, and evaluated for periplasmic expression, glycosylation, and assembly. Nanoparticles were characterized and examined under electron microscopy and in vitro studies were conducted. Representative examples are provided below.
  • GSGGGDQNATGSGGG SEQ ID NO: 36
  • GSGDQNATGSG SEQ ID NO: 31
  • additional rounds of mutagenesis in the available and selected single site variants were performed as needed.
  • flanking sequence of between 1-5 amino acid residues may be added to either or both sides of the consensus sequence DQNAT (SEQ ID NO: 32), when added internally, or at the N- or C- terminus.
  • E2p representative examples showing expression, assembly, and glycosylation in a single step (e.g. based on SDS-PAGE and Western blot characterization) are shown in FIG. 11A for Spl2F and in FIG. 12A-12D for KpO3b.
  • ferritin nanoparticles from Helicobacter pylori and Pseudomonas aeruginosa revealed that suitable positions for engineering glycosites are the N-terminus and the 4 loops connecting the helices: A and B (loop 1), B and C (loop 2), C and D (loop 3) and D and E (loop 4).
  • Ferritin C-terminus is not solvent accessible and therefore not suitable for addition of glycosites.
  • For each loop a set of 4-6 glycosite variants was designed by varying the residues that were mutated to a glycosite.
  • FIGs. 3A - 8 and 13A-14C Representative examples showing expression, assembly, and glycosylation in a single step (e.g. based on SDS-PAGE and Western blot characterization) are shown in FIGs. 3A - 8 and 13A-14C. These examples show assembly and glycosylation of nanoparticles expressed in E. coli for different glyco-antigens, for N- based glycosylation sites, including Sp 12F and KpO3b.
  • glycosites were engineered at the N-terminus and in 3 internal positions including residues 12-16, 75-79 and 118. These residues were substituted with DQNAT (SEQ ID NO: 32). These residues appeared to undergo weak glycosylation. Results are not shown.
  • coli strains are derivatives of strain W3110, which include a deletion in the lipopolysaccharide O-antigen ligase gene waaL, the deletion or replacement of the 016 O-antigen cluster rfb and the replacement of a genomic cluster with the cluster responsible for the biosynthesis of the wanted recombinant glycan (e.g. Klebsiella pneumoniae O-antigen (KpO-antigen), 5pl2F, and 5p33F capsular polysaccharides.
  • KpO-antigen Klebsiella pneumoniae O-antigen
  • 5pl2F e.g. Klebsiella pneumoniae O-antigen (KpO-antigen)
  • 5p33F capsular polysaccharides e.g. Klebsiella pneumoniae O-antigen (KpO-antigen)
  • the cluster of genes for the biosynthesis of Kp05 glycan was integrated in the 016 locus.
  • the cluster of genes for the biosynthesis of /?O3b glycan was integrated into the 016 locus as well.
  • manB phosphomannomutase
  • manC mannose-l-phosphate guanyly transferase
  • the cluster responsible for the biosynthesis of colanic acid has been replaced by the S.pneumoniae serotype 33F capsular polysaccharide biosynthesis cluster (cpsSp33Y).
  • the O-antigen ligase-encoding gene waaL has been replaced by the wchA gene and the cluster responsible for the biosynthesis of the 016 O- antigen polysaccharide rbf) has been replaced by the cassette containing the genes wbbH-gnd.
  • Periplasmic extracts and IMAC-enriched periplasmic extracts were prepared using the same protocol also used for determining a level of expression. For example, the samples were analyzed by SDS-PAGE, anti-His Western blot and anti-glycan Western blots. The read-out by Western blot or SDS-PAGE showed increased molecular weight of glyco-conjugates suggesting higher sugar to protein ratio due to increased number of glycosites.
  • a library of 23 bacterial signal sequences was used.
  • the library contained the following signal sequences: ArgT, BtuB, DsbA (SEQ ID NO: 10), Figi (SEQ ID NO: 42), OmpAVl, 0mpAV2, OmpC, OmpT, PhoA, TolB (SEQ ID NO: 41), DegP, FhuA, Hla, Ltllb (SEQ ID NO: 46), LutA, MalE (SEQ ID NO: 44), OmpA (SEQ ID NO: 43), PelB (SEQ ID NO: 45), RBP, SipA (SEQ ID NO: 48), Sufi, TorA, and XynA (SEQ ID NO: 47).
  • Results Representative SDS-PAGE and/or Western blots for Spl2F ferritin Hp nanoparticles are shown in FIG. 3B, 3D, 4B, 5A.
  • results For Spl2F ferritin Hp, representative examples include SDS-PAGE (FIG. 5A), DLS (FIG. 5C) showing an average particle diameter of 30 nm and an electron micrograph (FIG. 5D).
  • KpO3b ferritin Hp representative examples include SDS-PAGE (FIG. 7A), DLS (FIG. 7C) showing an average particle diameter of about 25 nm, and an electron micrograph (FIG. 7D).
  • SDS-PAGE For Spl2F-dodecin, representative examples include SDS-PAGE (FIG. 9D).
  • KpO3b- dodecin representative examples include SDS-PAGE (FIG. 10A), DLS (FIG. 10C) showing an average particle diameter of about 17 nm, and an electron micrograph (FIG. 10D).
  • representative examples include SDS-PAGE (FIG. 12A), DLS (FIG. 12C) showing an average particle diameter of about 42 nm and an electron micrograph (FIG. 12D).
  • Periplasmic extracts were analysed by immunoblots against polysaccharide attached to the modified nanoparticle subunit. SDS-PAGE analysis was carried out on IMAC enriched periplasmic extract of E.coli strains producing antigen polysaccharide and expressing PglB.
  • the hydrodynamic diameter of the assembled nanoparticles in solution was measured with dynamic light scattering (DLS). Protocols for DLS are known in the art.
  • Dodecin modified amino acid sequence (signal sequence, N term glycotag sequence, (G55N, V80T, A83T corresponding to G25N, V50T, A53T of SEQ I D NO:2)) and Histidine tag
  • Dodecin modified amino acid sequence (signal sequence, (G44N, V69T, A72T corresponding to G25N,
  • Ferritin H. pylori modified amino acid sequence N-terminus glycotag sequence
  • Histidine tag N-terminus glycotag sequence
  • Ferritin H. pylori modified amino acid sequence (signal sequence, N + L2 glycosites) and Histidine tag
  • Ferritin H. pylori modified amino acid sequence (signal sequence, N + L4 glycosites) and Histidine tag
  • Ferritin H. pylori modified amino acid sequence (signal sequence, N + L2 + L4 glycosites) and Histidine tag
  • E2p modified amino acid sequence (signal sequence N-terminus glycotag sequence) and Histidine tag

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Abstract

La présente invention concerne le domaine des nanoparticules modifiées, des compositions immunogènes et des vaccins comprenant les nanoparticules modifiées, leur fabrication et l'utilisation de telles compositions en médecine.
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